A rapid procedure for the isolation of cyoplasmic membrane vesicles of Bacillus subtilis is described in Chapter II; the method circumvents the protoplast formation step and consists in essence of treatment of the cells with lysozyme in a hypnotic medium. ...

Zie: Summary

Bisschop, Arnold,

Bisschop, Arnold

ARTS repository - University of Groningen

   University of GroningenActive transport in cutoplasmic membrane vesicles of Bacillus subtilisBisschop, ArnoldIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1976Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Bisschop, A. (1976). Active transport in cutoplasmic membrane vesicles of Bacillus subtilis. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 13-02-2023ESUI{MARYA  r a p i d  p r o c e d u r e  f o r  t h e  i s o l a t i o n  o f  c y t o p l a s m i c  m e m b r a n e  v e s i c l e s  o f  B a -c z l l u s  s u b t i L i s  i s  d e s c r i b e d  i n  C h a p t e r  I  l ;  t h e  m e t h o d  c i r c u m v e n t s  t h e  p r o t o -p l a s t  f o r m a t i o n  s t e p  a n d  c o n s i s t s  i n  e s s e n c e  o f  t r e a t m e n t  o f  t h e  c e l l s  w i t hi y r o r y * "  i n  a  h y p o t o n i c  m e d i u m .  M e m b r a n e  v e s i c l e s  o b t a i n e d  i n  t h i s  w a y  h a v e  al o w  e n d o g e n o u s  o x y g e n  c o n s u m p t i o n  a n d  a  l o w  e n d o g e n o u s  a c t i v i t y  f o r  t r a n s p o r to f  s e v e r a l  s o l u t e s .  T h e  m e m b r a n e  v e s i c l e s  a r e  d e v o i d  o f  o t h e r  c e l l  c o m P o n e n t sa s  j u d g e d  b y  e l e c t r o n  m i c r o s c o p i c a l  e x a m i n a t i o n  o f  t h i n  s e c t i o n s  o f  t h e  v e s -í c l e s .  F r e e z e - e t c h  s t u d i e s  o f  i n t a c t  c e l l s ,  p r o t o p l a s t s ,  a n d  m e m b r a n e  v e s i c l e ss h o w  t h a t  t h e  o r i e n t a t i o n  o f  t h e  v e s i c l e  m e m b r a n e  i s  i d e n t i c a l  t o  t h a t  o f  t h e: ; t o p l a s m i c  m e m b r a n e  i n  i n t a c t  c e l  l s  a n d  p r o t o p l a s t s .A c t i v e  t r a n s p o r t  o f  s o l u t e s  i n  m e m b r a n e  v e s i c l e s  f r o n  B .  s u b t í L í s  i s  c o u p l e d' :  e l e c t r o n  t r a n s f e r  i n  t h e  r e s p i r a t o r y  c h a i n  w i t h  o x y g e n  a s  t e r m i n a l  e l e c t r o n+ : : e f , t o r .  T h e  b e s t  e l e c t r o n  d o n o r s  f o r  t h e  e n e r g i z a t i o n  o f  t r a n s p o r t  a r e  r e -i - : e c : - n i c o t i n a m i d e - a d e n i n e - d i n u c l e o r i d e  ( N A D H )  a n d  t h e  n o n - p h y s i o l o g i c a l  e -= : : - o n  d o n o r  a s c o r b a t e + p h e n a z i n e m e t h o s u l f a t e  ( A s c - P M S ) .  L ' i t h  A s c - P M S  a s  e l e c -- - : ^  d o n o r  t h e  t r a n s p o r t  a c t i v i t i e s  o f  t h e  m e m b r a n e  v e s i c l e s  a r e  c o m p a r a b l e.  : ^  t h o s e  o f  i n t a c t  c e l l s  a n d  t h e s e  a c t i v i t i e s  a r e  r e t a i n e d  f o r  a  p r o l o n g e d; < -  c i  o f  t i m e .: ' e c t r o n  t r a n s f e r  f r o m  N A D H  t o  f e r r i c y a n i d e  u n d e r  a n a e r o b i c  c o n d i t i o n s  a l s o: : - : e n e r a t e  e n e r g y  f o r  a c t i v e  t r a n s p o r t  i n  m e m b r a n e  v e s i c l e s  o f  t h e  p r e d o m i -- : - :  l r , ,  a e r o b i c  b a c t e r i u n  B .  s u b t í L í s .  Í h e  m e m b r a n e - i m p e r m e a b l e  e c t r o n  a c -: í ; : o r  f e r r i c y a n i d e ,  a c c e p t s  e l e c t r o n s  f r o m  c y t o c h r o m e s  a t  t h e  t e r m i n a l: '  : 3 s e - s i d e  o f  t h e  r e s p i r a t o r y  c h a i n  a n d  n o t  a t  a l l  f r o m  N A D H  d e h y d r o g e n a s e:  - € : i l y  ( C h ê p t e r  l l l ) .  C o n c i u s i o n s  a b o u t  t h e  l o c a ' l i z a t i o n  o f  d e h y d r o g á n " r " ,-  : r e  m e m b r a n e  a n d  a b o u t  t h e  o r i e n t a t i o n  o f  t h e  v e s i c l e  m e m b r a n e  b a s e d  o n  a c -'  .  : y  m e a s u r e m e n t s  w i t h  f e r r i c y a n i d e  a r e  t h e r e f o r e  n o t  j u s t i f i e d .Y : - b r ê n e  v e s i c l e s  o f  B .  s u b t i L i s  p e r f o r m  a c t i v e  t r a n s p o r t  o f  a m i n o - ,  m o n o - ,:  - .  a n d  t r i c a r b o x y l i c  a c i d s .  T r a n s p o r t  o f  t h e  d i c a r b o x y l i c  a m í n o  a c i d s  g l u; - :  a s p  i n  t h e s e  v e s i c l e s  i s  m e d i a t e d  b y  a  s i n g l e ,  h i g h l y  s p e c i f i c ,  h i g h  a f -:  - ; l y  s y s t e m .  T h e  s y s t e m  h a s  n o  a f f i n i t y  f o r  o t h e r  n a t u r a l  a m i n o  a c i d s  o r  f o r; : . . - a l  m o n o -  a n d  d i c a r b o x y l  i c  a c i d s  ( C h a p t e r  l V )  .- : e  d i c a r b o x y l  i c  a c i d s  L - m a l a t e ,  f u m a r a t e  a n d  s u c c i n a t e  a r e  a l s o  t r a n s p o r t e d: ,  : ' r e  h i g h l y  s p e c i f i c ,  h i g h  a f f i n i t y  t r a n s p o r t  s y s t e m  l n  B .  s u b t í L i s  \ J 2 3.--: .àe!e\ \ \ .  \ \ \s s\ste(\ se.e(\s to be consti tut ive and has aff ini ty for qlu andasp, but these d\carboxl\\c cnniro ac\Às are not Lranspor\eè b1 \\ \s s)s\es\. \ \i s  s u g g e s t e d  t h a t  g l u  a n d  a s p  b i n d  a t  a  s i t e  o n  t h i s  c a r r ' i e r  d i s t i n c t  f r o m  t h es i t e  w h e r e  t h e  t r a n s p o r t  s o l u t e s  a r e  b o u n d .M e m b r a n e  v e s i c l e s  i s o l a t e d  f r o m  c e l l s  a t  d i f f e r e n t  s t a g e s  o f  g r o w t h  o x i d i z eN A D H  a t  d i f f e r e n t  r a t e s .  D u r i n g  t h e  e x p o n e n t i a l  g r o w t h  p h a s e ,  a  3 - 4  f o l d  i n -c r e a s e  i s  o b s e r v e d  a n d  m a x i m a l  l e v e l s  ê r e  f o u n d  i n  v e s i c l e s  i s o l a t e d  f r o m  s t a -t i o n a r y  p h a s e  c e l l s .  I n i t i a l  r a t e s  o f  a m i n o  a c i d  t r a n s p o r t '  e n e r g i z e d  b y  N A D H ,c l o s e l y  p a r a l  l e l s  t h e  i n c r e a s e  i n  N A D H  o x i d a t i o n  r a t e .  A t  a l l  r a t e s  o f  N A D Ho x i d a t i o n  t h e  t r a n s p o r t  o f  o n e  m o l e c u l e  o f  a  g i v e n  a m i n o  a c i d  r e q u i r e s  t h eo x i d a t i o n  o f  a  c o n s t a n t  n u m b e r  o f  N A D H  m o l e c u l e s  ( C h a p t e r  V l ) .  T h i s  n u m b e rv a r i e s  f r o m  1 5 0 - 2 6 0  f o r  t h e  d i f f e r e n t  a m i n o  a c i d s .M e n a q u i  n o n e s  ê r e  e s s e n t  i a  I  e l e c t r o n  c a r r i e r s  f u n c t  i o n  i  n g  b e t w e e n  d e h y d r o g e n -a s e s  a n d  c y t o c h r o m e s  i n  t h e  r e s p i r a t o r y  c h a i n  o f  B .  s u b t i L í s .  l í e m b r a n e  v e s -i c l e s  f r o m  t h e  m e n a q u i n o n e - d e f i c i e n t  m u t a n t  B .  s u b t í L í s  a t ' o  D  o x i d i z e  N A D H  a ta  l o w  r a t e .  l n  t h e s e  v e s i c l e s  t h e  N A D H  o x i d a s e  a c t i v i t y  c a n  b e  r e c o n s t i t u t e db y  t h e  i n c o r p o r a t i o n  o f  m e n a d i o n e  ( a  m e n a q u i n o n e - a n a l o g u e )  t o  e v e n  h i g h e ró)r a t e s  t h a n  o b s e r v e d  i n  w i l d  t y p e  v e s i c l e s .  T h e  N A D H  o x i d a s e  ê c t i v i i . ,  : j  : - *m e m b r a n e  v e s i c l e s  i n c r e a s e s  I  i n e a r l y  v í i t h  t h e  m e n a d i o n e  c o n t e n t  o .  : - Ê  - r - -b r a n e  a n d  a  3 5 - f o l d  s t i m u l a t i o n  i s  o b t a i n e d  i n  f u l l y  r e c o n s t i t u t e C  , e i ' : : :( c h a p t e r  V t t ) .I n  a g r e e m e n t  w i t h  t h e s e  o b s e r v a t i o n s  m e m b r a n e  v e s i c l e s  f  r o m  : .  . - . . .  :  'D  a c c u m u l a t e  a m i n o  a c i d s  i n  t h e  p r e s e n c e  o f  A s c - P M S ,  b u t  n o t  w i t h  t i i l -e v e r ,  m e m b r a n e  v e s i c l e s  f r o m  t h i s  m u t ê n t ,  r e c o n s t i t u t e d  w i t h  m e n a d i o r e ,  : : -p l a y  N A D H - d r i v e n  t r a n s p o r t  a c t i v i t y .  T h i s  a c t i v i t y  i n c r e a s e s  I  i n e a r l . .  , . - : -t h e  N A D H  o x i d a s e  a c t i v i t y ,  b u t  m a x i m a l  t r a n s p o r t  a c t i v i t i e s  a r e  r e a c n e :  _ - : r .c o n d i t i o n s  w h e r e  t h e  N A D H  o x i d a s e  a c t i v i t y  i s  n o t  y e t  m a x i m a l .  T h i s  i ^ : ' : : : t rt h a t  t h e  r a t e  o f  e n e r g y  s u p p l y  i s  t h e  l i m i t i n g  f a c t o r  f o r  t r a n s p o r r  a . -  ' : , .N A D H  o x i d a s e  a c t i v i t i e s  a n d  t h a t  t h e  t r a n s p o r t  s y s t e m s  t h e m s e ' l v e s  b e c c - eI  i m i t i n g  u n d e r  c o n d i t i o n s  o f  h i g h  N A D H  o x i d a s e  a c t i v i t i e s .  U n d e r  e n e r c . -I i m i t i n g  c o n d i t i o n s  1 3 5 - 2 3 5  m o l e c u l e s  o f  N A D H  h a v e  t o  b e  o x i d i z e d  i n  o - : e .  : :t r a n s p o r t  o n e  m o l e c u ' l e  o f  a m i n o  a c i d .  A t  a l l  l e v e l s  o f  e n e r g y  s u p p l y . c : : , * -p e t i t i o n  b y  t h e  d i f f e r e n t  a m i n o  a c i d  t r a n s p o r t  s y s t e m s  i s  o b s e r v e d  í c r : - ea v a i l a b l e  e n e r g y .  T h e s e  o b s e r v a t i o n s  i n d i c a t e  t h a t  o n l y  a  f r a c t i o n  o f  : - ee n e r g y ,  g e n e r a t e d  b y  t h e  r e s p i r a t o r y  c h a i n ,  i s  u s e d  f o r  t h e  t r a n s p o r t : j : -a m i n o  a c i d  a n d  t h a t  t h e  b u l k  o f  t h e  e n e r g y  d i s s i p a t e s  u i a  o t h e r  c h a n n e t s  -t h e  m e m b r a n e  v e s i c l e s .86

Breton

Active transport in cutoplasmic membrane vesicles of Bacillus subtilis

   University of GroningenActive transport in cutoplasmic membrane vesicles of Bacillus subtilisBisschop, ArnoldIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1976Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Bisschop, A. (1976). Active transport in cutoplasmic membrane vesicles of Bacillus subtilis. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 09-09-2023ESUI{MARYA  r a p i d  p r o c e d u r e  f o r  t h e  i s o l a t i o n  o f  c y t o p l a s m i c  m e m b r a n e  v e s i c l e s  o f  B a -c z l l u s  s u b t i L i s  i s  d e s c r i b e d  i n  C h a p t e r  I  l ;  t h e  m e t h o d  c i r c u m v e n t s  t h e  p r o t o -p l a s t  f o r m a t i o n  s t e p  a n d  c o n s i s t s  i n  e s s e n c e  o f  t r e a t m e n t  o f  t h e  c e l l s  w i t hi y r o r y * "  i n  a  h y p o t o n i c  m e d i u m .  M e m b r a n e  v e s i c l e s  o b t a i n e d  i n  t h i s  w a y  h a v e  al o w  e n d o g e n o u s  o x y g e n  c o n s u m p t i o n  a n d  a  l o w  e n d o g e n o u s  a c t i v i t y  f o r  t r a n s p o r to f  s e v e r a l  s o l u t e s .  T h e  m e m b r a n e  v e s i c l e s  a r e  d e v o i d  o f  o t h e r  c e l l  c o m P o n e n t sa s  j u d g e d  b y  e l e c t r o n  m i c r o s c o p i c a l  e x a m i n a t i o n  o f  t h i n  s e c t i o n s  o f  t h e  v e s -í c l e s .  F r e e z e - e t c h  s t u d i e s  o f  i n t a c t  c e l l s ,  p r o t o p l a s t s ,  a n d  m e m b r a n e  v e s i c l e ss h o w  t h a t  t h e  o r i e n t a t i o n  o f  t h e  v e s i c l e  m e m b r a n e  i s  i d e n t i c a l  t o  t h a t  o f  t h e: ; t o p l a s m i c  m e m b r a n e  i n  i n t a c t  c e l  l s  a n d  p r o t o p l a s t s .A c t i v e  t r a n s p o r t  o f  s o l u t e s  i n  m e m b r a n e  v e s i c l e s  f r o n  B .  s u b t í L í s  i s  c o u p l e d' :  e l e c t r o n  t r a n s f e r  i n  t h e  r e s p i r a t o r y  c h a i n  w i t h  o x y g e n  a s  t e r m i n a l  e l e c t r o n+ : : e f , t o r .  T h e  b e s t  e l e c t r o n  d o n o r s  f o r  t h e  e n e r g i z a t i o n  o f  t r a n s p o r t  a r e  r e -i - : e c : - n i c o t i n a m i d e - a d e n i n e - d i n u c l e o r i d e  ( N A D H )  a n d  t h e  n o n - p h y s i o l o g i c a l  e -= : : - o n  d o n o r  a s c o r b a t e + p h e n a z i n e m e t h o s u l f a t e  ( A s c - P M S ) .  L ' i t h  A s c - P M S  a s  e l e c -- - : ^  d o n o r  t h e  t r a n s p o r t  a c t i v i t i e s  o f  t h e  m e m b r a n e  v e s i c l e s  a r e  c o m p a r a b l e.  : ^  t h o s e  o f  i n t a c t  c e l l s  a n d  t h e s e  a c t i v i t i e s  a r e  r e t a i n e d  f o r  a  p r o l o n g e d; < -  c i  o f  t i m e .: ' e c t r o n  t r a n s f e r  f r o m  N A D H  t o  f e r r i c y a n i d e  u n d e r  a n a e r o b i c  c o n d i t i o n s  a l s o: : - : e n e r a t e  e n e r g y  f o r  a c t i v e  t r a n s p o r t  i n  m e m b r a n e  v e s i c l e s  o f  t h e  p r e d o m i -- : - :  l r , ,  a e r o b i c  b a c t e r i u n  B .  s u b t í L í s .  Í h e  m e m b r a n e - i m p e r m e a b l e  e c t r o n  a c -: í ; : o r  f e r r i c y a n i d e ,  a c c e p t s  e l e c t r o n s  f r o m  c y t o c h r o m e s  a t  t h e  t e r m i n a l: '  : 3 s e - s i d e  o f  t h e  r e s p i r a t o r y  c h a i n  a n d  n o t  a t  a l l  f r o m  N A D H  d e h y d r o g e n a s e:  - € : i l y  ( C h ê p t e r  l l l ) .  C o n c i u s i o n s  a b o u t  t h e  l o c a ' l i z a t i o n  o f  d e h y d r o g á n " r " ,-  : r e  m e m b r a n e  a n d  a b o u t  t h e  o r i e n t a t i o n  o f  t h e  v e s i c l e  m e m b r a n e  b a s e d  o n  a c -'  .  : y  m e a s u r e m e n t s  w i t h  f e r r i c y a n i d e  a r e  t h e r e f o r e  n o t  j u s t i f i e d .Y : - b r ê n e  v e s i c l e s  o f  B .  s u b t i L i s  p e r f o r m  a c t i v e  t r a n s p o r t  o f  a m i n o - ,  m o n o - ,:  - .  a n d  t r i c a r b o x y l i c  a c i d s .  T r a n s p o r t  o f  t h e  d i c a r b o x y l i c  a m í n o  a c i d s  g l u; - :  a s p  i n  t h e s e  v e s i c l e s  i s  m e d i a t e d  b y  a  s i n g l e ,  h i g h l y  s p e c i f i c ,  h i g h  a f -:  - ; l y  s y s t e m .  T h e  s y s t e m  h a s  n o  a f f i n i t y  f o r  o t h e r  n a t u r a l  a m i n o  a c i d s  o r  f o r; : . . - a l  m o n o -  a n d  d i c a r b o x y l  i c  a c i d s  ( C h a p t e r  l V )  .- : e  d i c a r b o x y l  i c  a c i d s  L - m a l a t e ,  f u m a r a t e  a n d  s u c c i n a t e  a r e  a l s o  t r a n s p o r t e d: ,  : ' r e  h i g h l y  s p e c i f i c ,  h i g h  a f f i n i t y  t r a n s p o r t  s y s t e m  l n  B .  s u b t í L i s  \ J 2 3.--: .àe!e\ \ \ .  \ \ \s s\ste(\ se.e(\s to be consti tut ive and has aff ini ty for qlu andasp, but these d\carboxl\\c cnniro ac\Às are not Lranspor\eè b1 \\ \s s)s\es\. \ \i s  s u g g e s t e d  t h a t  g l u  a n d  a s p  b i n d  a t  a  s i t e  o n  t h i s  c a r r ' i e r  d i s t i n c t  f r o m  t h es i t e  w h e r e  t h e  t r a n s p o r t  s o l u t e s  a r e  b o u n d .M e m b r a n e  v e s i c l e s  i s o l a t e d  f r o m  c e l l s  a t  d i f f e r e n t  s t a g e s  o f  g r o w t h  o x i d i z eN A D H  a t  d i f f e r e n t  r a t e s .  D u r i n g  t h e  e x p o n e n t i a l  g r o w t h  p h a s e ,  a  3 - 4  f o l d  i n -c r e a s e  i s  o b s e r v e d  a n d  m a x i m a l  l e v e l s  ê r e  f o u n d  i n  v e s i c l e s  i s o l a t e d  f r o m  s t a -t i o n a r y  p h a s e  c e l l s .  I n i t i a l  r a t e s  o f  a m i n o  a c i d  t r a n s p o r t '  e n e r g i z e d  b y  N A D H ,c l o s e l y  p a r a l  l e l s  t h e  i n c r e a s e  i n  N A D H  o x i d a t i o n  r a t e .  A t  a l l  r a t e s  o f  N A D Ho x i d a t i o n  t h e  t r a n s p o r t  o f  o n e  m o l e c u l e  o f  a  g i v e n  a m i n o  a c i d  r e q u i r e s  t h eo x i d a t i o n  o f  a  c o n s t a n t  n u m b e r  o f  N A D H  m o l e c u l e s  ( C h a p t e r  V l ) .  T h i s  n u m b e rv a r i e s  f r o m  1 5 0 - 2 6 0  f o r  t h e  d i f f e r e n t  a m i n o  a c i d s .M e n a q u i  n o n e s  ê r e  e s s e n t  i a  I  e l e c t r o n  c a r r i e r s  f u n c t  i o n  i  n g  b e t w e e n  d e h y d r o g e n -a s e s  a n d  c y t o c h r o m e s  i n  t h e  r e s p i r a t o r y  c h a i n  o f  B .  s u b t i L í s .  l í e m b r a n e  v e s -i c l e s  f r o m  t h e  m e n a q u i n o n e - d e f i c i e n t  m u t a n t  B .  s u b t í L í s  a t ' o  D  o x i d i z e  N A D H  a ta  l o w  r a t e .  l n  t h e s e  v e s i c l e s  t h e  N A D H  o x i d a s e  a c t i v i t y  c a n  b e  r e c o n s t i t u t e db y  t h e  i n c o r p o r a t i o n  o f  m e n a d i o n e  ( a  m e n a q u i n o n e - a n a l o g u e )  t o  e v e n  h i g h e ró)r a t e s  t h a n  o b s e r v e d  i n  w i l d  t y p e  v e s i c l e s .  T h e  N A D H  o x i d a s e  ê c t i v i i . ,  : j  : - *m e m b r a n e  v e s i c l e s  i n c r e a s e s  I  i n e a r l y  v í i t h  t h e  m e n a d i o n e  c o n t e n t  o .  : - Ê  - r - -b r a n e  a n d  a  3 5 - f o l d  s t i m u l a t i o n  i s  o b t a i n e d  i n  f u l l y  r e c o n s t i t u t e C  , e i ' : : :( c h a p t e r  V t t ) .I n  a g r e e m e n t  w i t h  t h e s e  o b s e r v a t i o n s  m e m b r a n e  v e s i c l e s  f  r o m  : .  . - . . .  :  'D  a c c u m u l a t e  a m i n o  a c i d s  i n  t h e  p r e s e n c e  o f  A s c - P M S ,  b u t  n o t  w i t h  t i i l -e v e r ,  m e m b r a n e  v e s i c l e s  f r o m  t h i s  m u t ê n t ,  r e c o n s t i t u t e d  w i t h  m e n a d i o r e ,  : : -p l a y  N A D H - d r i v e n  t r a n s p o r t  a c t i v i t y .  T h i s  a c t i v i t y  i n c r e a s e s  I  i n e a r l . .  , . - : -t h e  N A D H  o x i d a s e  a c t i v i t y ,  b u t  m a x i m a l  t r a n s p o r t  a c t i v i t i e s  a r e  r e a c n e :  _ - : r .c o n d i t i o n s  w h e r e  t h e  N A D H  o x i d a s e  a c t i v i t y  i s  n o t  y e t  m a x i m a l .  T h i s  i ^ : ' : : : t rt h a t  t h e  r a t e  o f  e n e r g y  s u p p l y  i s  t h e  l i m i t i n g  f a c t o r  f o r  t r a n s p o r r  a . -  ' : , .N A D H  o x i d a s e  a c t i v i t i e s  a n d  t h a t  t h e  t r a n s p o r t  s y s t e m s  t h e m s e ' l v e s  b e c c - eI  i m i t i n g  u n d e r  c o n d i t i o n s  o f  h i g h  N A D H  o x i d a s e  a c t i v i t i e s .  U n d e r  e n e r c . -I i m i t i n g  c o n d i t i o n s  1 3 5 - 2 3 5  m o l e c u l e s  o f  N A D H  h a v e  t o  b e  o x i d i z e d  i n  o - : e .  : :t r a n s p o r t  o n e  m o l e c u ' l e  o f  a m i n o  a c i d .  A t  a l l  l e v e l s  o f  e n e r g y  s u p p l y . c : : , * -p e t i t i o n  b y  t h e  d i f f e r e n t  a m i n o  a c i d  t r a n s p o r t  s y s t e m s  i s  o b s e r v e d  í c r : - ea v a i l a b l e  e n e r g y .  T h e s e  o b s e r v a t i o n s  i n d i c a t e  t h a t  o n l y  a  f r a c t i o n  o f  : - ee n e r g y ,  g e n e r a t e d  b y  t h e  r e s p i r a t o r y  c h a i n ,  i s  u s e d  f o r  t h e  t r a n s p o r t : j : -a m i n o  a c i d  a n d  t h a t  t h e  b u l k  o f  t h e  e n e r g y  d i s s i p a t e s  u i a  o t h e r  c h a n n e t s  -t h e  m e m b r a n e  v e s i c l e s .86

University of Groningen Research Database

  
 University of Groningen
Active transport in cutoplasmic membrane vesicles of Bacillus subtilis
Bisschop, Arnold
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
1976
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Bisschop, A. (1976). Active transport in cutoplasmic membrane vesicles of Bacillus subtilis s.n.
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 11-02-2018
E
SUI{MARY
A  r a p i d  p r o c e d u r e  f o r  t h e  i s o l a t i o n  o f  c y t o p l a s m i c  m e m b r a n e  v e s i c l e s  o f  B a -
c z l l u s  s u b t i L i s  i s  d e s c r i b e d  i n  C h a p t e r  I  l ;  t h e  m e t h o d  c i r c u m v e n t s  t h e  p r o t o -
p l a s t  f o r m a t i o n  s t e p  a n d  c o n s i s t s  i n  e s s e n c e  o f  t r e a t m e n t  o f  t h e  c e l l s  w i t h
i y r o r y * "  i n  a  h y p o t o n i c  m e d i u m .  M e m b r a n e  v e s i c l e s  o b t a i n e d  i n  t h i s  w a y  h a v e  a
l o w  e n d o g e n o u s  o x y g e n  c o n s u m p t i o n  a n d  a  l o w  e n d o g e n o u s  a c t i v i t y  f o r  t r a n s p o r t
o f  s e v e r a l  s o l u t e s .  T h e  m e m b r a n e  v e s i c l e s  a r e  d e v o i d  o f  o t h e r  c e l l  c o m P o n e n t s
a s  j u d g e d  b y  e l e c t r o n  m i c r o s c o p i c a l  e x a m i n a t i o n  o f  t h i n  s e c t i o n s  o f  t h e  v e s -
í c l e s .  F r e e z e - e t c h  s t u d i e s  o f  i n t a c t  c e l l s ,  p r o t o p l a s t s ,  a n d  m e m b r a n e  v e s i c l e s
s h o w  t h a t  t h e  o r i e n t a t i o n  o f  t h e  v e s i c l e  m e m b r a n e  i s  i d e n t i c a l  t o  t h a t  o f  t h e
: ; t o p l a s m i c  m e m b r a n e  i n  i n t a c t  c e l  l s  a n d  p r o t o p l a s t s .
A c t i v e  t r a n s p o r t  o f  s o l u t e s  i n  m e m b r a n e  v e s i c l e s  f r o n  B .  s u b t í L í s  i s  c o u p l e d
' :  e l e c t r o n  t r a n s f e r  i n  t h e  r e s p i r a t o r y  c h a i n  w i t h  o x y g e n  a s  t e r m i n a l  e l e c t r o n
+ : : e f , t o r .  T h e  b e s t  e l e c t r o n  d o n o r s  f o r  t h e  e n e r g i z a t i o n  o f  t r a n s p o r t  a r e  r e -
i - : e c : - n i c o t i n a m i d e - a d e n i n e - d i n u c l e o r i d e  ( N A D H )  a n d  t h e  n o n - p h y s i o l o g i c a l  e -
= : : - o n  d o n o r  a s c o r b a t e + p h e n a z i n e m e t h o s u l f a t e  ( A s c - P M S ) .  L ' i t h  A s c - P M S  a s  e l e c -
- - : ^  d o n o r  t h e  t r a n s p o r t  a c t i v i t i e s  o f  t h e  m e m b r a n e  v e s i c l e s  a r e  c o m p a r a b l e
.  : ^  t h o s e  o f  i n t a c t  c e l l s  a n d  t h e s e  a c t i v i t i e s  a r e  r e t a i n e d  f o r  a  p r o l o n g e d
; < -  c i  o f  t i m e .
: ' e c t r o n  t r a n s f e r  f r o m  N A D H  t o  f e r r i c y a n i d e  u n d e r  a n a e r o b i c  c o n d i t i o n s  a l s o
: : - : e n e r a t e  e n e r g y  f o r  a c t i v e  t r a n s p o r t  i n  m e m b r a n e  v e s i c l e s  o f  t h e  p r e d o m i -
- : - :  l r , ,  a e r o b i c  b a c t e r i u n  B .  s u b t í L í s .  Í h e  m e m b r a n e - i m p e r m e a b l e  e c t r o n  a c -
: í ; : o r  f e r r i c y a n i d e ,  a c c e p t s  e l e c t r o n s  f r o m  c y t o c h r o m e s  a t  t h e  t e r m i n a l
: '  : 3 s e - s i d e  o f  t h e  r e s p i r a t o r y  c h a i n  a n d  n o t  a t  a l l  f r o m  N A D H  d e h y d r o g e n a s e
:  - € : i l y  ( C h ê p t e r  l l l ) .  C o n c i u s i o n s  a b o u t  t h e  l o c a ' l i z a t i o n  o f  d e h y d r o g á n " r " ,
-  : r e  m e m b r a n e  a n d  a b o u t  t h e  o r i e n t a t i o n  o f  t h e  v e s i c l e  m e m b r a n e  b a s e d  o n  a c -
'  
.  : y  m e a s u r e m e n t s  w i t h  f e r r i c y a n i d e  a r e  t h e r e f o r e  n o t  j u s t i f i e d .
Y : - b r ê n e  v e s i c l e s  o f  B .  s u b t i L i s  p e r f o r m  a c t i v e  t r a n s p o r t  o f  a m i n o - ,  m o n o - ,
:  - .  a n d  t r i c a r b o x y l i c  a c i d s .  T r a n s p o r t  o f  t h e  d i c a r b o x y l i c  a m í n o  a c i d s  g l u
; - :  a s p  i n  t h e s e  v e s i c l e s  i s  m e d i a t e d  b y  a  s i n g l e ,  h i g h l y  s p e c i f i c ,  h i g h  a f -
:  - ; l y  s y s t e m .  T h e  s y s t e m  h a s  n o  a f f i n i t y  f o r  o t h e r  n a t u r a l  a m i n o  a c i d s  o r  f o r
; : . . - a l  m o n o -  a n d  d i c a r b o x y l  i c  a c i d s  ( C h a p t e r  l V )  .
- : e  d i c a r b o x y l  i c  a c i d s  L - m a l a t e ,  f u m a r a t e  a n d  s u c c i n a t e  a r e  a l s o  t r a n s p o r t e d
: ,  : ' r e  h i g h l y  s p e c i f i c ,  h i g h  a f f i n i t y  t r a n s p o r t  s y s t e m  l n  B .  s u b t í L i s  \ J 2 3
.--: .àe!e\ \ \ .  \ \ \s s\ste(\ se.e(\s to be consti tut ive and has aff ini ty for qlu and
asp, but these d\carboxl\\c cnniro ac\Às are not Lranspor\eè b1 \\ \s s)s\es\. \ \
i s  s u g g e s t e d  t h a t  g l u  a n d  a s p  b i n d  a t  a  s i t e  o n  t h i s  c a r r ' i e r  d i s t i n c t  f r o m  t h e
s i t e  w h e r e  t h e  t r a n s p o r t  s o l u t e s  a r e  b o u n d .
M e m b r a n e  v e s i c l e s  i s o l a t e d  f r o m  c e l l s  a t  d i f f e r e n t  s t a g e s  o f  g r o w t h  o x i d i z e
N A D H  a t  d i f f e r e n t  r a t e s .  D u r i n g  t h e  e x p o n e n t i a l  g r o w t h  p h a s e ,  a  3 - 4  f o l d  i n -
c r e a s e  i s  o b s e r v e d  a n d  m a x i m a l  l e v e l s  ê r e  f o u n d  i n  v e s i c l e s  i s o l a t e d  f r o m  s t a -
t i o n a r y  p h a s e  c e l l s .  I n i t i a l  r a t e s  o f  a m i n o  a c i d  t r a n s p o r t '  e n e r g i z e d  b y  N A D H ,
c l o s e l y  p a r a l  l e l s  t h e  i n c r e a s e  i n  N A D H  o x i d a t i o n  r a t e .  A t  a l l  r a t e s  o f  N A D H
o x i d a t i o n  t h e  t r a n s p o r t  o f  o n e  m o l e c u l e  o f  a  g i v e n  a m i n o  a c i d  r e q u i r e s  t h e
o x i d a t i o n  o f  a  c o n s t a n t  n u m b e r  o f  N A D H  m o l e c u l e s  ( C h a p t e r  V l ) .  T h i s  n u m b e r
v a r i e s  f r o m  1 5 0 - 2 6 0  f o r  t h e  d i f f e r e n t  a m i n o  a c i d s .
M e n a q u i  n o n e s  ê r e  e s s e n t  i a  I  e l e c t r o n  c a r r i e r s  f u n c t  i o n  i  n g  b e t w e e n  d e h y d r o g e n -
a s e s  a n d  c y t o c h r o m e s  i n  t h e  r e s p i r a t o r y  c h a i n  o f  B .  s u b t i L í s .  l í e m b r a n e  v e s -
i c l e s  f r o m  t h e  m e n a q u i n o n e - d e f i c i e n t  m u t a n t  B .  s u b t í L í s  a t ' o  D  o x i d i z e  N A D H  a t
a  l o w  r a t e .  l n  t h e s e  v e s i c l e s  t h e  N A D H  o x i d a s e  a c t i v i t y  c a n  b e  r e c o n s t i t u t e d
b y  t h e  i n c o r p o r a t i o n  o f  m e n a d i o n e  ( a  m e n a q u i n o n e - a n a l o g u e )  t o  e v e n  h i g h e r
ó)
r a t e s  t h a n  o b s e r v e d  i n  w i l d  t y p e  v e s i c l e s .  T h e  N A D H  o x i d a s e  ê c t i v i i . ,  : j  : - *
m e m b r a n e  v e s i c l e s  i n c r e a s e s  I  i n e a r l y  v í i t h  t h e  m e n a d i o n e  c o n t e n t  o .  : - Ê  - r - -
b r a n e  a n d  a  3 5 - f o l d  s t i m u l a t i o n  i s  o b t a i n e d  i n  f u l l y  r e c o n s t i t u t e C  , e i ' : : :( c h a p t e r  V t t ) .
I n  a g r e e m e n t  w i t h  t h e s e  o b s e r v a t i o n s  m e m b r a n e  v e s i c l e s  f  r o m  : .  . - . . .  :  '
D  a c c u m u l a t e  a m i n o  a c i d s  i n  t h e  p r e s e n c e  o f  A s c - P M S ,  b u t  n o t  w i t h  t i i l -
e v e r ,  m e m b r a n e  v e s i c l e s  f r o m  t h i s  m u t ê n t ,  r e c o n s t i t u t e d  w i t h  m e n a d i o r e ,  : : -
p l a y  N A D H - d r i v e n  t r a n s p o r t  a c t i v i t y .  T h i s  a c t i v i t y  i n c r e a s e s  I  i n e a r l . .  , . - : -
t h e  N A D H  o x i d a s e  a c t i v i t y ,  b u t  m a x i m a l  t r a n s p o r t  a c t i v i t i e s  a r e  r e a c n e :  _ - : r .
c o n d i t i o n s  w h e r e  t h e  N A D H  o x i d a s e  a c t i v i t y  i s  n o t  y e t  m a x i m a l .  T h i s  i ^ : ' : : : t r
t h a t  t h e  r a t e  o f  e n e r g y  s u p p l y  i s  t h e  l i m i t i n g  f a c t o r  f o r  t r a n s p o r r  a . -  ' : , .
N A D H  o x i d a s e  a c t i v i t i e s  a n d  t h a t  t h e  t r a n s p o r t  s y s t e m s  t h e m s e ' l v e s  b e c c - e
I  i m i t i n g  u n d e r  c o n d i t i o n s  o f  h i g h  N A D H  o x i d a s e  a c t i v i t i e s .  U n d e r  e n e r c . -
I i m i t i n g  c o n d i t i o n s  1 3 5 - 2 3 5  m o l e c u l e s  o f  N A D H  h a v e  t o  b e  o x i d i z e d  i n  o - : e .  : :
t r a n s p o r t  o n e  m o l e c u ' l e  o f  a m i n o  a c i d .  A t  a l l  l e v e l s  o f  e n e r g y  s u p p l y . c : : , * -
p e t i t i o n  b y  t h e  d i f f e r e n t  a m i n o  a c i d  t r a n s p o r t  s y s t e m s  i s  o b s e r v e d  í c r : - e
a v a i l a b l e  e n e r g y .  T h e s e  o b s e r v a t i o n s  i n d i c a t e  t h a t  o n l y  a  f r a c t i o n  o f  : - e
e n e r g y ,  g e n e r a t e d  b y  t h e  r e s p i r a t o r y  c h a i n ,  i s  u s e d  f o r  t h e  t r a n s p o r t : j : -
a m i n o  a c i d  a n d  t h a t  t h e  b u l k  o f  t h e  e n e r g y  d i s s i p a t e s  u i a  o t h e r  c h a n n e t s  -
t h e  m e m b r a n e  v e s i c l e s .
86


NARCIS 

University of Groningen Digital Archive

University of Groningen

   University of GroningenActive transport in cutoplasmic membrane vesicles of Bacillus subtilisBisschop, ArnoldIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1976Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Bisschop, A. (1976). Active transport in cutoplasmic membrane vesicles of Bacillus subtilis. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-11-2019ESUI{MARYA  r a p i d  p r o c e d u r e  f o r  t h e  i s o l a t i o n  o f  c y t o p l a s m i c  m e m b r a n e  v e s i c l e s  o f  B a -c z l l u s  s u b t i L i s  i s  d e s c r i b e d  i n  C h a p t e r  I  l ;  t h e  m e t h o d  c i r c u m v e n t s  t h e  p r o t o -p l a s t  f o r m a t i o n  s t e p  a n d  c o n s i s t s  i n  e s s e n c e  o f  t r e a t m e n t  o f  t h e  c e l l s  w i t hi y r o r y * "  i n  a  h y p o t o n i c  m e d i u m .  M e m b r a n e  v e s i c l e s  o b t a i n e d  i n  t h i s  w a y  h a v e  al o w  e n d o g e n o u s  o x y g e n  c o n s u m p t i o n  a n d  a  l o w  e n d o g e n o u s  a c t i v i t y  f o r  t r a n s p o r to f  s e v e r a l  s o l u t e s .  T h e  m e m b r a n e  v e s i c l e s  a r e  d e v o i d  o f  o t h e r  c e l l  c o m P o n e n t sa s  j u d g e d  b y  e l e c t r o n  m i c r o s c o p i c a l  e x a m i n a t i o n  o f  t h i n  s e c t i o n s  o f  t h e  v e s -í c l e s .  F r e e z e - e t c h  s t u d i e s  o f  i n t a c t  c e l l s ,  p r o t o p l a s t s ,  a n d  m e m b r a n e  v e s i c l e ss h o w  t h a t  t h e  o r i e n t a t i o n  o f  t h e  v e s i c l e  m e m b r a n e  i s  i d e n t i c a l  t o  t h a t  o f  t h e: ; t o p l a s m i c  m e m b r a n e  i n  i n t a c t  c e l  l s  a n d  p r o t o p l a s t s .A c t i v e  t r a n s p o r t  o f  s o l u t e s  i n  m e m b r a n e  v e s i c l e s  f r o n  B .  s u b t í L í s  i s  c o u p l e d' :  e l e c t r o n  t r a n s f e r  i n  t h e  r e s p i r a t o r y  c h a i n  w i t h  o x y g e n  a s  t e r m i n a l  e l e c t r o n+ : : e f , t o r .  T h e  b e s t  e l e c t r o n  d o n o r s  f o r  t h e  e n e r g i z a t i o n  o f  t r a n s p o r t  a r e  r e -i - : e c : - n i c o t i n a m i d e - a d e n i n e - d i n u c l e o r i d e  ( N A D H )  a n d  t h e  n o n - p h y s i o l o g i c a l  e -= : : - o n  d o n o r  a s c o r b a t e + p h e n a z i n e m e t h o s u l f a t e  ( A s c - P M S ) .  L ' i t h  A s c - P M S  a s  e l e c -- - : ^  d o n o r  t h e  t r a n s p o r t  a c t i v i t i e s  o f  t h e  m e m b r a n e  v e s i c l e s  a r e  c o m p a r a b l e.  : ^  t h o s e  o f  i n t a c t  c e l l s  a n d  t h e s e  a c t i v i t i e s  a r e  r e t a i n e d  f o r  a  p r o l o n g e d; < -  c i  o f  t i m e .: ' e c t r o n  t r a n s f e r  f r o m  N A D H  t o  f e r r i c y a n i d e  u n d e r  a n a e r o b i c  c o n d i t i o n s  a l s o: : - : e n e r a t e  e n e r g y  f o r  a c t i v e  t r a n s p o r t  i n  m e m b r a n e  v e s i c l e s  o f  t h e  p r e d o m i -- : - :  l r , ,  a e r o b i c  b a c t e r i u n  B .  s u b t í L í s .  Í h e  m e m b r a n e - i m p e r m e a b l e  e c t r o n  a c -: í ; : o r  f e r r i c y a n i d e ,  a c c e p t s  e l e c t r o n s  f r o m  c y t o c h r o m e s  a t  t h e  t e r m i n a l: '  : 3 s e - s i d e  o f  t h e  r e s p i r a t o r y  c h a i n  a n d  n o t  a t  a l l  f r o m  N A D H  d e h y d r o g e n a s e:  - € : i l y  ( C h ê p t e r  l l l ) .  C o n c i u s i o n s  a b o u t  t h e  l o c a ' l i z a t i o n  o f  d e h y d r o g á n " r " ,-  : r e  m e m b r a n e  a n d  a b o u t  t h e  o r i e n t a t i o n  o f  t h e  v e s i c l e  m e m b r a n e  b a s e d  o n  a c -'  .  : y  m e a s u r e m e n t s  w i t h  f e r r i c y a n i d e  a r e  t h e r e f o r e  n o t  j u s t i f i e d .Y : - b r ê n e  v e s i c l e s  o f  B .  s u b t i L i s  p e r f o r m  a c t i v e  t r a n s p o r t  o f  a m i n o - ,  m o n o - ,:  - .  a n d  t r i c a r b o x y l i c  a c i d s .  T r a n s p o r t  o f  t h e  d i c a r b o x y l i c  a m í n o  a c i d s  g l u; - :  a s p  i n  t h e s e  v e s i c l e s  i s  m e d i a t e d  b y  a  s i n g l e ,  h i g h l y  s p e c i f i c ,  h i g h  a f -:  - ; l y  s y s t e m .  T h e  s y s t e m  h a s  n o  a f f i n i t y  f o r  o t h e r  n a t u r a l  a m i n o  a c i d s  o r  f o r; : . . - a l  m o n o -  a n d  d i c a r b o x y l  i c  a c i d s  ( C h a p t e r  l V )  .- : e  d i c a r b o x y l  i c  a c i d s  L - m a l a t e ,  f u m a r a t e  a n d  s u c c i n a t e  a r e  a l s o  t r a n s p o r t e d: ,  : ' r e  h i g h l y  s p e c i f i c ,  h i g h  a f f i n i t y  t r a n s p o r t  s y s t e m  l n  B .  s u b t í L i s  \ J 2 3.--: .àe!e\ \ \ .  \ \ \s s\ste(\ se.e(\s to be consti tut ive and has aff ini ty for qlu andasp, but these d\carboxl\\c cnniro ac\Às are not Lranspor\eè b1 \\ \s s)s\es\. \ \i s  s u g g e s t e d  t h a t  g l u  a n d  a s p  b i n d  a t  a  s i t e  o n  t h i s  c a r r ' i e r  d i s t i n c t  f r o m  t h es i t e  w h e r e  t h e  t r a n s p o r t  s o l u t e s  a r e  b o u n d .M e m b r a n e  v e s i c l e s  i s o l a t e d  f r o m  c e l l s  a t  d i f f e r e n t  s t a g e s  o f  g r o w t h  o x i d i z eN A D H  a t  d i f f e r e n t  r a t e s .  D u r i n g  t h e  e x p o n e n t i a l  g r o w t h  p h a s e ,  a  3 - 4  f o l d  i n -c r e a s e  i s  o b s e r v e d  a n d  m a x i m a l  l e v e l s  ê r e  f o u n d  i n  v e s i c l e s  i s o l a t e d  f r o m  s t a -t i o n a r y  p h a s e  c e l l s .  I n i t i a l  r a t e s  o f  a m i n o  a c i d  t r a n s p o r t '  e n e r g i z e d  b y  N A D H ,c l o s e l y  p a r a l  l e l s  t h e  i n c r e a s e  i n  N A D H  o x i d a t i o n  r a t e .  A t  a l l  r a t e s  o f  N A D Ho x i d a t i o n  t h e  t r a n s p o r t  o f  o n e  m o l e c u l e  o f  a  g i v e n  a m i n o  a c i d  r e q u i r e s  t h eo x i d a t i o n  o f  a  c o n s t a n t  n u m b e r  o f  N A D H  m o l e c u l e s  ( C h a p t e r  V l ) .  T h i s  n u m b e rv a r i e s  f r o m  1 5 0 - 2 6 0  f o r  t h e  d i f f e r e n t  a m i n o  a c i d s .M e n a q u i  n o n e s  ê r e  e s s e n t  i a  I  e l e c t r o n  c a r r i e r s  f u n c t  i o n  i  n g  b e t w e e n  d e h y d r o g e n -a s e s  a n d  c y t o c h r o m e s  i n  t h e  r e s p i r a t o r y  c h a i n  o f  B .  s u b t i L í s .  l í e m b r a n e  v e s -i c l e s  f r o m  t h e  m e n a q u i n o n e - d e f i c i e n t  m u t a n t  B .  s u b t í L í s  a t ' o  D  o x i d i z e  N A D H  a ta  l o w  r a t e .  l n  t h e s e  v e s i c l e s  t h e  N A D H  o x i d a s e  a c t i v i t y  c a n  b e  r e c o n s t i t u t e db y  t h e  i n c o r p o r a t i o n  o f  m e n a d i o n e  ( a  m e n a q u i n o n e - a n a l o g u e )  t o  e v e n  h i g h e ró)r a t e s  t h a n  o b s e r v e d  i n  w i l d  t y p e  v e s i c l e s .  T h e  N A D H  o x i d a s e  ê c t i v i i . ,  : j  : - *m e m b r a n e  v e s i c l e s  i n c r e a s e s  I  i n e a r l y  v í i t h  t h e  m e n a d i o n e  c o n t e n t  o .  : - Ê  - r - -b r a n e  a n d  a  3 5 - f o l d  s t i m u l a t i o n  i s  o b t a i n e d  i n  f u l l y  r e c o n s t i t u t e C  , e i ' : : :( c h a p t e r  V t t ) .I n  a g r e e m e n t  w i t h  t h e s e  o b s e r v a t i o n s  m e m b r a n e  v e s i c l e s  f  r o m  : .  . - . . .  :  'D  a c c u m u l a t e  a m i n o  a c i d s  i n  t h e  p r e s e n c e  o f  A s c - P M S ,  b u t  n o t  w i t h  t i i l -e v e r ,  m e m b r a n e  v e s i c l e s  f r o m  t h i s  m u t ê n t ,  r e c o n s t i t u t e d  w i t h  m e n a d i o r e ,  : : -p l a y  N A D H - d r i v e n  t r a n s p o r t  a c t i v i t y .  T h i s  a c t i v i t y  i n c r e a s e s  I  i n e a r l . .  , . - : -t h e  N A D H  o x i d a s e  a c t i v i t y ,  b u t  m a x i m a l  t r a n s p o r t  a c t i v i t i e s  a r e  r e a c n e :  _ - : r .c o n d i t i o n s  w h e r e  t h e  N A D H  o x i d a s e  a c t i v i t y  i s  n o t  y e t  m a x i m a l .  T h i s  i ^ : ' : : : t rt h a t  t h e  r a t e  o f  e n e r g y  s u p p l y  i s  t h e  l i m i t i n g  f a c t o r  f o r  t r a n s p o r r  a . -  ' : , .N A D H  o x i d a s e  a c t i v i t i e s  a n d  t h a t  t h e  t r a n s p o r t  s y s t e m s  t h e m s e ' l v e s  b e c c - eI  i m i t i n g  u n d e r  c o n d i t i o n s  o f  h i g h  N A D H  o x i d a s e  a c t i v i t i e s .  U n d e r  e n e r c . -I i m i t i n g  c o n d i t i o n s  1 3 5 - 2 3 5  m o l e c u l e s  o f  N A D H  h a v e  t o  b e  o x i d i z e d  i n  o - : e .  : :t r a n s p o r t  o n e  m o l e c u ' l e  o f  a m i n o  a c i d .  A t  a l l  l e v e l s  o f  e n e r g y  s u p p l y . c : : , * -p e t i t i o n  b y  t h e  d i f f e r e n t  a m i n o  a c i d  t r a n s p o r t  s y s t e m s  i s  o b s e r v e d  í c r : - ea v a i l a b l e  e n e r g y .  T h e s e  o b s e r v a t i o n s  i n d i c a t e  t h a t  o n l y  a  f r a c t i o n  o f  : - ee n e r g y ,  g e n e r a t e d  b y  t h e  r e s p i r a t o r y  c h a i n ,  i s  u s e d  f o r  t h e  t r a n s p o r t : j : -a m i n o  a c i d  a n d  t h a t  t h e  b u l k  o f  t h e  e n e r g y  d i s s i p a t e s  u i a  o t h e r  c h a n n e t s  -t h e  m e m b r a n e  v e s i c l e s .86

https://pure.rug.nl/ws/files/14626316/Bisschop.PDF

Active transport in cutoplasmic membrane vesicles of Bacillus subtilis

Abstract

Similar works

Full text

Available Versions

ARTS repository - University of Groningen

ARTS repository - University of Groningen

University of Groningen Research Database

NARCIS

University of Groningen Digital Archive

University of Groningen