Immunological and enzymological approaches to evolution of enzymes and nucleic acid

Allison, W. S.

Kaplan, N. O.

Levine, L.

English

NASA Technical Reports Server

BRANDEZS UNIVERSITY 
Grant NsG -375 
Semi-annual Progress Report covering 
the period 5-1-67 - 11-30-67 
By: Nathan 0. Kaplan, 
Lawrence Levine, and 
William S. Allison 
https://ntrs.nasa.gov/search.jsp?R=19680004511 2020-03-12T12:10:50+00:00Z
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Progress  Report  (May 1, 1967 through November 30, 1967) 
Immunological Approach -- Antibodies  t o  a h igh ly  ac id ,  b ra in-  
s p e c i f i c  p r o t e i n  four,d i n  ve r t eb ra t e s ,  t h e  5-100 p ro te in ,  were obta ined  
wi th  t h e  p u r i f i e d  bovine S-100 complexed w i t h  methylated bovine serum 
albumin as immunogen. 
e x t r a c t s  from a number of v e r t e b r a t e s  measured by a q u a n t i t a t i v e  com- 
plement ( C ' )  f i x a t i o n  technique i n d i c a t e d . a n  unusual ly  c l o s e  s e r o l o g i c a l  
r e l a t i o n s h i p  among t h e s e  S-100 p ro te ins .  
The presence of  a n t i g e n i c  a c t i v i t y  i n  b r a i n  
When t h e  p u r i f i e d  bovine S-100 p r o t e l n  w a s  heate: i n  a t r i s - s a l i n e  
b u f f e r  (pH 7.4) f o r  10 min a t  temperatures from 20-60 , t h e  per  c e n t  C '  
f i x a t i o n ,  measured a t  2-4 , gradual ly  decreased and t h e  an t igen  concen- 
t r a t i o n  g iv ing  maximum C '  f i x a t i o n  increased.  
h ighe r ,  no a n t i g e n i c  a c t i v i t y  was de tec t ed .  
M EDTA, 2-mercaptoethanol o r  CaCl , t h e  p r o t e i n  w a s  p ro t ec t ed  from thermal 
dena tu ra t ion .  Divalent  ca t ions  ozher  than  Ca* d i d  no t  e x h i b i t  t h i s  pro- 
t e c t i v e  e f f e c t .  
0 
Afte r  h e a t i n g  a t  70' or-3 
In t h e  presence of 1 x 10 
S tud ie s  of t h e  o p t i c a l  r o t a t o r y  d i s p e r s i o n  (ORD) of bovine S-100 
s o l u t i o n s  i n  t r i s - s a l i n e  bu f fe r  (pH 7.4) a t  va r ious  temperatures  i n d i -  
c a t e d  t h a t  t h e  d i s p e r s i o n  curve d i sp lays  a t rough wi th  a minimum a t  a 
wavelength of 233 nip and t h a t  t h i s  t rough va lue  becomes l e s s  nega t ive  
as t h e  temperature  inc reases .  This may be i n t e r p r e t e d  as t h e  des t ruc -  
t i o n  o f  a h e l i c a l  s t r u c t u r e  as t h e  temperature  i s  increased .  
t h e  hea ted  s o l u t i o n ,  t h e  [m'] t h e  reduced mean r e s i d u e  r o t a t i o n  a t  
233 mp, r e v e r t e d  t o  t h e  o r i g i n  '2  ml*:,lue only  i f  EDTA was present ,  t o  a 
lesser ex ten t  i n  t h e  presence  of mercaptoethanol o r  C a C l  
i n  t r i s - s a l i n e  bu f fe r .  
Upon coo l ing  
and only  p a r t l y  2' 
When t h e  p r o t e i n  w a s  d issolved i n  d i s t i l l e d  water, ORD measurements 
showed t h a t  a t  20°, t h e  ORD curve had a maximum [m'] o f  29,500 a t  198 mp 
and a minimum of  about  -6,000 a t  233 mp. Among computer generated ORD 
curves  r ep resen t ing  varying proport ions of  Q: h e l i x ,  fl and random chain,  
the one w i t h  40% a h e l i x ,  30% /3, and 30% random s t r u c t u r e  most c l o s e l y  
resembled bovine  S-100 i n  d i s t i l l e d  water  a t  20'. A s  t h e  p r o t e i n  so lu-  
t i o n  w a s  he'ated t h e  [ m ' ]  198 mp va lue  decreased and t h e  [ m ' ]  233 m p  b ecanie 
less l evoro ta to ry ,  i n d i c a t i n g  d e s t r u c t i o n  of t h e  a h e l i x .  However, w i t h  
the r i s e  i n  temperature ,  a peak g radua l ly  appeared a t  about  190 rap. This  
w a s  i n t e r p r e t e d  as t h e  formation of a new p r o t e i n  conformation a t  high 
temperature ,  perhaps t h e  p a r a l l e l  f3 s t r u c t u r e .  So lu t ions  t h a t  have been 
h e a t e d  t o  in t e rmed ia t e  temperatures and then cooled r e t a i n  a small amount 
o f  t h e  190 q i  peak and p a r t l y  rega in  t h e  198 mp peak. Af t e r  90' incuba- 
peak, b u t  r e -  t i o n ,  t h e  cooled p r o t e i n  r e t a ined  none of t h e  [m/ ]  
v e r t e d  t o  about  25% Q: h e l i x .  
190 mp 
Enzymological&psoach. Aldolase -- Our s t u d i e s  on the  heteroge-  
n e i t y  of t h e  m u l t i p l e  forms of a l d o l a s e  i:i chicken t i s s u e s  have demon- 
s t r a t e d  t h a t  a t h i r d  p a r e n t a l  type ( a ldo la se  C) occurs  i n  chicken t i s s u e s  
a long  wi th  i t s  hybrid forms. These s t u d i e s  showed t1,at t h r e e  independent 
3 
genes t h a t  l ead  t o  t h e  syn thes i s  of a l d o l a s e  A, B, and C po lypept ide  
cha ins  a r e  r e spons ib l e  f o r  t h e  m u l t i p l e  forms of a l d o l a s e  found i n  
chicken t i s s u e s .  S tudies  on chicken embryos have shown t h a t  dur ing  
t h e  i n i t i a l  s t a g e s  of development, t h e  a c t i v i t y  of t h e  A and C-type 
a l d o l a s e s  are  present ,  b u t  the p r i n c i p a l  embryonic form i s  a l d o l a s e  C. 
The a c t i v i t y  of  B-type a l d o l a s e  appears  i n  l i v e r  and kidney du r ing  t h e  
la ter  s t a g e s  of development. 
, 
Aspartate Aminotransferase -- The s o l u b l e  a s p a r t a t e  aminotrans- 
Both t h e  halo-and apo- 
The p r o p e r t i e s  of t h e  chicken h e a r t  
f e r a s e  from chicken h e a r t  has  been p u r i f i e d .  
enzymes have been c r y s t a l l i z e d .  
enzyme have been compared t o  those r epor t ed  f o r  t he  enzyme i s o l a t e d  
from p i g  h e a r t ,  rat  l i ve r ,  and ox heart. 
Arginine Kinases -- Our work on a r g i n i n e  k inase  has cons i s t ed  of 
comparative s t u d i e s  of t h e  enzyme i n  va r ious  ar thropod spec ie s .  
p u r i f i e d  a r g i n i n e  k inase  from th ree  c rus tacean  spec ies ,  t h e  l o b s t e r ,  
b lue  crab,  and t h e  hermit  crab.  I n  add i t ion ,  two e l e c t r o p h o r e t i c a l l y  
d i s t i n c t  forms have been p u r i f i e d  from the  muscle of t he  horseshoe crab, 
Limulus polyphemus. S imi la r  isoenzymic forms were separa ted  i n  e x t r a c t s  
from several s p i d e r  spec ies .  In sec t  a r g i n i n e  k inases  have been s t u d i e d  
wi th  crude e x t r a c t s  and p a r t l y  p u r i f i e d  systems. The p u r i f i e d  prepara-  
t i o n s  have many p r o p e r t i e s  i n  common. 
s imi l a r .  Each conta ins  f i v e  cys t e ine  and no cys t ine ,  and a l l  a re  inh i -  
b i t e d  by su l fhydry l  reagents .  
t h e  a n a l y t i c a l  u l t r a c e n t r i f u g e  and by chromatography on Sephadex G-100 
range between 35-4.0,OOO. 
t h e  l o b s t e r  enzyme can b e  r e a c t i v a t e d  by d i l u t i o n  i n t o  b u f f e r  conta in ing  
a t h i o l ,  such as P-mercaptoethanol. L-arg in ine  and ATP a l s o  promote 
r e a c t i v a t i o n  b u t  ADP i s  less e f f e c t i v e  i n  t h i s  regard.  Magnesium and 
o t h e r  d i v a l e n t  m e t a l  ions  i n h i b i t  substrate-promoted r e a c t i v a t i o n .  
S imi l a r  s t u d i e s  on r e a c t i v a t i o n  a f t e r  t reatment  wi th  8 M urea  have been 
conducted w i t h  t h e  o the r  p u r i f i e d  a r thropod a r g i n i n e  k inases .  
a r g i n i n e  k inase  isoenzymes i s o l a t e d  from Limulus muscle d i f f e r  i n  t h e i r  
s t a b i l i t y  a t  h igh  temperatures,  b u t  they  are  very  s imi l a r  i n  t h e i r  physi-  
c a l  and c a t a l y t i c  p rope r t i e s .  
t rea tment  wi th  8 M  urea  and r e a c t i v a t i o n ,  and C '  f i x a t i o n  experiments i n -  
d i c a t e  d e f i n i t e  s t r u c t u r a l  d i f f e rences  between t h e  two forms. 
W e  have 
Thei r  amino a c i d  compositions a r e  
Their  molecular weights as measured i n  
Af te r  i n a c t i v a t i o n  by exposure t o  8 M urea,  
The two 
The two forms a r e  not  i n t e rconve r t ed  by 
Sequence of Dogfish M Lac ta t e  Dehydrogenase (LDH) -- The determi- 4 n a t i o n  of t h e  t o t a l  amino a c i d  sequence of dogf i sh  M LDH has  progressed  
i n  t h e  fol lowing way. 
enzyme have been reso lved  by a combination of g e l  f i l t r a t i o n ,  paper e lec-  
t r o p h o r e s i s  a t  va r ious  pH values,  and paper chromatography. The amino 
a c i d  compositions of t hese  pept ides  have been determined and, i n  some 
have been determined. 
4 A l l  o f  the pept ides  i n  a t r y p t i c  d i g e s t  o f  t h e  
c a s e s i  the N - t r e r m i r ? a l  amino a c i d  and parti,l SeqlJenCEis of these peptides 
LDH from t h e  t a i l  muscle of t he  American ___- l o b s t e r  (Homarus Americanus) 
has been p u r i f i e d  t o  homogeneity as determined by u l t r a c e n t r i f u g a l  a n a l y s i s  
and s t a r c h  g e l  e l ec t rophores i s .  The phys ica l  P r o p e r t i e s  of t h e  enzyme have 
been cha rac t e r i zed  and a re  i n  general ,  similar t o  LDH's from v e r t e b r a t e  and 
, .  * .  
o t h e r  i n v e r t e b r a t e  sources .  The c a t a l y t i c  parameters,  K tu rnover  
number, optimal pyruvate  concentrat ion,  and s u b s t  a te  i z f b i t i o n ,  f o r  
t h e  r eve r se  r e a c t i o n  (F'y + DPNH- l a c t a t e  + DPN ) have been determined 
and are s imi l a r  t o  LDII's repor ted  i n  t h e  l i t e r a t u r e .  However, t h e  ca t a -  
l y t i c  p r o p e r t i e s  f o r  the  forward r e a c t i o n  ( l a c t a t e  + DP$ -2 Py + DPNH) 
d i f f e r  s i g n i f i c a n t l y  from LDH's h i t h e r t o  s tud ied .  
t h e  product  DPNH inc reases  t h e  ra te  of t he  r e a c t i o n  and a t  h igh  concentra-  
t i o n s  i n h i b i t s  i t;  t h e  i n h i b i t i o n  i s  compet i t ive  w i t h  r e s p e c t  t o  DP@. 
The product  a c t i v a t i o n  causes  the r a t e  E s u b s t r a t e  concen t r a t ion  p l o t s  
t o  assume a sigmoidal shape. 
be l i eved  t o  be  caused by conformational changes i n  t h e  p r o t e i n  brought 
about  by t h e  binding of DPNKj which enhances t h e  a f f i n i t y  of t he  enzyme 
f o r  DPN+. 
f: 
- - A t  low concent ra t ions ,  
The a c t i v a t i o n  of t he  enzyme by DPNH is  
Pseudomonas Mutant Enzymes -- Several  enzymes i s o l a t e d  from Pseudo- 
monas t e s t o s t e r o n i  mutant strains have behaved very  d i f f e r e n t l y  from t h e  
wi ld  type. 
c rude  e x t r a c t  from t h e  mutant s t r a i n  showed abso lu te  migra t ion  toward the  
anode, whereas t h e  wi ld  type  p ro te ins  migrated both toward t h e  anode and 
cathode. 
c i n i c  dehydrogenase, glutamate dehydrogenase, malate dehydrogenase, s t e r o i d  
dehydrogenase, i s o c i t r a t e  dehydrogenase, glucose 6-phosphate dehydrogenase, 
showed more a c i d i c  p r o p e r t i e s  than the  wild type  did.  
w a s  observed while  t h e  mutant was being grown, showed e x a c t l y  t h e  same 
p r o p e r t i e s  as the  wild type  did.  
t a i l  t h e  p r o p e r t i e s  of the p ro te ins  from t h e s e  t h r e e  r e l a t e d  s t r a i n s  and 
e v e n t u a l l y  t o  s tudy  t h e  d i f f e r e n c e s  i n  t h e i r  gene t i c  codes by sequencing 
e i t h e r  t h e i r  DNA o r  RNA. 
On e l ec t rophores i s ,  i t  was found t h a t  t h e  p r o t e i n s  i n  the  
A l l  t h e  enzymes i s o l a t e d  from the  mutant s t r a i n ,  such as suc- 
Another s t r a i n  t h a t  
We a r e  t r y i n g  t o  compare i n  g r e a t e r  de- 
I 
I 
Pub l i ca t ions  supported by NSG-375 f o r  t h e  per iod  February 1, 1967 
through January 31, 1968 
1. G. B. Ki t to ,  M. E. Kottke, L. H. Bertland, W. H. Murphy, and N. 0. 
Kaplan. 
f e r a s e s  from Neurospora c rassa .  Arch. Biochem. Biophys. 121, 224, 1967. 
S tudies  on Malate Dehydrogenases and Aspa r t a t e  Aminotrans- 
2. L. Corman and N.  0. Kaplan. K i n e t i c  S tud ie s  of Dogfish L ive r  Glutamate 
Dehydrogenase wi th  Diphosphopyridine Nucleot ide and t h e  E f f e c t  of Added 
S a l t s .  Biochemistry 242, 2840, 1967. 
3. S. L. Blethen and N. 0. Kaplan. P u r i f i c a t i o n  of Arginine Kinase from 
Bio- Lobs ter  and a Study of  Some Fac to r s  Af fec t ing  Its Reac t iva t ion .  
chemistry s, 1413, 1967. 
4 .  L. H. Ber t land  and N. 0. Kaplan. Chicken Heart  Solubil:e Aspa r t a t e  
Aminotransferase:  P u r i f i c a t i o n  and P rope r t i e s .  Biochemistry, i n  press .  
5. N. 0. Kaplan. Concepts and S ign i f i cance  of Enzyme Evolut ion.  Collo- 
quium on Biochemical Evolution, Par i s ,  March 28-30, 1967, i n  p re s s .  
(P rep r in t s  enclosed) .  
6. L. Levine. Immunochemical Approaches t o  t h e  Study of t h e  Nervous 
. System. I n  "The Nervous System," F. 0. Schmidt, ed. New York, The 
Rockefe l le r  Un ive r s i ty  Press, i n  press .  
7 .  C. J. Masters. Characteristics of Aldolase Variformity. Biochem. 
1 Biophys. Res. Commun. 28, 978, 1967. 
8. C. J. Masters, J. J. Herskovits, and N. 0 .  Kaplan. The Developmental 
Multiplicity of Chicken Aldolase. Submitted for publication. 
9. R. von Fellenberg and L. Levine. Proxiinity of the Enzyme Active 
Center and an Antigenic Determinant of Lysozyme. 
- 4, 363, 1967. 
Immunochemistry 
10. D. Kessler, L. Levine and G. D. Fasman. Some Conformational and 
Immunological Properties of a Bovine Brain Acidic Protein (S-100). 
Biochemistry, in press. 


A comparative study of the evolution of enzymes and nucleic acids Semiannual progress report, 1 May - 30 Nov. 1967

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