Fries, M.

Scriba, Peter Christian

English

Universität München: Elektronischen Publikationen

© 1967 Nature Publishing Group
NATURE, VOL, 214. APRIL I, 1967 
Primary Fractions Sucrose Gradient Hypo-Osmotlc 
80 (MIT) Shock 
(MIT) 
70 
60 
'" j 50 
]! 40 
'Ci 
P SO 
20 
10 
o 
NUC MIT MIC SUP ABC 0 E MI M2 M3 
Fig.2. Effeet of'Triton X-100' on unmasking oflatent phosphodiester-
ase activity in various subcellular fractiofls. }i'ractions were prepared 
as u8ual and each fraction was divided into two, one serving as control; 
the other was dJluted wlth 0·32 molar 5uerose to a protein concentration 
of 5 mg/mI. and then treated with 0'2 per cent 'Triton X-IOO' (final eon-
centr~tion). Whit.e block, control; solid block , 'Triton'. Separate 
experIments estabhshed that thls eoneen tratlOn of 'Triton' exposed all 
latent activity (Cheung, to be published) . Both sets of sam pies were 
kept at 00 lor 30 min before enzymatic assay. The activity ofeach fraction 
is expressed as unitsjg tissllc. The total activity in the conlrol cxh ibited 
77 unitsjg tisslle while that treated with 0·2 per cent 'Triton' 122 units/g 
tissue. Itesults are average of two experiments uslng a pool uf ten brains 
in @ach experiment. Recovery of phosphodiesterase from the various 
fractions in these experiments was 80-85 per cent. 
activities. With the exceptions of SUP and M 3, all 
fractions display an increase in activity. MIC was most 
pronounced, giving a 4-fold increase. The increase in all 
other fractions could be caused by the small contamination 
by microsomes wh ich would be expected. In the presence 
of 'Triton', MIC accounts for more than half the activity 
in the whole brain. The fact that there was no increase of 
activity in the soluble enzyme in SUP and M 3 would 
suggest that 'Triton' did not activate the enzyme as such, 
but possibly exposed the enzyme which would otherwise 
have been inaccessible during the assay. In order to see 
whether phosphodiesterase was associated with lysosome-
type particles, we submitted the microsomal fraction to 
vigorous m echanical disruption in a 'VirTis 45' homo-
genizer for times up to 9 min. SampIes were taken at 
various intervals and no increase was observed in any of 
them. Repeated freezing and thawing of the microsomal 
fraction also caused no appreciable increase. More than 
90 per cent of the activity was still attached to the sediment 
(lOO,OOOg x 45 min) after such physical disruption. 
Parallel treatment of the microsomes with 'Triton' gave 
the usual burst of activity. It seems that the latent enzyme 
might be embedded in the lipoprotein matrix of micro-
somes in such a manner that it could only be exposed by 
the detergent. In this respect, the behaviour of phos-
phodiesterase is different from acid phosphatase, a typical 
lysosomal enzyme, the latent activity of which could be 
released by all the treatments d escribed above". It is also 
different from cholinesterase in that the latter is a 
microsomal enzyme but shows no latent activity 13. 
Both MIC and M, are composed ofmembrane fragments, 
accounting for more or less the same amount of pro tein. 
Whereas there is virtually no effect of 'Triton' on the 
activity associated with M" the increase of activity in 
MIC amounts to 400 per cent, suggesting that phospho-
diesterase might be preferentially distributed in certain 
m embrane structures. 
Drummond and Perrott-Yee14 reported that a cyclic 
3',5' -nucleotide phosphodiesterase from rabbit brain was 
localized entirely in the 100,000g supernatant. Nair'5 
noted that a similar enzyme prepared from frozen dog 
heart was present cxclusively in the supernatant. These 
observations are at variance with our data and those of 
Butcher and Sutherland" who found that phospho-
diesterase from beef heart is mostly particulate. The 
reason for such a discrepancy is not apparent to uso 
91 
Our results indicate that phosphodiesterase as frac-
tionated in isotonic sucrose solution does not exhibit its 
full potential activity. The majority of the activity in the 
microsomes is latent and is unmasked by the action of 
'Triton', but not by mechanical disruption or repeated 
freezing and thawing. In the presence of 'Triton', micro-
somes account for more than half the total activity. The 
remaining activity distributes about equally among 
synaptoplasm and a 100,000g supernatant. 
We thank Professor B. Chance for his interest and 
encouragement throughout the course of this investigation. 
We also thank Mrs. A. E. Strichartz for technical assistance. 
This work was supported by the U.S. Public Health Ser-
vice. 
Johnson R esearch Foundation, 
University of Pennsylvania, 
Philadelphia, Pennsylvania. 
WAl YIU CHEUNG 
LEON SALGANICOFF* 
• Fellow of the Consejo Nacional de Investigaciones Cientifieas y Teenicas 
Republiea Argentlna. 
• Rall , T. W., a nd Sutherland , E. W., J. Biol. Chem., 237 , 1228 (1962). 
'Sutherland, E. W., and RaH, T. W., J. Bio!. Chem., 232,1077 (1958). 
'Sutherland, E. W., RaH, T. W., and Menon, T., J. Biol. Chem., 237, 1220 
(1962). 
• Butcher, R. W ., and Sutherland, E . W. , J. Biol. Chem., 237, 1244 (1962). 
' Cheung, W. Y .. Biochem. Biophys. Res. Commun., 23,214 (1966). 
• De Robertis, E.,l'ellegrino de Iraldi, A., Rodriguez de LoresArnaiz, G., and 
Salganicoff, L., J. Neurochem., 9,23 (1962). 
, De Robertis, E.. Salganicoff, L., Zieher, L . M., Rodriguez de Lores Amai?, 
G., J. Neurochem., 10,255 (1963). 
'Fiske, C. H. ,andSubbarow, Y., J. Biol. Chem., 66, 375 (1925). 
• Lowry, O. H. , and Lope?, J . A., J. Biol. Chem., 162. 421 (1946). 
10 De Robertis, E., in Hislophysiology 0/ Synapses and Neurostelion, 19 " 
(Pergamon Press, New York. 1964). 
.. Salganieoff, L., and De Roberti., E., J. Neurochem.,12. 287 (1965)_ 
" Sellinger, O. Z. , Rucker, D. L .• and De Balbian Verster, J. Neurochem .. l1. 
271 (1964). 
lS Toschi. G., Exp. Gell Res., 16, 232 (1959) . 
.. Drummond. G. I., and l'errott-Yee, S., J. Biol. Chem., 236,1126 (1961). 
I ,' ~air, K. G., Biochemistry. 5, 150 (1965). 
Aminoacyltransferase Stimulation of 
Protein Synthesis by Pig Adrenal Polysomes 
ADMINISTRATION of adrenocorticotrophin in viva increases 
the incorporation of 14C-glycine into protein by the 
supernatant of adrenal homogenates centrifuged at 
15,000g'. A factor of the soluble cell fraction (105,000g 
supernatant) haI> been shown to be rate limiting for in 
vitra proteiD synthesis and its activity is doubled by ACTH 
administration in rats. This factor was reported to be 
proteinaceous; its elution pattern from DEAE-cellulose 
columns' was identical with liver aminoacyltransfcrase3 • 
The increased activity of the microsomal fraction by 
treatment with ACTH was repol'ted to be delayed compared 
with the soluble eell fraction'. Thus it was of interest to 
find wh ether polysomes, which are polymeres of ribosomes 
and messenger-RNA5 .• , or the protein from the soluble 
adrenal cell fraction were rate limiting for protein syn-
thesis. 
When polysomes wcre incubated with constant amounts 
of soluble cell fraction, the incorporation of 14C-glycine 
increased only slightly and by no means in proportion to 
the amount of polysomes added. Thus incubation of 
polysomes with 0·4 ml. of 105,000g supernatant (80 mg-
equivalents) showed the following incorporations. Poly-
somes: 0·1 ml. (240 c.p.m.); 0·2 ml. (269 c.p.m.); 0·4 ml. 
(337 c.p.m.); pclysomes omitted (179 c.p.m.) (für incuba-
tion conditions see Table 1). W e conclude that the amount 
ofpolysomes and therefore the amount ofmessenger RNA 
present in the incubation mixture did not limit thc rate of 
amino-acid incorporation. 
The protein factor prepared from pig soluble adrenal 
cell fraction (Fig. 1) and likely to be aminoacyltransferase' 
did, however, stimulate the incorporation of 14C-glycine by 
constant amounts of polysomes in approximate proportion 
to the amount added. Sampies VI and VII (Fig. 1) con-
© 1967 Nature Publishing Group
92 
'fable 1. INCORPORATION Ol!' "C-GLYCINE BY ADRENAL POLYSOMES AS STIMl" 
LATED BY AMINOACYLTRANSFERASE 
SampIe 
(Fig.l) 
V 
VI 
VII 
VIII 
IX 
0·2 mI. 
270 
615 
754 
210 
255 
0·4ml. 
208 
1,288 
1,835 
311 
Polysomes were prepared from fresh plg adrenals··' and 0·1 mL = 160 mg-
equivalents (containing 0·78 mg protein' and approxlmately 1·8 mg RNA') 
were incubated with 0·3 mI. 105,OOOg supernatant of fresh pig adrenals 
(60 ml(-equivalent). "C-glycine. ATP. GTP, PEP and P·kinase were added 
as In Fig. 1. In thls system 0'2 or 0'4 mJ. of sampIes V-IX (Fig. 1) were 
assayed for stimulation of "C·glyclne Incorporation in a total volumc of 
1'05 mJ. (60 min, 37° Cl. The complete system wlth no addition showed an 
incorporation of 209 c.p.m.; 105,OOOg supernatant omitted and 5 c.p.m. 
when polysomes were omitted. Addition of 0'2 ml. of sampIes produced the 
followlng Incorporations: VI, 176 c.p.m.; VII, 342 c.p.m.; VIII, 108 c.p.m.; 
IX, 206 c.p.m. 
tained aminoacyltransferase and 
incorporation when they were 
mixtures (Table 1). 
20 , 
stimulated amino·acid 
added to incubation 
NATURE. VOL. 214, APRIL I, 1967 
IMMUNOLOGY 
Inter Heavy-Light Chain Disulphide Bridge 
in Immune Globulins 
THE immune globulins are a heterogeneous family of 
proteins made up of light and heavy* chains joined by 
disulphide bridges. Immunological teehniques1,t and 
chemical investigation3 indieate that most or all of the 
normal population of light chains is a mixture of two types 
of polypeptide-x and A. The majority of heavy chains 
can be classified as y, (1. or ce. In humans, the y chains 
have been further subdivided, making use of common 
antigenie determinants, into four major sub-classes: 
y2a (or Ne), y2b (or We), y2c (or Vi), and y2d (or Ge)4,5. 
Combination of either x or A chains with y, JL or ce 
ehains gives rise to immune globulins IgG (gamma· 
globnlins), IgM (maeroglobulins), or IgA. It has been 
20,000 
&lABIEN' • VI.vm 
IHKCI .. 
• I 
! 
<= 
~ ... 
Po< 
10 
15,000 
10,000 Ei 
P-
o 
5,000 
'" 8 -"i:.=::..... ... __ ..L. 
postulated3 that a single disulphide 
bridge linking x or A chains to heavy 
chains oecurs through the light chain 
C-terminal or next to C·terminal eys-
teine residue, and this has been based 
mainly on the arrangements of disul-
phide bridges in light chain monomers 
and dimers produced in the urine of 
myeloma patients (Bence-Jones pro· 
teins). A direct demonstration that 
this is the case has now been obtained 
by isolating disulphide-bridged peptides 
containing the C-terminal peptide of 
the light chain and a half-cystine 
peptide from the heavy chain of 
different types of human pathological 
immune globulins. In addition, a 
radioactive technique for the isolation 
of the corresponding carboxymethyl-
cysteine peptides has been developed. 
This communication presents a com· 
parison between peptides derived from 
different classes and sub·classes. Pooled 
normal IgG is shown to contain in good 
yields the peptides found in the two 
chief sub-classes of y-chains. 
0 o m m~~ m m 
I-+-u---f'm+IV+V+VI-tVn+-Vm-+IX+-X 
SampIe 
Fig. 1. Preparat.lon of aminoacyltransferase from pig adrenals. 13· 7 g 
fresh pig adrenals were homogenized in 68·5 ml. medium Ä (0'25 molar 
sucrose, 0'025 molar potassium chloride, 0·005 molar magnesium 
chloride, 0'05 molar tri. hydrochloric acid bulfer, pB 7'5) aß deBcribed' 
and the 105,OOOu supernatant was passed through dextran gel ('Sephadex 
G-25'). It was then concentrated by ultrailltration and subjected to anion 
exchange chromatography on a DEAE·cellulose column using a Iincar 
gradient made from I: 0'03 molar potassium chloride, 0·02 molar Iris 
hydrochlorlc acid bulfer, pH 7'5, and II: 0·6 molar potasslum chloride, 
0·02 molar tris hydrochlorlc acid bulfer, pB 7'5 (ref. 2). The eluate was 
recombined into sampies I-X as indieated, eoncentrated by uUra-
filtration, dialYBed againBt medium A and portions assayed for stimula· 
tion of protein Bynthesls in a celHree system. This contained 1 pc. "C· 
glyclne (0'014 pmoles), 10 pmoles phosphoenol·pyruvate, 1 pmole ATP. 
0·25 pmoles GTP, 60 Pfl: pyruvate kinase and 80 mg-equivalent of 
15,OOOg aupernatant of plg adrenal homogenate in a total volume of 
1·05 ml. (370 C, 60 min). The assay of 14C-glycine incorporated into 
pro tein was described earller'. The data represent the total stimulation of 
Incorporatlon In each sampIe (c.p.m.) 
o 
Disulphide-bridged peptides were 
isolated by the performic acid oxidation 
diagonal technique6 ,7 after peptic (1/20 
enzyme-substrate ratio in 5 per cent 
formic acid, 14 h at 37° C) and then 
Adrenocorticotrophin administered to ra.ts leads to 
increased protein synthesis in a cell-free system because of 
olevated aetivity or amount of aminoacyltransterase2• 
Onlyafter eontinued treatment with ACTH is the activity 
of polysomes stimulated4, but aminoacyltransferase 
remains the rate limiting factor for in vitro protein 
synthesis. 
This investigation was supported by the Deutsche 
Forsehungsgemeinschaft.. 
tryptic digestion (1/50 enzyme-substrate ratio in pH 
7'0, 0·1 molar ammonium acetate buffer, 6 h at 37° Cl. 
Peptides were purified by paper electrophoresis at pH 
6'5, and pH 3'5 before and after oxidation; with the 
macroglobulin Ale, a preliminary fractionation of peptides 
on a 'Sephadex G-25' column in 2 per cent acetic acid was 
necessary. Peptides with the sequenees shown in Table 1 
were isolated from three different pathological immune 
globulins. 
PETER C. SCRIBA 
MARIANNE FRIES 
II. Medizinische Klinik der Universitat, 
Munieh. 
Received N ovem ber 9, 1966. 
I Farese, R. V., and Reddy, W. J .. Endocrinology, 73, 294 (1963). 
'Seriba, P. C., anel Redcly, W. J ., Endocrinology, 78, 745 (1965). 
• Takanami, 111., Biochim. Biophys. Acta, 61 , 85 (1961). 
• Farese, R. V., Endocrinology, 78, 125 (1966). 
• Wettstein, F. 0., Staehelln, 'r., and Noll, JI., Nature, 197,430 (1963). 
• GIerer, A., J. Mol. Bioi., 6, 148 (1963), 
I Farese, R. V., EndocrinoloUlI, 77, 128 (1965). 
• Lowry, O. R., Rosenbrough, N. J., Farr, A. L., and RandalI, R. V., J. Biol. 
allem., 193, 265 (1951). 
• Dlache, Z., in The Nucleic Acid8 (edlt. by Chargaff, E., and Davidson, J. N.), 
301 (Mademic Press, New York, 1955). 
'fable 1. OYSTINE PEPTIDES CONTAINING TBE DISULPIDDE BOND BBTWIiEN 
LIGHT oI.ND HEAVY OHAINS OF SOME PoI.THOLOGlCAL IMMUNE GLOBULINS 
Beavy Light 
Protein chaln chain 
Ale 
Fle 
Viu 
type type 
)' 
v 
Pro.Leu.VaJ.Ser.Cys.GIx.ABx.Ser(Asp,Ser .. ThJ,Pro) 
I 
" Gly .Glu.Cys 
Ser.Cys.Asp.Lys 
I 
" Gly. Glu. Cys 
Pro.Leu.Ala(Cys,Ser,Pro) 
I 
Pro.Thr.Glu.Cys.Ser 
The occurrence of the sequences derived from Fis and 
Vin in pooled normal IgG and in other myeloma proteins 
• The nomenclature used for Immune globulin molecules, ohalD& and 
fragments is that recommended in ref. 24. 


Aminoacyltransferase Stimulation of Protein Synthesis by Pig Adrenal Polysomes

Open Access LMU ( Ludwig-Maximilians-Univ. München)

© 1967 Nature Publishing GroupNATURE, VOL, 214. APRIL I, 1967 Primary Fractions Sucrose Gradient Hypo-Osmotlc 80 (MIT) Shock (MIT) 70 60 '" j 50 ]! 40 'Ci P SO 20 10 o NUC MIT MIC SUP ABC 0 E MI M2 M3 Fig.2. Effeet of'Triton X-100' on unmasking oflatent phosphodiester-ase activity in various subcellular fractiofls. }i'ractions were prepared as u8ual and each fraction was divided into two, one serving as control; the other was dJluted wlth 0·32 molar 5uerose to a protein concentration of 5 mg/mI. and then treated with 0'2 per cent 'Triton X-IOO' (final eon-centr~tion). Whit.e block, control; solid block , 'Triton'. Separate experIments estabhshed that thls eoneen tratlOn of 'Triton' exposed all latent activity (Cheung, to be published) . Both sets of sam pies were kept at 00 lor 30 min before enzymatic assay. The activity ofeach fraction is expressed as unitsjg tissllc. The total activity in the conlrol cxh ibited 77 unitsjg tisslle while that treated with 0·2 per cent 'Triton' 122 units/g tissue. Itesults are average of two experiments uslng a pool uf ten brains in @ach experiment. Recovery of phosphodiesterase from the various fractions in these experiments was 80-85 per cent. activities. With the exceptions of SUP and M 3, all fractions display an increase in activity. MIC was most pronounced, giving a 4-fold increase. The increase in all other fractions could be caused by the small contamination by microsomes wh ich would be expected. In the presence of 'Triton', MIC accounts for more than half the activity in the whole brain. The fact that there was no increase of activity in the soluble enzyme in SUP and M 3 would suggest that 'Triton' did not activate the enzyme as such, but possibly exposed the enzyme which would otherwise have been inaccessible during the assay. In order to see whether phosphodiesterase was associated with lysosome-type particles, we submitted the microsomal fraction to vigorous m echanical disruption in a 'VirTis 45' homo-genizer for times up to 9 min. SampIes were taken at various intervals and no increase was observed in any of them. Repeated freezing and thawing of the microsomal fraction also caused no appreciable increase. More than 90 per cent of the activity was still attached to the sediment (lOO,OOOg x 45 min) after such physical disruption. Parallel treatment of the microsomes with 'Triton' gave the usual burst of activity. It seems that the latent enzyme might be embedded in the lipoprotein matrix of micro-somes in such a manner that it could only be exposed by the detergent. In this respect, the behaviour of phos-phodiesterase is different from acid phosphatase, a typical lysosomal enzyme, the latent activity of which could be released by all the treatments d escribed above". It is also different from cholinesterase in that the latter is a microsomal enzyme but shows no latent activity 13. Both MIC and M, are composed ofmembrane fragments, accounting for more or less the same amount of pro tein. Whereas there is virtually no effect of 'Triton' on the activity associated with M" the increase of activity in MIC amounts to 400 per cent, suggesting that phospho-diesterase might be preferentially distributed in certain m embrane structures. Drummond and Perrott-Yee14 reported that a cyclic 3',5' -nucleotide phosphodiesterase from rabbit brain was localized entirely in the 100,000g supernatant. Nair'5 noted that a similar enzyme prepared from frozen dog heart was present cxclusively in the supernatant. These observations are at variance with our data and those of Butcher and Sutherland" who found that phospho-diesterase from beef heart is mostly particulate. The reason for such a discrepancy is not apparent to uso 91 Our results indicate that phosphodiesterase as frac-tionated in isotonic sucrose solution does not exhibit its full potential activity. The majority of the activity in the microsomes is latent and is unmasked by the action of 'Triton', but not by mechanical disruption or repeated freezing and thawing. In the presence of 'Triton', micro-somes account for more than half the total activity. The remaining activity distributes about equally among synaptoplasm and a 100,000g supernatant. We thank Professor B. Chance for his interest and encouragement throughout the course of this investigation. We also thank Mrs. A. E. Strichartz for technical assistance. This work was supported by the U.S. Public Health Ser-vice. Johnson R esearch Foundation, University of Pennsylvania, Philadelphia, Pennsylvania. WAl YIU CHEUNG LEON SALGANICOFF* • Fellow of the Consejo Nacional de Investigaciones Cientifieas y Teenicas Republiea Argentlna. • Rall , T. W., a nd Sutherland , E. W., J. Biol. Chem., 237 , 1228 (1962). 'Sutherland, E. W., and RaH, T. W., J. Bio!. Chem., 232,1077 (1958). 'Sutherland, E. W., RaH, T. W., and Menon, T., J. Biol. Chem., 237, 1220 (1962). • Butcher, R. W ., and Sutherland, E . W. , J. Biol. Chem., 237, 1244 (1962). ' Cheung, W. Y .. Biochem. Biophys. Res. Commun., 23,214 (1966). • De Robertis, E.,l'ellegrino de Iraldi, A., Rodriguez de LoresArnaiz, G., and Salganicoff, L., J. Neurochem., 9,23 (1962). , De Robertis, E.. Salganicoff, L., Zieher, L . M., Rodriguez de Lores Amai?, G., J. Neurochem., 10,255 (1963). 'Fiske, C. H. ,andSubbarow, Y., J. Biol. Chem., 66, 375 (1925). • Lowry, O. H. , and Lope?, J . A., J. Biol. Chem., 162. 421 (1946). 10 De Robertis, E., in Hislophysiology 0/ Synapses and Neurostelion, 19 " (Pergamon Press, New York. 1964). .. Salganieoff, L., and De Roberti., E., J. Neurochem.,12. 287 (1965)_ " Sellinger, O. Z. , Rucker, D. L .• and De Balbian Verster, J. Neurochem .. l1. 271 (1964). lS Toschi. G., Exp. Gell Res., 16, 232 (1959) . .. Drummond. G. I., and l'errott-Yee, S., J. Biol. Chem., 236,1126 (1961). I ,' ~air, K. G., Biochemistry. 5, 150 (1965). Aminoacyltransferase Stimulation of Protein Synthesis by Pig Adrenal Polysomes ADMINISTRATION of adrenocorticotrophin in viva increases the incorporation of 14C-glycine into protein by the supernatant of adrenal homogenates centrifuged at 15,000g'. A factor of the soluble cell fraction (105,000g supernatant) haI> been shown to be rate limiting for in vitra proteiD synthesis and its activity is doubled by ACTH administration in rats. This factor was reported to be proteinaceous; its elution pattern from DEAE-cellulose columns' was identical with liver aminoacyltransfcrase3 • The increased activity of the microsomal fraction by treatment with ACTH was repol'ted to be delayed compared with the soluble eell fraction'. Thus it was of interest to find wh ether polysomes, which are polymeres of ribosomes and messenger-RNA5 .• , or the protein from the soluble adrenal cell fraction were rate limiting for protein syn-thesis. When polysomes wcre incubated with constant amounts of soluble cell fraction, the incorporation of 14C-glycine increased only slightly and by no means in proportion to the amount of polysomes added. Thus incubation of polysomes with 0·4 ml. of 105,000g supernatant (80 mg-equivalents) showed the following incorporations. Poly-somes: 0·1 ml. (240 c.p.m.); 0·2 ml. (269 c.p.m.); 0·4 ml. (337 c.p.m.); pclysomes omitted (179 c.p.m.) (für incuba-tion conditions see Table 1). W e conclude that the amount ofpolysomes and therefore the amount ofmessenger RNA present in the incubation mixture did not limit thc rate of amino-acid incorporation. The protein factor prepared from pig soluble adrenal cell fraction (Fig. 1) and likely to be aminoacyltransferase' did, however, stimulate the incorporation of 14C-glycine by constant amounts of polysomes in approximate proportion to the amount added. Sampies VI and VII (Fig. 1) con-© 1967 Nature Publishing Group92 'fable 1. INCORPORATION Ol!' "C-GLYCINE BY ADRENAL POLYSOMES AS STIMl" LATED BY AMINOACYLTRANSFERASE SampIe (Fig.l) V VI VII VIII IX 0·2 mI. 270 615 754 210 255 0·4ml. 208 1,288 1,835 311 Polysomes were prepared from fresh plg adrenals··' and 0·1 mL = 160 mg-equivalents (containing 0·78 mg protein' and approxlmately 1·8 mg RNA') were incubated with 0·3 mI. 105,OOOg supernatant of fresh pig adrenals (60 ml(-equivalent). "C-glycine. ATP. GTP, PEP and P·kinase were added as In Fig. 1. In thls system 0'2 or 0'4 mJ. of sampIes V-IX (Fig. 1) were assayed for stimulation of "C·glyclne Incorporation in a total volumc of 1'05 mJ. (60 min, 37° Cl. The complete system wlth no addition showed an incorporation of 209 c.p.m.; 105,OOOg supernatant omitted and 5 c.p.m. when polysomes were omitted. Addition of 0'2 ml. of sampIes produced the followlng Incorporations: VI, 176 c.p.m.; VII, 342 c.p.m.; VIII, 108 c.p.m.; IX, 206 c.p.m. tained aminoacyltransferase and incorporation when they were mixtures (Table 1). 20 , stimulated amino·acid added to incubation NATURE. VOL. 214, APRIL I, 1967 IMMUNOLOGY Inter Heavy-Light Chain Disulphide Bridge in Immune Globulins THE immune globulins are a heterogeneous family of proteins made up of light and heavy* chains joined by disulphide bridges. Immunological teehniques1,t and chemical investigation3 indieate that most or all of the normal population of light chains is a mixture of two types of polypeptide-x and A. The majority of heavy chains can be classified as y, (1. or ce. In humans, the y chains have been further subdivided, making use of common antigenie determinants, into four major sub-classes: y2a (or Ne), y2b (or We), y2c (or Vi), and y2d (or Ge)4,5. Combination of either x or A chains with y, JL or ce ehains gives rise to immune globulins IgG (gamma· globnlins), IgM (maeroglobulins), or IgA. It has been 20,000 &lABIEN' • VI.vm IHKCI .. • I ! <= ~ ... Po< 10 15,000 10,000 Ei P-o 5,000 '" 8 -"i:.=::..... ... __ ..L. postulated3 that a single disulphide bridge linking x or A chains to heavy chains oecurs through the light chain C-terminal or next to C·terminal eys-teine residue, and this has been based mainly on the arrangements of disul-phide bridges in light chain monomers and dimers produced in the urine of myeloma patients (Bence-Jones pro· teins). A direct demonstration that this is the case has now been obtained by isolating disulphide-bridged peptides containing the C-terminal peptide of the light chain and a half-cystine peptide from the heavy chain of different types of human pathological immune globulins. In addition, a radioactive technique for the isolation of the corresponding carboxymethyl-cysteine peptides has been developed. This communication presents a com· parison between peptides derived from different classes and sub·classes. Pooled normal IgG is shown to contain in good yields the peptides found in the two chief sub-classes of y-chains. 0 o m m~~ m m I-+-u---f'm+IV+V+VI-tVn+-Vm-+IX+-X SampIe Fig. 1. Preparat.lon of aminoacyltransferase from pig adrenals. 13· 7 g fresh pig adrenals were homogenized in 68·5 ml. medium Ä (0'25 molar sucrose, 0'025 molar potassium chloride, 0·005 molar magnesium chloride, 0'05 molar tri. hydrochloric acid bulfer, pB 7'5) aß deBcribed' and the 105,OOOu supernatant was passed through dextran gel ('Sephadex G-25'). It was then concentrated by ultrailltration and subjected to anion exchange chromatography on a DEAE·cellulose column using a Iincar gradient made from I: 0'03 molar potassium chloride, 0·02 molar Iris hydrochlorlc acid bulfer, pH 7'5, and II: 0·6 molar potasslum chloride, 0·02 molar tris hydrochlorlc acid bulfer, pB 7'5 (ref. 2). The eluate was recombined into sampies I-X as indieated, eoncentrated by uUra-filtration, dialYBed againBt medium A and portions assayed for stimula· tion of protein Bynthesls in a celHree system. This contained 1 pc. "C· glyclne (0'014 pmoles), 10 pmoles phosphoenol·pyruvate, 1 pmole ATP. 0·25 pmoles GTP, 60 Pfl: pyruvate kinase and 80 mg-equivalent of 15,OOOg aupernatant of plg adrenal homogenate in a total volume of 1·05 ml. (370 C, 60 min). The assay of 14C-glycine incorporated into pro tein was described earller'. The data represent the total stimulation of Incorporatlon In each sampIe (c.p.m.) o Disulphide-bridged peptides were isolated by the performic acid oxidation diagonal technique6 ,7 after peptic (1/20 enzyme-substrate ratio in 5 per cent formic acid, 14 h at 37° C) and then Adrenocorticotrophin administered to ra.ts leads to increased protein synthesis in a cell-free system because of olevated aetivity or amount of aminoacyltransterase2• Onlyafter eontinued treatment with ACTH is the activity of polysomes stimulated4, but aminoacyltransferase remains the rate limiting factor for in vitro protein synthesis. This investigation was supported by the Deutsche Forsehungsgemeinschaft.. tryptic digestion (1/50 enzyme-substrate ratio in pH 7'0, 0·1 molar ammonium acetate buffer, 6 h at 37° Cl. Peptides were purified by paper electrophoresis at pH 6'5, and pH 3'5 before and after oxidation; with the macroglobulin Ale, a preliminary fractionation of peptides on a 'Sephadex G-25' column in 2 per cent acetic acid was necessary. Peptides with the sequenees shown in Table 1 were isolated from three different pathological immune globulins. PETER C. SCRIBA MARIANNE FRIES II. Medizinische Klinik der Universitat, Munieh. Received N ovem ber 9, 1966. I Farese, R. V., and Reddy, W. J .. Endocrinology, 73, 294 (1963). 'Seriba, P. C., anel Redcly, W. J ., Endocrinology, 78, 745 (1965). • Takanami, 111., Biochim. Biophys. Acta, 61 , 85 (1961). • Farese, R. V., Endocrinology, 78, 125 (1966). • Wettstein, F. 0., Staehelln, 'r., and Noll, JI., Nature, 197,430 (1963). • GIerer, A., J. Mol. Bioi., 6, 148 (1963), I Farese, R. V., EndocrinoloUlI, 77, 128 (1965). • Lowry, O. R., Rosenbrough, N. J., Farr, A. L., and RandalI, R. V., J. Biol. allem., 193, 265 (1951). • Dlache, Z., in The Nucleic Acid8 (edlt. by Chargaff, E., and Davidson, J. N.), 301 (Mademic Press, New York, 1955). 'fable 1. OYSTINE PEPTIDES CONTAINING TBE DISULPIDDE BOND BBTWIiEN LIGHT oI.ND HEAVY OHAINS OF SOME PoI.THOLOGlCAL IMMUNE GLOBULINS Beavy Light Protein chaln chain Ale Fle Viu type type )' v Pro.Leu.VaJ.Ser.Cys.GIx.ABx.Ser(Asp,Ser .. ThJ,Pro) I " Gly .Glu.Cys Ser.Cys.Asp.Lys I " Gly. Glu. Cys Pro.Leu.Ala(Cys,Ser,Pro) I Pro.Thr.Glu.Cys.Ser The occurrence of the sequences derived from Fis and Vin in pooled normal IgG and in other myeloma proteins • The nomenclature used for Immune globulin molecules, ohalD& and fragments is that recommended in ref. 24. 

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Aminoacyltransferase Stimulation of Protein Synthesis by Pig Adrenal Polysomes

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