Gel Electrophoresis of Chloroplast Polypeptides: Comparison of One-Dimensional and Two-Dimensional Gel Analyses of Chloroplast Polypeptides From Euglena gracilis

Euglena chloroplast polypeptides are resolved by an adaptation of the two-dimensional gel electrophoretic technique of O'Farrell (1975 J Biol Chem 250: 4007-4021). The present results are compared with those obtained by our earlier two-dimensional gel analyses as well as those obtained by one-dimensional gel analyses. Up to 75 micrograms of Euglena chloroplast polypeptides are resolved on one-dimensional sodium dodecylsulfate linear gradient 7.5 to 15% polyacrylamide gels into 43 stained polypeptide bands compared to only 33 bands resolved on a similar gel containing only 10% polyacrylamide. In contrast, two-dimensional gel electrophoresis (isoelectric focusing for the first dimension, sodium dodecylsulfate gel electrophoresis for the second dimension) further improves the resolution of the chloroplast polypeptides and especially so when a linear gradient gel is used for the second dimension. Delipidation of Euglena chloroplasts with acetone-ether and subsequent solubilization of polypeptides with Triton X-100 followed by sonication are all necessary for successful resolution of chloroplast polypeptides on two-dimensional gels. Up to 300 micrograms of chloroplast polypeptides can be clearly resolved into 56 to 59 stainable spots by the present two-dimensional gel technique when a linear gradient gel is used for the second dimension. Thus, about 30% of the polypeptide bands on a one-dimensional gel are separated into multiple polypeptides on a two-dimensional gel. The use of two-dimensional gels to separate labeled polypeptides with subsequent detection of labeled spots by autoradiography or fluorography again improves the resolution of the chloroplast polypeptides. For example, when ³⁵S-labeled chloroplast polypeptides are separated by the present two-dimensional gel technique with a linear gradient polyacrylamide gel in the second dimension, autoradiography or fluorography detects over 80 individual polypeptide spots. This is about twice the number resolved by our previous analyses which used a 10% polyacrylamide gel in the second dimension. Polypeptides detected range in molecular weight from about 8.5 to about 145 kilodaltons with apparent isoelectric points from pH 4.5 to 8.0. Fluorography provides rapid detection of labeled polypeptides and is 10 times more sensitive than autoradiography.     

Two-dimensional separation of chloroplast membrane proteins by isoelectri focusing and electrophoresis in sodium dodecyl sulphate

Proteins of chloroplast subfragments enriched in Photosystem I and Photosystem II electron flow activity have been analyzed by two-dimensional polyacrylamide gel electrophoresis. In the first dimension, polyacrylamide gel isoelectric focusing (pH 5–7) was used in the presence of Triton X-100, followed at right angle by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Characteristic fingerprints were obtained for the Photosystem I and II fractions and a correlation between the major proteins separated by isoelectric focusing and the major polypeptides separated by undimensional SDS electrophoresis was established. Two dominant spots of 68 000 and 60 000 daltons appeared in the two-dimensional patterns of Photosystem I fractions $\text{p}\text{I}$ values about 5.6; two spots with molecular weights of 33 000 and 23 000 were characteristics for Photosystem II fractions $\text{p}\text{I}$ values about 5.3 and 6.3). Photosystem I fractions were furthermore characteristics by a series of spots in the 44 000–33 000 range $\text{p}\text{I}$ values from about 5.9 to 6.8). The two-dimensional system revealed that (a) several SDS-polypeptides have multiple forms differing in charge only, (b) some proteins separated by isoelectric focusing are resolved in the second dimensional into polypeptides of different size. The two-dimensional method combining Triton X-100 isoelectric focusing' and SDS electrophoresis provides a higher degree of resolution than either of the unidimensional methods thus allowing a detailed analysis of chloroplast membrane proteins.     

Alkaline urea solubilization, two-dimensional electrophoresis and lectin staining of mammalian cell plasma membrane and plant seed proteins

Plasma membranes, isolated from Chinese hamster ovary cells and seed proteins from Arachis hypogaea (L.) were analyzed by two-dimensional electrophoresis. Polypeptides were solubilized without employing sodium dodecyl sulfate (SDS), using in its place 5 mm K₂CO₃ and 9.5 m urea. After addition of dithiothreitol and the nonionic detergent Nonidet P-40, more than 95% of the total protein remained in the supernatant fraction after the preparation was centrifuged at 100,000 g. The solubilization was comparable to that achieved with boiling SDS solution. This soluble material could be used directly for either isoelectric focusing or nonequilibrium pH gradient electrophoresis in narrow bore, tubular, polyacrylamide gels crosslinked by means of N,N′-diallyltartardiamide. Up to 750 μg of protein could be analyzed in one such 3 mm gel. Electrophoresis in polyacrylamide slab gels containing SDS was used for separations in the second dimension. The method allows large amounts of both basic and acidic insoluble proteins to be solubilized and then analyzed without employing SDS as a solubilizing agent. Classes of glycoproteins on the gels were detected by incubating with small volumes of ¹²⁵I-lectins in heat-sealed plastic bags. CHO cells contain several high molecular weight acidic glycoproteins that bind wheat germ agglutinin, but which do not stain with Coomassie blue. Several of the storage polypeptides in peanut seeds were also shown to bind wheat germ agglutinin and are probably, therefore, glycoproteins containing N-acetyl d-glucosamine.     

Development of a dedicated two-dimensional gel electrophoresis system that provides optimal pattern reproducibility and polypeptide resolution.

The development of a dedicated two-dimensional gel electrophoresis system is described that provides superior performance in terms of high resolving power and enhanced gel-to-gel reproducibility. Isoelectric focusing is performed in a 1-mm capillary tube with a 0.08-mm thread, optimized for this application, incorporated along its length prior to polymerization of the gel matrix. The isoelectric focusing gel is 4% T, 2.6% C to minimize sieving of proteins and promote adhesion of the gel to the thread. The thread incorporated in the isoelectric focusing matrix prevents gel stretching and breakage during its application to the second dimension. An optimum ampholyte pH range has been defined based on 1600 polypeptides present in a transformed fibroblast cell lysate and verified using a variety of other cell types. The length of time required to complete an electrophoretic separation in the second dimension was found to depend on buffer conductivity establishing the importance of high quality electrophoresis grade reagents devoid of contaminating salts. To ensure reproducibility of electrophoretic separations, it is critical to maintain a strict control of temperature during the second dimension separation. This prevents altered migration of some polypeptides relative to neighboring polypeptides that have constant Rfs over a broad temperature range. It was also determined that to obtain the maximum information from a complex protein mixture it is critical to use a large format 22- x 22-cm two-dimensional electrophoretic system. Using the optimized two-dimensional electrophoretic system and computerized gel analysis, it was determined that molecular weight estimates of polypeptides differed by approximately 350 daltons between gels, while isoelectric point estimates differed by approximately 0.03 pH units between gels. Using the two-dimensional electrophoresis system described, approximately 1000 polypeptides can be routinely detected from silver-stained 10% polyacrylamide gels or 1600 polypeptides from autoradiographs of 35S-methionine-labeled polypeptides.     

A Comparison of the Effect of Salt on Polypeptides and Translatable mRNAs in Roots of a Salt-Tolerant and a Salt-Sensitive Cultivar of Barley

The effect of salt stress on polypeptide and mRNA levels in roots of two barley (Hordeum vulgare L.) cultivars differing in salt tolerance (cv CM 72, tolerant; cv Prato, sensitive) was analyzed using two-dimensional polyacrylamide gel electrophoresis. Preliminary experiments indicated that germination of Prato was inhibited significantly in the presence of NaCl, but growth of the surviving Prato seedlings was not substantially different from that of CM 72. Fluorographs of two-dimensional gels containing in vivo labeled polypeptides or in vitro translation products were computer analyzed to identify and quantitate changes that resulted when plants were grown in the presence of 200 millimolar NaCl for 6 days. The patterns of in vivo labeled polypeptides and in vitro products of CM 72 and Prato were qualitatively the same. Salt caused quantitative changes in numerous polypeptides and translatable mRNAs, but, overall, the changes were relatively small. Salt did not induce the synthesis of unique polypeptides or translatable mRNAs and did not cause any to disappear. Because of the similarities of the two cultivars with respect to growth and polypeptide patterns and the slight changes in polypeptide and translation product levels caused by salt, specific polypeptides or translatable mRNAs that are related to salt tolerance in barley could not be identified.     

Comparison of malate dehydrogenase isoenzymes in someAllium species by isoelectric focusing

Malate dehydrogenase isoenzymes were studied in tenAllium species and in six cultivars ofA. cepa by isoelectric focusing in polyacrylamide gel with Ampholine pH 3.5–10.0. Using this method better resolution was obtained than by polyacrylamide gel electrophoresis. The number of MDH isoenzymes obtained by isoelectric focusing is from five to ten in the range of pH 3.65 to 6.75. MDH isoenzymes can be used for characterization on the level of species and cultivars (inA. cepa), but its use on the level of sections and subgenera is questionable.     

Subunit Composition of Glutamine Synthetase Isozymes from Root Nodules of Bean (Phaseolus vulgaris L.)

Glutamine synthetase from bean nodules can be separated into two isoforms, GS_n1 and GS_n2. A purification protocol has been developed. It included protamine sulfate precipitation, ammonium sulfate fractionation, anthranilate-affinity chromatography, Dye-Matrex (Orange A) chromatography, and diethylaminoethyl-cellulose ion-exchange chromatography. GS_n1 and GS_n2 have been purified to homogeneity. Subunit structure analysis using two-dimensional polyacrylamide gel electrophoresis revealed that GS_n1 was composed of two different types of subunit polypeptides. They differed in isoelectric points (6.0 and 6.3) but had the same molecular weights (46,000 Daltons). GS_n2 was composed of only one type of subunit polypeptide. It had an isoelectric point of 6.0 and a molecular weight of 46,000 Daltons. It was apparently identical to one of the polypeptides found in GS_n1. Glutamine synthetase holoenzyme consisted of eight subunits. In the nodule there are two different types of glutamine synthetase subunit polypeptides. Random combinations of the polypeptides should generate nine different isozymes. Our electrophoretic analysis revealed that GS_n2 was but one of the isozymes, and GS_n1 was a composite of the other eight. Hence, nodule glutamine synthetase isozymes were homo-octameric as well as hetero-octameric.     

Schistosoma mansoni: One- and two-dimensional electrophoresis of proteins synthesized in vitro by males,females, and juveniles

Polyacrylamide gel electrophoresis coupled with fluorography is a sensitive method for visualizing individual gene products synthesized in vitro by Schistosoma mansoni (K. Atkinson and B. G. Atkinson 1980, Nature (London)283, 478–479). In vitro labelling with radioactive amino acids ensures that the proteins are of parasite origin and fluorography permits detection of minute amounts of newly synthesized, electrophoretically separated gene products. One-dimensional electrophoretic separation in polyacrylamide gels with sodium dodecyl sulphate and fluorography of juvenile and adult proteins reveal that juveniles produce most adult proteins. Although similar studies with proteins from sexed adults imply that analogous gene products are elaborated by both sexes, a number of sex-specific gene products are resolvable by more rigorous two-dimensional electrophoretic separations. The homogametic male produces 5 polypeptides not produced by the heterogametic female. Three outstanding male-specific gene products include a polypeptide with a molecular weight (MW) of 88 kilodaltons (kd) and an isoelectric point (pI) of 5.65, one with an MW of 66 kd and a pI of 5.25, and one with an MW of 58 kd and a pI of 5.25. Other, readily detectable male-specific polypeptides include one which coelectrophoreses with β-actin and one which coelectrophoreses with β-tropomyosin. The female synthesizes 4 specific polypeptides which have isoelectric points between 4.3 and 4.7, are of low molecular weight, and are resolvable only with 12% acrylamide gels. Two-dimensional electrophoresis resolves 74 major polypeptides synthesized by adult worms, and a code is presented which identifies each polypeptide by sex specificity, isoelectric point, and molecular weight.     

Characterization of isoperoxidase activity in Arachis hypogaea cultivars

Isoperoxidases (both anodic and cathodic) from individual seeds of several peanut cultivars (Arachis hypogaea), after ammonium sulfate precipitation of the major storage protein, arachin, from the extracts were characterized by polyacrylamide gel electrophoresis with respect to seed development, maturity and germination and the geographic areas where grown. All cultivars had a major cathodic isozyme near the origin of the gels. Much anodic intra- and inter-varietal isozyme polymorphism was observed in mature seeds collected from different geographic areas. These polymorphic isozymes were consistently present during the earlier stages of seed development (the activities decreased quantitatively and became variable during the later stages of maturation), and most were observed in each peanut upon germination, the latter showing quantitative increases in activity.     

Effect of fungal-isolate aggressivity on the biosynthesis of symbiosis-related polypeptides in differentiating eucalypt ectomycorrhizas

Changes in protein biosynthesis were examined during the early stages of differentiation of Eucalyptus grandis-Pisolithus tinctorius ectomycorrhizas by two-dimensional polyacrylamide gel electrophoresis of ³⁵S-labelled proteins. Three distinct isolates of P. tinctorius Coker & Couch were chosen based on the rate of ectomycorrhizal formation (i.e. infectivity) with E. grandis W. Hill ex Maiden. The isolate H506 was not able to induce mycorrhiza, isolate 441 showed moderate infectivity and isolate H2144 exhibited a very high infectivity. Mycorrhiza were produced in vitro in a system where seeds were germinated in the presence of fungal mycelium and exudates. The non-mycorrhizal isolate caused no changes in root protein biosynthesis as analyzed by two-dimensional polyacrylamide gel electrophoresis, whereas drastic alterations in protein biosynthesis were observed from initial contact with the aggressive mycobionts. During mycorrhizal development, there was a marked inhibition of plant polypeptides synthesis, enhanced accumulation of some fungal polypeptides and the emergence of symbiosis-specific polypeptides, the so-called ectomycorrhizins. The major changes were observed in a group of fungal acidic polypeptides (apparent molecular weight 28–32 kDa) including the ectomycorrhizin E₃₂. These polypeptides first appeared at contact and their synthesis increased during mycorrhizal formation, suggesting a role in mycorrhizal development, most likely as structural proteins. Up-regulation of the synthesis of fungal symbiosis-related polypeptides was tightly correlated to the infectivity of the strain.Abbreviations FW fresh weight - MW molecular weight - pI isoelectric point - SR-polypeptides symbiosis-related polypeptides This work was supported by a research grant from the Eureka-Eurosilva programme (Changes in Gene Expression during Ectomycorrhiza Differentiation and Function) to F.M. and a Murdoch University Special Research Grant to B.D; T.B. was a recipient of a Doctoral Fellowship from the INRA and an Australian Postgraduate Scholarship. We would like to thank Dr Denis Tagu and Dulcinéia de Carvalho (Institut National de la Recherche Agronomique, Nancy, France) for helpful discussions.     

A comparative study of the polypeptides of three iridescent viruses by N-terminal analysis,amino acid analysis,and surface labeling

Polyacrylamide gel analysis of the structural proteins of three types of iridescent viruses (2, 6, and 9) demonstrated that the purified virions had one major and more than 20 minor polypeptides. Surface labeling procedures performed on pure intact virions, using ¹²⁵I in the presence of lactoperoxidase and chloramine T (at low iodine concentrations), demonstrated that the major and two or three minor polypeptides were located on the outside. The major structural polypeptide was isolated from each virus type by preparative polyacrylamide gel electrophoresis. Amino acid analysis indicated that this protein was very similar in the three iridescent viruses. The three polypeptides had an identical N terminal (proline). While the major polypeptide of each virus has a slightly different molecular weight as determined by polyacrylamide gel electrophoresis, the similarities in iodine labeling, N terminals, and amino acids suggests a common function for this protein.     

The Glycoproteins of Plant Seeds : ANALYSIS BY TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS AND BY THEIR LECTIN-BINDING PROPERTIES

Protein from the jack bean, peanut, soybean and kidney bean seeds were extracted with a solution containing 9.3 molar urea, 5 millimolar K₂CO₃, 0.5% dithiothreitol and 2% Nonidet P-40 and then subjected to two-dimensional gel electrophoresis. After electrophoresis, the slab gels were stained with a variety of ¹²⁵I-labeled lectins and the lectin-binding proteins were identified after autoradiography. Incubation of slab gels of jack bean with concanavalin A, peanut with peanut agglutinin, soybean with soybean agglutinin, and kidney bean with phytohemagglutinin showed that the majority of the polypeptides in each seed type were able to bind to their homologous lectins. Control slab gels in which incubations were carried out with identical amounts of proteins, ¹²⁵I-lectin and an appropriate sugar inhibitor showed little or no lectin binding to the polypeptides. Additionally, incubation of slab gels of peanut proteins with ¹²⁵I-ricin, ¹²⁵I-wheat germ agglutinin, ¹²⁵I-concanavalin A, and ¹²⁵I-soybean agglutinin each revealed a clearly distinct binding pattern compared to the one observed with the peanut agglutinin. The results demonstrate that a large number of legume seed polypeptides are glycoproteins and that the carbohydrate groups within a seed species are heterogeneous in structure, thus indicating the existence of complex glycosylating enzyme systems in legume seeds. It is suggested that the high degree of binding between seed proteins and their homologous lectins might have some functional significance in maintaining large aggregates of protein in compact, insoluble form.     

Polypeptides associated with the induction of direct somatic embryogenesis in Camellia japonica leaves I. Identification of embryo-specific polypeptides

Embryogenic and non-embryogenic induced leaves of Camellia japonica,with the same shoot origin and submitted to the same cultureconditions, were used to study protein changes during the inductionof direct embryogenesis. The analysis of protein changes inthese samples, based on two-dimensional polyacrylamide gel electrophoresis,revealed that 91% and 66.2% of the polypeptides detected, respectively,in embryogenic and non-embryogenic induced leaves, were alreadypresent in the non-induced control leaves. The results of thedifferential expression of eight selected polypeptides detectedin induced and non-induced leaves are presented. A spotlistreport was obtained for the 9% (43) embryogenesis-associatedleaf polypeptides. From these polypeptides, two polypeptideswere identified which seem to be specifically associated withsomatic embryogenesis in C. japonica: E1 (pl 5.6; mol. wt. 43.5)and E2 (pl 6.0; mol. wt. 25.7). Key words: Camellia japonica, embryogenesis-associated polypeptides, somatic embryogenesis, two-dimensional polyacrylamide gel electrophoresis     

Proteins and polypeptides of envelope membranes from spinach chloroplasts. I. Isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis separations

Polypeptides of spinach chloroplast envelopes were separated by electrophoresis in an SDS-polyacrylamide gradient gel. At least 37 polypeptides were resolved; nine were prominent. Two (M_r 54 000 and 16 000) were also found in the stroma fraction and identified by peptide mapping and isoelectric focusing in the second dimension as the large and small subunits of ribulose-1,5-bisphosphate carboxylase. Proteins of the chloroplast envelope were also separated by isoelectric focusing. An adaptation of a previous method (Ames, G.F.L. and Nikaido, K. (1976) Biochemistry 15, 616ndash;623), using solubilization in SDS and isoelectric focusing in the presence of a high concentration of Nonidet P-40, gave the best separation and resolved the envelope membranes into at least 21 proteins. The major band (pI 6.85) contained both subunits of the carboxylase and at least two additional polypeptides which corresponded to the prominent bands found in SDS gel electrophoresis of chloroplast envelopes.     

Purification and characterization of mojave (Crotalus scutulatus scutulatus) toxin and its subunits

An acidic lethal protein, Mojave toxin, has been isolated from the venom of Crotalus scutulatus scutulatus. The purified toxin had an i.v. LD₅₀ of 0.056 μg/g in white mice. Disc polycrylamide gel electrophoresis at pH values of 9.6 and 3.8 and isoelectric focusing in polyacrylamide gels with a pH 3.5–10 Ampholyte gradient were used to establish the presence of one major protein band. The pI of the most abundant form of the toxin was determined to be 5.5 by polyacrylamide gel isoelectric focusing experiments. The molecular weight was established to be 24,310 daltons from amino acid composition data. Mojave toxin was shown to consist of two subunits, one acidic and one basic with isoelectric point (pI) values of 3.6 and 9.6, respectively. Amino acid analyses established molecular weights of 9593 for the acidic component and 14,673 for the basic component. The acidic subunit consisted of three peptide chains intermolecularly linked by cystine residues. The basic subunit was a single polypeptide chain with six intramolecular disulfide bonds. The basic subunit was lethal to test animals with an intravenous LD₅₀ of 0.58 μg/g. Following recombination of the subunits a recombinant toxin was isolated which was identical to the native toxin by comparisons of electrophoretic mobility and toxicities. Comparisons of circular dichroism spectra also indicated reassociation to the native toxin structure. Phospholytic activity was associated with Mojave toxin and the basic subunit was responsible for this enzymic activity. Phospholipase activity of the basic subunit was inhibited by addition of the acidic subunit.     

Isolation and properties of cytochrome c-553, cytochrome c-550, and cytochrome c-549, 554 from Nitrobacter agilis

Three c-type cytochromes isolated from Nitrobacter agilis were purified to apparent homogeneity: cytochrome c-553, cytochrome c-550 and cytochrome c-549, 554. Their amino acid composition and other properties were studied. Cytochrome c-553 was isolated as a partially reduced form and could not be oxidized by ferricyanide. The completely reduced form of the cytochrome had absorption maxima at 419, 524 and 553 nm. It had a molecular weight of 25 000 and dissociated into two polypeptides of equal size of 11 500 during SDS gel electrophoresis. The isoelectric point of cytochrome c-553 was pH 6.8. The ferricytochrome c-550 exhibited an absorption peak at 410 nm and the ferrocytochrome c showed peaks at 416, 521 and 550 nm. The molecular weight of the cytochrome estimated by gel filtration and by SDS gel electrophoresis was 12 500. It had an E_m(7) value of 0.27 V and isoelectric point pH 8.51. The N-terminal sequence of cytochrome c-550 showed a clear homology with the corresponding portions of the sequences of other c-type cytochromes. Cytochrome c-549, 554 possessed atypical absorption spectra with absorption peaks at 402 nm as oxidized form and at 419, 523, 549 and 554 nm when reduced with Na₂S₂O₄. Its molecular weight estimated by gel filtration and SDS polyacrylamide gel electrophoresis was 90 000 and 46 000, respectively. The cytochrome had an isoelectric point of pH 5.6. Cytochrome c-549, 554 was highly autoxidizable.     

Detection of elastase activity with a zymogram method after isoelectric focusing in polyacrylamide gel

A zymogram method for detecting elastase activity following isoelectric focusing in polyacrylamide gel is described. After enzyme activity has been visualized, the gel itself is available for protein staining and for analysis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in second dimension. The zymogram method is suitable for detecting microgram amounts of elastase and has one step only. It can be used with the purified enzyme as well as with crude extracts of tissue containing elastases showing activity toward succinyl-(Ala)₃-p-nitroanilide. By this method a major component of elastase in both porcine and rat pancreas was detected. In addition, two forms of elastase with isoelectric points of 8.2 and 8.8, respectively, were identified in rat leukocyte extracts.     

Use of esterase isoenzymes revealed by gel isoelectric focusing as an aid in chemotaxonomical study of the genusAllium

Eleven species of the genusAllium belonging to the sectionCepa, Phyllodolon, Rhizirideum, Melanocrommyum andAlliun ware investigated as to the presence of esterase isoenzymes by means of the gal isoelectric focusing which shows better resolution than polyaorylamide gel electrophoresis. In all examined species twenty six isoenzymes were found. The individual sections are well characterized by esterase isoenzymes revealed by this method, on the other hand, the differences, as shown by the Jaceard index, between the subganera are insignificant. Investigation of seven cultivars ofAllium cepa shows the isolated position of the cultivar Ka?tická. These results are in full agreement with those found with use of the polyacrylamide gel electrophoresis and show the usefulness of gel isoelectric focusing for the solution of the chemotaxonomical problems.     

Electrophoretic Comparison of Mitochondrial Polypeptides from Maize Lines Susceptible and Resistant to Helminthosporium maydis Race T

The mitochondrial polypeptides from maize lines susceptible and resistant to Helminthosporium maydis were studied by single- and two-dimensional gel electrophoresis. Approximately 120 polypeptides were detected by two-dimensional gel electrophoresis. However, it was not possible to detect qualitative differences between the mitochondrial polypeptides of the two inbreds. These observations are discussed with reference to the putative mechanism of action of the pathotoxin.     

Analysis of the major polypeptides of spectrin by tryptic digestion

The two major polypeptides of erythrocyte membrane spectrin have been isolated by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The tryptic peptide maps of the two polypeptides have been prepared by thin-layer chromatography and electrophoresis. Radioactive peptides have been prepared by ¹⁴C-carboxymethylation and chloramine T-catalysed ¹²⁵I iodination. Maps of both sets of peptides demonstrate a marked similarity between the two parent polypeptides.