Characterization of Vicilin Seed Storage Protein of Chickpea (Cicer arietinum L.)

Vicilin, one of the major storage proteins of chickpea (Cicer arietinum L.) was purified and characterized during seed development. Vicilin was purified by zonal isoelectric precipitation followed by chromatography on DEAE-cellulose column. Vicilin on SDS-PAGE resolved into 5 major bands ranging in mol wt from 14 to 66 kD. More heterogenous pattern emerged on isoelectric focussing. This protein had high amount of amides and low amount of sulphur containing amino acids.     

Cloning and sequencing of a cDNA encoding the major storage proteins of Theobroma cacao

The major storage proteins, polypeptides of 31 and 47 kilodaltons (kDa), from the seeds of cocoa (Theobroma cacao L.), have been identified and partially purified by preparative gel electrophoresis. The polypeptides were both N-terminally blocked, but some N-terminal amino-acid sequence was obtained from a cyanogen bromide peptide common to both polypeptides, permitting the construction of an oligonucleotide probe. This probe was used to isolate the corresponding copy-DNA (cDNA) clone from a library made from poly(A)⁺ RNA from immature cocoa beans. The cDNA sequence has a single major open reading frame, that translates to give a 566-amino-acid polypeptide of M_r 65 612. The existence of a common precursor to the 31- and 47-kDa polypeptides of this size was confirmed by immunoprecipitation from total poly(A)⁺RNA translation products. The precursor has an N-terminal hydrophobic sequence which appears to be a typical signal sequence, with a predicted site of cleavage 20 amino acids after the start. This is followed by a very hydrophilic domain of 110 amino acids, which, by analogy with the cottonseed -globulin, is presumed to be cleaved off to leave a domain of approx. 47 kDa, very close to the observed size of the mature polypeptide. Like the hydrophilic domain of the cottonseed -globulin the cocoa hydrophilic domain is very rich in glutamine and charged residues (especially glutamate), and contains several Cys-X-X-X-Cys motifs. The cyanogen-bromide peptide common to the 47-kDa and 31-kDa polypeptides is very close to the proposed start of the mature domain, indicating that the 31-kDa polypeptide arises via further C-terminal processing. The polypeptide sequence is homologous to sequences of the vicilin class of storage proteins, previously found only in legumes and cotton. Most of these proteins have a mature polypeptide size of approx. 47 kDa, and are synthesised as precursors only slightly larger than this. Some, however, are larger polypeptides (e.g. -conglycinin from soybean is 72 kDa), usually due to an additional N-terminal domain. In cottonseed the situation appears to parallel that in cocoa in that the vicilin is synthesised as an approx. 70-kDa precursor and then processed to a 47-kDa (and in the case of cocoa also a 31-kDa) mature protein. In this context it is interesting that cotton is closer in evolutionary terms to cocoa than are the legumes, both cotton and cocoa being in the order Malvales.Abbreviations A absorbance - cDNA copy DNA - IgG immunoglobulin G - kb kilobase pairs - kDa kilodaltons - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacylamide gel electrophoresis The authors are very grateful to Dr R. Jennings of the Virology Department, Sheffield University Medical School, for help in raising antibodies.     

Purification and partial characterization of a 31-kDa cysteine endopeptidase from germinated barley

Proteolytic enzymes hydrolyze cereal seed storage proteins into small peptides and amino acids, which are very important for seed germination and the malting process. A cysteine-class endopeptidase was purified from 4-d-germinated barley (Hordeum vulgare L. cv. Morex). Four purification steps were used, carboxymethyl cellulose cation-exchange chromatography, chromatofocusing, size-exclusion chromatography, and electroelution from a polyacrylamide gel. The endopeptidase was most active at pH 4.5. It's isoelectric point (pI) was 4.4, as determined by isoelectric focusing, and it's SDS-PAGE molecular size was 31 kDa. The enzyme specifically hydrolyzed peptide bonds when the S₂ site contained relatively large hydrophobic amino acids. The N-terminal amino acid sequence residues (1–9) of the 31-kDa endopeptidase had high homology to those of the EP-A and EP-B cysteine proteinases reported previously. The 31-kDa endopeptidase had a hydrolytic specificity similar to that of the Morex green malt 30-kDa endopeptidase we characterized previously, and also reacted with the antibody raised against the purified 30-kDa proteinase, but the two had different mobilities on non-denaturing PAGE. The hydrolytic specificities of both 30- and 31-kDa endopeptidases are such that both would very quickly cleave hordein (barley storage) proteins to small glutamine- and proline-rich peptides that could be quickly degraded to amino acids by barley exopeptidases.Abbreviations CMC carboxymethyl cellulose - E-64 transepoxysuccinyl-l-leucylamido-(4-guanidino)butane - EMI N-ethylmaleimide - IEF isoelectricfocusing - Phen 1,10-phenanthroline - PI isoelectric point - PMSF phenylmethylsulfonyl fluoride We thank the American Malting Barley Association for partially funding this work. Germinated barley seeds were kindly prepared by Eddie D. Goplin. Special thanks to Laurie Marinac for her excellent technical assistance.     

Isolation and comparative biochemical properties of the major internal polypeptides of equine infectious anemia virus.

We describe procedures for the large-scale production of equine infectious anemia virus (EIAV) and for the isolation of the four major non-glycosylated virion proteins, designated p26, p15, p11, and p9. Comparisons of the purified proteins by peptide mapping procedures and by enzyme-linked immunosorbent assays demonstrated the unrelatedness of the four proteins. The characteristic properties of each purified protein were examined by determining isoelectric points and amino acid compositions. We found that EIAV p26 and p9 focus at pI values of 6.2 and 5.0, respectively, and that these proteins contain no unusual amino acids. In contrast, EIAV p15 reproducibly displayed a heterogeneous isoelectric focusing pattern, with major pI values ranging from 5.7 to 8.3. This charge variation evidently correlated with different levels of phosphorylated serine or threonine or both, which could be detected by an amino acid analysis of purified p15. EIAV p11 apparently focused at a pI of greater than 10, reflecting its high content of basic amino acids. Moreover, localization experiments indicated that all four nonglycosylated proteins constitute the internal components of the virus, with all of the virion p11 closely associated with the viral RNA genome. Thus, our results demonstrated that EIAV, a lentivirus, contains structural polypeptides which are analogous to the structural polypeptides described previously in prototype C oncoviruses.     

Biochemistry of Fern Spore Germination: Globulin Storage Proteins in Matteuccia struthiopteris L

Two globulin storage proteins have been identified in spores of the ostrich fern, Matteuccia struthiopteris (L.) Todaro. The two proteins comprise a significant amount of the total spore protein, are predominantly salt-soluble, and can be extracted by other solvents to a limited extent. The large 11.3 Svedberg unit (S) globulin is composed of five polypeptides with molecular weights of 21,000, 22,000, 24,000, 28,000 and 30,000. Each polypeptide has several isoelectric point (pI) variants between pH 5 and 7. The small 2.2S storage protein has a pI > 10.5 and is composed of at least two major polypeptides of 6,000 and 14,000 M_r. The amino acid composition of both storage proteins reveals that the 11.3S protein is particularly rich in aspartic and glutamic acid, while the 2.2S protein has few acidic amino acids. During imbibition and germination the globulin fraction declines rapidly, with a corresponding degradation of individual polypeptides of each protein. Polyclonal antibodies against each of the two proteins were produced and used for immunolocalization to determine the site of storage protein deposition within the quiescent spore. The proteins were sequestered in protein bodies of 2 to 10 micrometers, that are morphologically similar to those found in the seeds of flowering plants. The results suggest that spore globulins are biochemically similar to seed globulins, especially those found in some cruciferous seeds.     

Prekeratin biosynthesis in human scalp epidermis.

Analysis of human scalp epidermal prekeratin polypeptides by two-dimensional gel electrophoresis revealed that each of the bands observed in one-dimensional electrophoresis consisted of three to five polypeptides of the same molecular weight but differing in isoelectric points. It was possible to divide the polypeptides into two families, with isoelectric points in the ranges pH 6.0-8.0 and pH 5.0-5.5 respectively. Incorporation of radiolabelled amino acids into freshly excised pieces of scalp epidermis showed that some of the polypeptides had relatively greater contents of glycine and serine than others. Radiolabelled methionine and leucine were, in contrast, incorporated more or less uniformly into all the polypeptides. After incubation with 32P-labelled orthophosphate, relatively more intense labelling by 32P was observed in the higher molecular weight bands of each family. The most basic of the isoelectric variants in each case did not take up phosphate, implying that at least some of the variation in charge was due to different degrees of phosphorylation. Polyadenylated RNA isolated from scalp epidermis was translated in an RNA-dependent reticulocyte haemolysate system followed by immunoprecipitation and electrophoresis. The polypeptides isolated by using anti-(human scalp prekeratin) immunoglobulin G had similar electrophoretic mobilities in sodium dodecyl sulphate/polyacrylamide gels to authentic prekeratin polypeptides, but had different isoelectric properties. This suggested that the products of keratin gene expression undergo post-translational modification.     

Purification and Partial Amino Acid Sequence of the Major 70,000-Dalton Heat Shock Protein in Neurospora crassa

Fracella, F., Mohsenzadeh, S., and Rensing, L. 1993. Purification and partial amino acid sequence of the major 70,000-Dalton heat shock protein in Neurospora crassa. Experimental Mycology , 17, 362-367. The major heat shock protein of 70 kDa (hsp70) from heat-shocked mycelial extracts of Neurospora crassa was purified to near homogeneity employing DEAE anion-exchange chromatography followed by affinity chromatography on ATP-agarose. The isolated hsp70 migrates as a single band on one-dimensional sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE), with a molecular mass of ∼69 kDa. On two-dimensional gels it is resolved into two polypeptides with isoelectric points in the acidic range of ∼pH 5.2. The first 53 amino terminal amino acids of the major protein were sequenced and compared with hsp70 of other species. The amino acids aspartic acid, arginine, and phenylalanine occur at positions 27, 28, and 44 (from the methionine terminus) in contrast to the main consensus sequence. These three differing amino acids are shared by yeast, and, in addition, the first two by Arabidopsis, petunia, and maize.     

Vegetative storage proteins in poplar : induction and characterization of a 32- and a 36-kilodalton polypeptide

Bark, wood, and root tissues of several Populus species contain a 32- and a 36-kilodalton polypeptide which undergo seasonal fluctuations and are considered to be storage proteins. These two proteins are abundant in winter and not detectable in summer as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection. An antibody raised against the 32-kilodalton storage protein of Populus trichocarpa (T. & G.) cross-reacts with the 36-kilodalton protein of this species. The synthesis of the 32- and 36-kilodalton proteins can be induced in micropropagated plants by short-day conditions in the growth chamber. These proteins are highly abundant in structural roots, bark, and wood and combined represent >25% of the total soluble proteins in these tissues. Nitrate concentration in the leaves and nitrate uptake rate decreased dramatically when LD plants were transferred to short-day conditions; the protein content in leaves was unaffected. A decrease of the 32- and 36-kilodalton polypeptides occurs after transferring induced plants back to LD conditions. Both polypeptides are glycosylated and can be efficiently purified by affinity chromatography using concanavalin A-Sepharose 4B. The 32- and the 36-kilodalton polypeptides have identical basic isoelectric points and both consist of at least three isoforms. The storage proteins show a loss in apparent molecular mass after deglycosylation with trifluoromethanesulfonic acid. It is concluded that the 32- and 36-kilodalton polypeptides are glycoforms differing only in the extent of glycosylation. The relative molecular mass of the native storage protein was estimated to be 58 kilodalton, using gel filtration. From the molecular mass and the elution pattern it is supposed that the storage protein occurs as a heterodimer composed of one 32- and one 36-kilodalton subunit. Preliminary data suggest the involvement of the phytochrome system in the induction process of the 32- and 36-kilodalton polypeptides.     

The Native Structure and Composition of the Cruciferin Complex in Brassica napus

Globulins are an important group of seed storage proteins in dicotyledonous plants. They are synthesized during seed development, assembled into very compact protein complexes, and finally stored in protein storage vacuoles (PSVs). Here, we report a proteomic investigation on the native composition and structure of cruciferin, the 12 S globulin of Brassica napus. PSVs were directly purified from mature seeds by differential centrifugations. Upon analyses by blue native (BN) PAGE, two major types of cruciferin complexes of ∼ 300–390 kDa and of ∼470 kDa are resolved. Analyses by two-dimensional BN/SDS-PAGE revealed that both types of complexes are composed of several copies of the cruciferin α and β polypeptide chains, which are present in various isoforms. Protein analyses by two-dimensional isoelectric focusing (IEF)/SDS-PAGE not only revealed different α and β isoforms but also several further versions of the two polypeptide chains that most likely differ with respect to posttranslational modifications. Overall, more than 30 distinct forms of cruciferin were identified by mass spectrometry. To obtain insights into the structure of the cruciferin holocomplex, a native PSV fraction was analyzed by single particle electron microscopy. More than 20,000 images were collected, classified, and used for the calculation of detailed projection maps of the complex. In contrast to previous reports on globulin structure in other plant species, the cruciferin complex of Brassica napus has an octameric barrel-like structure, which represents a very compact building block optimized for maximal storage of amino acids within minimal space.     

Characterization of vacuolar polypeptides of barley mesophyll cells by two-dimensional gel electrophoresis and by their affinity to lectins

Vacuoles were isolated from primary leaves of barley (Hordeum vulgare L.) by mechanical breakage of protoplasts, and their polypeptide composition analyzed by two-dimensional gel electrophoresis. Vacuoplasts which consist of the vacuole, a portion of the plasmalemma and of the cytoplasma were prepared from protoplasts by ultracentrifugation. By comparing the vacuolar polypeptide pattern with polypeptide patterns of isolated chloroplasts and of vacuoplasts, vacuolar polypeptides could clearly be distinguished from polypeptides derived from cross-contaminating cell compartments. At least 14 polypeptides of apparent molecular mass between 12 and 76 kilodaltons and an isoelectric point between 4.5 and 7.6 could be attributed to the tonoplast fraction of the vacuole, and 35 polypeptides to the soluble fraction of the vacuole. Several lectins with different specificity were employed to characterize the degree and nature of glycosylation of vacuolar polypeptides. Concanavalin A bound to a large number of polypeptides. Three out of the 14 tonoplast polypeptides exhibited detectable carbohydrate moieties and almost two-thirds of the surveyed soluble polypeptides were glycosylated.Abbreviations IEF isoelectric focussing - kDa kilodalton - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

The amino acid sequence of the polypeptide segment which regulates membrane adhesion (grana stacking) in chloroplasts

A positively charged amino acid sequence, located on the NH2 terminus of the polypeptides of the chlorophyll a/b light harvesting complex, stabilizes thylakoid membrane adhesion. Threonine residues in this segment are the site of light-induced, reversible phosphorylation; this covalent modification results in changes in excitation-energy distribution in chloroplast membranes. Removal of the positively charged peptide by treatment with trypsin or chemical modification of amino acids in the sequence disrupts thylakoid adhesion and inhibits regulation of excitation-energy distribution. Purified preparations of the chlorophyll a/b light harvesting complex consist of 2 major polypeptides of 27 and 26 kDa and 2 minor polypeptides of 29 and 25 kDa (based upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Trypsin treatment of the isolated chlorophyll proteins decreases the apparent molecular mass of the 27- and 26-kDa polypeptides by 1-1.5 kDa and releases 3 peptides; [Lys, Arg], Ser-Ala-Thr-Thr-Lys-Lys, and Ser-Ala-Thr-Thr-Lys. These peptides probably form the overlap sequence, [Lys, Arg]-Ser-Ala-Thr-Thr-Lys-Lys. The polypeptides of the chlorophyll a/b light-harvesting complex were separated by isoelectric focusing into 5 chlorophyll protein fractions which had isoelectric points between 4.0 and 4.55. The 27-kDa polypeptides had an isoelectric point of 4.3, and bound 11 chlorophyll molecules/polypeptide.     

Identification of a novel metal binding protein, segon, in plasma of the eastern oyster, Crassostrea virginica

The second most abundant protein of eastern oyster plasma was purified, characterized and named segon. The 39 kDa protein as determined by SDS-PAGE under reducing conditions made up about 17% of plasma proteins and was found in extrapallial fluid. RACE reactions with primers designed from an EST sequence identified by BLAST search in GenBank using the N-terminal amino acid sequence obtained by Edman degradation of the purified protein, predicted a 997 bp complete cDNA that encoded 277 amino acids including a 16-residue signal peptide at the N-terminus. The deduced mature protein, composed of 261 amino acids, had a calculated molecular mass of 30,483.9 Da which was lower than the molecular mass of the purified protein measured by MALDI. The difference was likely due to post-translational modifications as the protein was predicted to have multiple sites for glycosylation and phosphorylation. The protein mRNA was detected in hemocytes by in situ hybridization and quantified in oyster tissues by RT-qPCR. Immunohistochemistry revealed that the protein was most abundant in tissues rich in blood sinuses like the gills and dorsally along the base of the mantle. ICP metal analysis of purified protein indicated highest association with zinc, calcium and iron and much greater metal content than in purified dominin, the most abundant protein of eastern oysters. Results of N-terminal and internal peptide sequencing of SDS-PAGE separated plasma proteins from Pacific, Suminoe and European flat oysters indicated that the second most abundant plasma protein is conserved. Several possible functions of segon in metal transport and detoxification, host defense, antioxidation and shell mineralization are proposed as they relate to its capacity to bind metals.     

Developmental Biochemistry of Cottonseed Embryogenesis and Germination : XII. PURIFICATION AND PROPERTIES OF PRINCIPAL STORAGE PROTEINS

The two principal storage proteins of the cottonseed have been purified and partially characterized. They have apparent molecular weights of 52,000 and 48,000 and are both comprised of sets of isoelectric isomers. Both sets have nearly identical solubility properties, ultraviolet absorption spectra, and contain similar proportions of amino acid residues. Furthermore, both sets share antigenic determinants suggesting their origin from a common ancestral gene. However, only the 52,000 set appears to be glycosylated.     

Identification of an Endogenous Inhibitor of the UDP-N-Acetylgalactosamine: GM3, N-Acetylgalactosaminyl Transferase as Apolipoprotein A1

A previously described inhibitor of the UDP-N-acetylgalactosamine: GM3, N-acetylgalactosaminyltransferase (GalNAc-T) (Quiroga et al., 1,2), was purified from chicken blood serum by a new procedure. When subjected to SDS-PAGE, two major polypeptides of 27 and 70 kDa were observed. When tested in vitro, only the 27 kDa polypeptide inhibited the GalNAc-T. When added to chick cerebral embryonic neurons in culture, both polypeptides inhibited neuritogenesis. Both the 27 kDa and the 70 kDa fractions were present in the cells at 3 h following their addition to the cultures; both polypeptides had aneuritogenic activity and both inhibited the incorporation of [³H]-galactose into the cell gangliosides modifying their labeling pattern to a similar extent. Sequencing of the amino terminal end of the polypeptides showed that 18 and 9 amino acids from, respectively, the 27 and the 70 kDa polypeptides, were 100% homologues with the corresponding region of chick apolipoprotein A1 (apo A1). After addition to cells in culture, no interconversion between the two polypeptides was detected after up to 20 h in culture. A monoclonal antibody that recognizes only the 70 kDa polypeptide, blocks its aneuritogenic effect without modifying that of the 27 kDa fraction. It is concluded that the endogenous inhibitor of GalNAc-T is apo A1.     

Purification of a 24 kD parasitism-specific hemolymph protein from pharate pupae of the caribbean fruit fly,Anastrepha suspensa

We report purification of a 24 kD parasitism-specific protein (24 kD PSP) from pharate pupal hemolymph of the Caribbean fruit fly, Anastrepha suspensa, after parasitization by the braconid wasp, Diachasmimorpha (= Biosteres) longicaudata. We previously utilized isoelectric focusing (IEF) and two-dimensional (2-D) gel electrophoresis to demonstrate that the 24 kD PSP consists of two variants with pl 6.7 (more abundant) and pl 6.3. Purification of the more abundant 24 kD PSP variant was accomplished by Concanavalin A (Con A) sepharose B affinity chromatography followed by DEAE column chromatography. A second protocol, utilizing wheat germ agglutinin (WGA) sepharose 6MB affinity chromatography between the ConA and DEAE chromatographic steps, resulted in the purification of a partially deglycosylated form of the 24 kD PSP which retained its immunore-activity with anti-PSP serum but which exhibited a greater relative migration in sodium dodecyl sulfate (SDS)-PAGE than the pl 6.7 24 kD PSP variant. For structural studies both 24 kD PSP variants were purified from whole hemolymph by flat bed IEF followed by SDS-PAGE. Peptide cleavage profiles in 1-D SDS-PAGE after treatment with BNPS-skatole, CNBr, and endproteinases Lys-C and Asp-N were identical for both 24 kD PSP variants. Primary N-terminus sequences of at least the first 20 amino acid residues of both variants were identical. A secondary sequence of five amino acids residues was detected in both variants at Thr, the seventh amino acid residue from the N-terminus of the primary sequence. These data indicate that both 24 kD PSPs are glycoforms of a branched, apparently homogeneous polypeptide. © 1994 Wiley-Liss, Inc.     

Storage protein accumulation during zygotic and somatic embryo development in Picea abies (Norway spruce)

Total protein was extracted from zygotic embryos and from somatic embryos of Picea abies (L.) Karst. (Norway spruce) cultured in vitro at different times during their development. An analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis of the protein extracts showed that protein composition and the temporal changes in protein abundance were very similar in the two embryo types. Both zygotic and somatic embryos accumulated storage proteins in abundance during their maturation phase of growth; the somatic embryos when cultured on medium containing 90 m M sucrose and 7.6 μ M ABA. The major storage proteins are composed of polypeptides with molecular masses of about 22, 28, 33 and 42 kDa and they are identical in both embryo types according to their molecular mass and average isoelectric points. These proteins are also the most abundant proteins in the female gametophytic tissue of the mature seed.     

Localization of the charge differences in the actins of rabbit skeletal muscle and chicken gizzard by two-dimensional gel electrophoretic analysis of tryptic fragments.

Partial tryptic cleavage products of pure actin from rabbit skeletal muscle and chicken gizzard are compared by two-dimensional electrophoresis in polyacrylamide gels with respect to isoelectric point and molecular weight. While the intact polypeptides (Mr 42,000) have different isoelectric points, two large cleavage products (Mr 35,000) generated from both both actin species have identical isoelectric points and identical molecular weights. These relatively trypsin-resistant cleavage products are presumably identical to the known "core actin" fragments which lack the aminoterminal region of the polypeptide chain. Therefore the differences that are responsible for the different isoelectric points of rabbit skeletal muscle actin and chicken gizzard actin seem to be restricted to the aminoterminal part of the actin polypeptide chains as was proposed on the basis of partial amino acid sequence data.     

In vivo and in vitro processing of seed reserve protein in the endoplasmic reticulum: evidence for two glycosylation steps

Cotyledons of the common bean (Phaseolus vulgaris L.) synthesize large amounts of the reserve protein phaseolin. The polypeptides are synthesized on membrane-bound polysomes, pass through the endoplasmic reticulum (ER) and accumulate in protein bodies. For a study of the biosynthesis and processing of phaseolin, developing cotyledons were labeled with radioactive amino acids, glucosamine and mannose, and isolated fractions (polysomal RNA, polysomes, and rough ER) were used for in vitro protein synthesis. Newly synthesized phaseolin present in the ER of developing cotyledons can be fractioned into four glycopolypeptides by SDS PAGE. In vitro synthesis with polysomal RNA results in the formation of two polypeptides by polysome run-off shows that glycosylation is a co-translational event. The two unglycosylated polypeptides formed by polysome run-off are slightly smaller than the two polypeptides formed by in vitro translation of isolated RNA, indicating that a signal peptide may be present on these polypeptides. Run-off synthesis with rough ER produces a pattern of four polypeptides similar to the one obtained by in vivo labeling. The two abundant glycopolypeptides formed by polysome run-off. This result indicates the existence of a second glycosylation event for the abundant polypeptides. Inhibition of glycosylation by Triton X-100 during chain-completion with rough ER was used to show that these two glycosylation steps normally occur sequentially. Both glycosylation steps are inhibited by tunicamycin. Analysis of carhohydrate to protein ratios of the different polypeptides and of trypsin digests of polypeptides labeled with [(3)H]glucosamine confirmed the conclusion that some glycosylated polypeptides contain two oligosaccharide chains, while others contain only one. An analysis of tryptic peptide maps shows that each of the unglycosylated polypeptides is the precursor for one glycosylated polypeptide with one oligosaccharide chain and one with two oligosaccharide chains.     

Comparison of the major polypeptides of the erythrocyte nuclear envelope

The three most abundant nonhistone polypeptides (molecular weights 75,000, 71,000 and 61,000) of the avian erythrocyte nucleus have previously been isolated in the nuclear envelope fraction. They have been separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis and peptide-mapped after limited enzymatic digestion. Three enzymes–chymotrypsin, papain and Staphylococcus aureus protease–were used. Results obtained with each enzyme indicate strong similarities between the three nuclear envelope polypeptides. The amino acid compositions of the two most abundant polypeptides (P75 and P71) have been determined and found to be similar. Further, they readily yield large fragments upon brief alkaline hydrolysis. For both P75 and P71 the degree and the pattern of alkaline fragmentation are almost identical. A 61,000-dalton polypeptide which appears to be P61 is obtained from P75 and P71 by mild acid hydrolysis. These results establish the close chemical similarity of these predominant polypeptides in the erythrocyte nucleus and suggest that they serve related functions.