豌豆球蛋白及其亚基的分离提纯

本文详细地叙述了提取、分离、提纯豌豆球蛋白及其亚基的方法:用硼酸钠缓冲液提取豌豆球蛋白,用85%饱和度硫酸铵沉淀除去其它蛋白质杂质,再用凝胶柱进一步提纯;提纯的豌豆球蛋白通过DE_(52)-纤维素阴离子树脂柱(8M尿素作为变性分离试剂)和S_(200)凝胶柱(70%甲酸作为变性分离试剂)则可分离出豌豆球蛋白的各个亚基;试验结果还表明:在pH8.5时,33,000的亚基携带少量负电荷;而12,000的亚基携带大量负电荷。     

Purification and characterization of high salt-soluble vicilin from mung bean (Vigna radiata)

We report a method for the purification of vicilin from mung bean (Vigna radiata) mainly on the basis of solubility of mung bean vicilin even in high salt. Mung bean vicilin remains in solution even after 90% relative saturation of ammonium sulphate. The resulting supernatant after dialysis was subjected to gel filtration (Sephadex G-150) to remove other contaminant polypeptides, and finally the protein was purified by DEAE cellulose chromatography. This purified fraction exhibited 3 bands on SDS-PAGE compared with vicilin from other legumes which exhibite more than 3 bands generally. The results raise the possibility that the presence of the two small polypeptides in vicilin preparations is the breakdown product of the major larger one of mol.wt. 52 K and that vicilin may be a tetramer of four subunits of Mr 52000. That the high salt-soluble protein containing 52 K subunit is vicilin has been determined by several criteria.     

Characterization of protein synthesis initiation factor 2 from Xenopus laevis oocytes

The protein synthesis initiation factor 2 (eIF2) from Xenopus laevis oocytes has been extensively purified and characterized. Depending upon the purification scheme, eIF2 containing three subunits (alpha, beta and gamma) with Mr of 160,000, or two subunits (alpha and gamma) with Mr 90,000 can be obtained. The key step for obtaining the three subunit factor is the addition of 30 mM benzamidine to the initial homogenization, since this compound protects the highly sensitive beta subunit from proteolytic degradation. Subunit alpha of the oocyte eIF2 can be phosphorylated by the specific kinase from rabbit reticulocytes, whereas subunit beta is phosphorylated by oocyte casein kinase II. The oocyte eIF2 has a KD of 7.2 X 10(-8) M for GDP and 3.8 X 10(-6) M for GTP. The purified three subunit eIF2 has 0.4 mol of GDP bound/mol of factor. The crude preparations of eIF2 are not affected by Mg2+ in their exchange of guanine nucleotides or in the formation of ternary complexes with GTP and methionyl-tRNA, but these reactions are strongly inhibited by Mg2+ when the highly purified preparations are used.     

Molecular heterogeneity and genetics of Vicia faba seed storage proteins

Summary Legumin and vicilin were purified from seeds of Vicia faba L. var. Scuro, characterized in different electrophoretic systems, and used to produce polyclonal antibodies in rabbits. Two-dimensional electrophoretic studies showed a wide range of heterogeneity in the subunits of both legumin and vicilin. Legumin was found to be composed of 29 disulphide-linked subunit pairs with different molecular weight and/or isoelectric point. Western blot analysis of legumin of several mutants revealed molecular polymorphism based on a corresponding gene family. Three different -major legumin patterns were found, and inheritance studies showed that the 34.3-kD legumin polypeptide is the product of one locus, Lg-1, which is the first legumin genetic locus described in Vicia faba. Vicilin was found to be composed of as many as 59 subunits distributed in a molecular weight range of 65.7 to 42.8 kD (major polypeptides) and 37.2 to 15.2 kD (minor polypeptides), with different isoelectric points. A model is proposed that explains the possible formation of the minor subunits and the major subunits of 48.2 and 46 kD molecular weight (MW) from proteolytic cleavages and/or glycosilation of precursor polypeptides. Ten different vicilin electrophoretic patterns were observed among the analyzed accessions, which showed large molecular polymorphism that proved to be under genetic control.Contribution no. 55 from the Center of Vegetable Breeding, Portici, Italy     

Phaseolus coccineus L. Storage Proteins. Extraction and Characterization

Phaseolus coccineus storage globulins were extracted from mature cotyledons, purified and characterized. Three major proteins were separated. A component showing erythroagglutinating activity was thoroughly purified by thyroglobulin-Sepharose chromatography. The relative molecular masses of the three fractions are Mr = 330, 178, and 500 kDa as determined by polyacrylamide gel electrophoresis (PAGE). They correspond to the proteins found in other systems and classified as phytohaemagglutinin (PHA), vicilin and legumin, respectively. Electrophoretic analyses under denaturating conditions (SDS-PAGE) evidenced the major subunits for the three proteins. Isoelectrofocusing of the isolated proteins indicated a large heterogeneity for vicilin. Part I.     

Baculovirus-directed expression of the human insulin receptor and an insulin-binding ectodomain

In this report we describe the use of the baculovirus expression system to overproduce the human insulin holoreceptor (HIR) and a truncated, secretory version of the HIR cDNA (HIRsec) consisting of the alpha subunit and the extracellular portion of the beta subunit (beta'). Sf9 cells infected with the full-length HIR viruses synthesize recombinant HIR (rHIR) with an insulin-binding alpha subunit of apparent Mr = 110,000 and a beta subunit of apparent Mr = 80,000. Uncleaved alpha beta proreceptor accumulates in infected cells. Both of these forms assemble into higher order disulfide-linked dimers or heterotetramers of apparent Mr greater than 350,000. Insulin-binding activity in cells infected with rHIR viruses is present predominantly on the extracellular aspect of the plasma membrane (greater than 80%). Insulin binding to the full-length rHIR occurs with typical complex kinetics with Kd1 = 0.5-1 x 10(-9) M and Kd2 = 10(-7) M and receptors are present in large amounts in infected cells (1 x 10(6) receptors/cell; 1-2 mg HIR/10(9) cells). The full-length rHIR undergoes insulin-dependent autophosphorylation; half-maximal activation of beta subunit autophosphorylation occurs at 1-2 x 10(-8) M. The alpha beta proreceptor also becomes phosphorylated in vitro. Analysis of tryptic phosphopeptides derived from in vitro autophosphorylated beta subunit and alpha beta proreceptor reveals a pattern of phosphorylation that is indistinguishable from that of authentic placental HIR. Sf9 cells infected with rHIRsec viruses synthesize and secrete an (alpha beta')2 heterotetrameric complex having an insulin-binding alpha subunit of apparent Mr = 110,000 and a truncated beta' subunit of apparent Mr = 45,000 that lacks kinase activity. The rHIRsec complex purified from the conditioned medium of infected cells binds insulin with high affinity (Kd = 10(-9) M).     

产肠毒素大肠埃希菌CfaB亚单位的免疫原性分析

目的表达并纯化产肠毒素大肠埃希菌（Enterotoxigenic Escherichia coli,ETEC）CFA／I定居因子的CfaB亚单位,分析其免疫原性。方法将不含信号肽序列的cfaB基因克隆到pQE一30上,构建重组质粒pQE-30-cfaB并转化EcoliM15,表达融合蛋白6xHis—CfaB,将表达的蛋白纯化和复性后免疫BALB／c小鼠,制备抗CfaB的抗血清,用ELISA法检测抗血清效价。结果6×His—CfaB融合蛋白高效表达,相对分子质量为15．5kD,纯化复性后免疫小鼠所得抗血清效价为1：125000。结论成功构建了高效表达6×His-CfaB融合蛋白的重组质粒,表达的融合蛋白免疫小鼠后获得了高效价的抗血清,为进一步研制以双歧杆菌为表达系统的新型ETEC亚单位口服疫苗奠定了基础。     

The purification and characterization of a third storage protein (convicilin) from the seeds of pea (Pisum sativum L.).

A third storage protein, distinct from legumin and vicilin, has been purified from the seeds of pea (Pisum sativum L.). This protein has been named 'convicilin' and is present in protein bodies isolated from pea seeds. Convicilin has a subunit mol.wt. of 71 000 and a mol.wt. in its native form of 290 000. Convicilin is antigenically dissimilar to legumin, but gives a reaction of identity with vicilin when tested against antibodies raised against both proteins. However, convicilin contains no vicilin subunits and may be clearly separated from vicilin by non-dissociating techniques. Unlike vicilin, convicilin does not interact with concanavalin A, and contains insignificant amounts of carbohydrates. Limited heterogeneity, as shown by isoelectric focusing, N-terminal analysis, and CNBr cleavage, is present in convicilin isolated from a single pea variety; genetic variation of the protein between pea lines has also been observed.     

Optimization of solubilization and purification procedures for the hydroxylase component of membrane-bound methane monooxygenase from Methylococcus capsulatus strain M

The hydroxylase component of membrane-bound (particulate) methane monooxygenase (pMMO) from Methylococcus capsulatus strain M was isolated and purified to homogeneity. The pMMO molecule comprises three subunits of molecular masses 47, 26, and 23 kD and contains three copper atoms and one iron atom. In solution the protein exists as a stable oligomer of 660 kD with possible subunit composition (alpha beta gamma)6. Mass spectroscopy shows high homology of the purified protein with methane monooxygenase from Methylococcus capsulatus strain Bath. Pilot screening of crystallization conditions has been carried out.     

Subunit and primary structure of a mouse alpha-macroglobulin, a human alpha 2-macroglobulin homologue

A mouse alpha-macroglobulin (AMG), a homologue of human alpha 2-macroglobulin (alpha 2 M), has been purified to homogeneity. In contrast to human and acute-phase rat alpha 2 M which contains subunits of about Mr 190 000, the mouse protein contains two major (Mr 163000 and 35000) and one minor (Mr 185000) subunits. Also unlike human alpha 2 M, which can be broken down into about 85000-dalton subunits when reacted with an endopeptidase, the native AMG is cleaved by trypsin into multiple components (Mr 86000, 63000, 61000 and 33000). Two-dimensional peptide map analysis of these various 125I-labeled subunit components reveals that the 185000- and 163000-dalton components are homologous proteins but only the 185000-dalton protein contains the 35000-dalton component. The 163000-dalton protein is cleaved by trypsin into 86000- and 63000-dalton components, and the 86-kDa component in turn can be broken down into 61000- and 33000-dalton fragments. Since the 35000-dalton component is serologically related to AMG but does not share any tryptic peptides with both the 163000- and 33000-dalton components, it is neither a copurified impurity nor a cleavage product of the major (163000-dalton) subunit. AMG, therefore, is composed of covalently linked subunits of Mr 163000 and 35000, and the 185000-dalton protein may be a variant subunit of AMG. Trypsin treatment of the [14C]methylamine-labeled AMG and alpha 2 M also sequentially generate subunit patterns indistinguishable from those of the unlabeled macroglobulins. The methylamine-sensitive site(s) of AMG is localized in the 63000-dalton peptide, which is rather resistant to trypsin digestion and to staining by Coomassie brillant blue. We conclude from this study that the mouse homologue has a subunit composition and primary structure distinctly different from those of human and rat alpha 2 M.     

Alpha 2-macroglobulin binding to cultured fibroblasts: identification by affinity chromatography of high-affinity binding sites

Binding sites having the properties of high-affinity receptors for activated alpha 2-macroglobulin (alpha 2M) have been purified over 100-fold from membranes of spontaneously transformed NIH-3T3 cells (J. A. Hanover, S.-y. Cheng, M. C. Willingham, and I. H. Pastan [1983] J. Biol. Chem. 258, 370-377). To identify the molecular species involved in high-affinity binding, the solubilized receptor has been purified 500-fold by conventional procedures and further purified by affinity chromatography. After radioiodination of the 500-fold-purified preparation, the detergent-solubilized extract was applied to alpha 2M-Sepharose and an 85,000 +/- 5000 Mr species was selectively retained by the column. Binding of the 85,000 +/- 5000 Mr species to the affinity resin was inhibited by EDTA and by excess alpha 2M. Elution from the affinity column could be accomplished with bacitracin, a competitive inhibitor of alpha 2M binding, or with EDTA. Consistent with the previously reported characteristics of the high-affinity alpha 2M receptor, the 85,000 Mr species bound much more efficiently to methylamine-activated alpha 2M-Affigel than to alpha 2M-Affigel which had not been amine-activated. The present data suggest that a protein with a subunit Mr of 85,000 +/- 5000 may represent a component of the high-affinity alpha 2M receptor present on cultured fibroblasts.     

Electrogenicity of phosphate transport by renal brush-border membranes.

An alpha 2-macroglobulin (alpha 2M)-like proteinase inhibitor from plasma of the crayfish Pacifastacus leniusculus was purified to apparent homogeneity by acid precipitation, hydrophobic interaction chromatography, affinity chromatography on concanavalin A-Sepharose and anion-exchange chromatography. The subunit Mr is about 190,000. Pore-size-limit electrophoresis proved the native protein to be a dimer. The purified protein resembled vertebrate alpha 2 Ms in that it protected trypsin from inhibition by soyabean trypsin inhibitor, and in its sensitivity to methylamine treatment. Methylamine also prevented the protein from being autolytically cleaved into Mr 60,000 and 140,000 fragments when subjected to heat treatment. The amino acid composition showed similarities with both human alpha 2 M and an alpha 2 M-like protein from the arthropod Limulus polyphemus. These data indicate that this Pacifastacus alpha 2M-like protein (P alpha 2M) may be a distantly related homologue of vertebrate alpha 2Ms.     

Deoxyribonuclease II purified from the isolated lysosomes of porcine spleen and from porcine liver homogenates. Comparison with deoxyribonuclease II purified from porcine spleen homogenates

Porcine spleen DNase II (EC 3.1.22.1), one of the best-characterized DNases II, is subcellularly located in lysosomes because the enzyme is co-sedimented with two of the lysosomal marker enzymes, cathepsin D and acid phosphatase. The physicochemical properties, including the subunit structure, sensitivity to iodoacetate inactivation, native molecular weight and chromatographic behavior, of the DNase II purified from the isolated lysosomes of porcine spleen are indistinguishable from those of the same enzyme purified from the whole porcine spleen homogenate. DNase II can also be extracted from porcine liver with 0.05 M H2SO4 or 0.1 M NaCl and purified from either extract by a series of column chromatographies. The purified liver DNase II from either extract has the same subunit structure (alpha-chain, Mr 35,000 and beta-chain, Mr 10,000) as the purified DNase II of porcine spleen. The two liver extracts as well as the extracts of spleen and gastric mucosa contain DNase II with very similar properties on Sephadex G-100 gel filtration, on acid polyacrylamide gel electrophoresis under non-denaturing conditions, and on isoelectric focusing. The data strongly suggest that, for the same species of animal, the DNase II activities in various tissues are associated with protein molecules of identical structure.     

Immunochemical characterization of myosin-specific phosphatase 1 regulatory subunits in bovine endothelium

We have previously shown that myosin-specific phosphatase 1 (PPase 1) activity is critical for maintaining endothelial cell barrier function (Verin et al. [1995] Am. J. Physiol. 269:L99-L108). To further characterize myosin-specific PPase 1 in endothelium, we generated antibodies specific to published sequences of the myosin-associated PPase 1 regulatory subunit (M110) from smooth muscle. Peptide antigens were designed based upon consensus sequences for a single ankyrin repeat (ANK 110) and a leucine zipper motif region (LZ 110), which represents putative sites for binding the PPase 1 catalytic subunit (CS1) and myosin, respectively. Our initial study demonstrated that each antibody immunoprecipitated 2 proteins with an apparent Mr of 110 and 70 kD on SDS-PAGE. The CS1delta isoform, which appeared to be characteristic for the myosin-specific phosphatase, was co-immunoprecipitated under non-denaturing conditions with ANK110 and LZ110 as was actin, myosin, and myosin light chain kinase (MLCK). Similarly, immunoprecipitation with specific anti-myosin or anti-MLCK antibodies under the same conditions, followed by immunostaining with either LZ110 or ANK110 revealed the same 110 and 70 kD protein bands. The 70 kD protein (p70) was immunoreactive with ANK 110 and LZ 110, was complexed with myosin and MLCK, and was detected in non-denaturing M110 immunoprecipitates. Consistent with these results, endothelial cell fractionation demonstrates the presence of p70 in both cytoskeletal and myosin-enriched fractions, but not in the myosin-depleted (cytosolic) fractions. These data suggest that endothelial cells may exhibit two distinct myosin-specific PPase 1 regulatory subunits which share certain structural features with the M110 regulatory subunit from smooth muscle and which are tightly associated with myosin and MLCK in a functional complex.     

Purification and properties of the catalytic subunit of the branched-chain alpha-keto acid dehydrogenase phosphatase from bovine kidney mitochondria

The catalytic subunit of the branched-chain alpha-keto acid dehydrogenase (BCKDH) phosphatase (Damuni, Z., Merryfield, M.L., Humphreys, J.S., and Reed, L.J., (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4335-4338) has been purified over 50,000-fold from extracts of bovine kidney mitochondria. The apparently homogeneous protein consists of a single polypeptide chain with an apparent Mr = approximately 33,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. BCKDH phosphatase, with an apparent Mr = 460,000, was dissociated to its catalytic subunit with no apparent change in activity, at an early stage in the purification procedure by treatment with 6 M urea. The specific activity of the catalytic subunit was 1,500-2,500 units/mg. The catalytic subunit exhibited approximately 10% maximal activity with 32P-labeled pyruvate dehydrogenase complex but was inactive with phosphorylase a and with p-nitrophenyl phosphate. The catalytic subunit, like the Mr = 460,000 species, was inhibited by nanomolar concentrations of BCKDH phosphatase inhibitor protein, was unaffected by protein phosphatase inhibitor 1 and inhibitor 2, and was inhibited by nucleoside tri- and diphosphates but not by nucleoside monophosphates.     

Distribution of Folate Derivatives and Enzymes for Synthesis of 10-Formyltetrahydrofolate in Cytosolic and Mitochondrial Fractions of Pea Leaves

Leaf extracts of 14-d-old pea (Pisum sativum L. cv Homesteader) seedlings were examined for folate derivatives and for 10-formyltetrahydrofolate synthetase (SYN), 5,10-methenyltetrahydrofolate cyclohydrolase (CYC), and 5,10-methylenetetrahydrofolate dehydrogenase (DHY) activities. Microbiological and enzyme assays showed that leaf folates SYN, CYC, and DHY were predominantly cytosolic. Extracts of Percoll gradient-purified mitochondria contained less than 1% of total leaf folate and less that 1% of each enzyme activity. Fractionation of whole-leaf homogenates resulted in the copurification of DHY and CYC (subunit 38 kD) and the isolation of a SYN protein (subunit 66 kD). Polyclonal antibodies were raised against purified cytosolic DHY-CYC (DHY-CYC-Ab) and cytosolic SYN (SYN-Ab), respectively. Immunoblots showed that DHY-CYC-Ab cross-reacted with a mitochondrial protein band (38 kD). Two mitochondrial protein bands (subunit Mr = 40,000 and 44,000) cross-reacted with SYN-Ab. Immunoaffinity chromatography (DHY-CYC-Ab as the immobile ligand) indicated that the bulk of mitochondrial SYN activity was not associated with mitochondrial DHY or CYC. When 9-d-old etiolated pea seedlings were exposed to light for up to 3 d, the specific enzyme activities of DHY-CYC in whole-leaf extracts rose 2-fold and more DHY-CYC-Ab cross-reacting protein was detected. In contrast, the specific activity of SYN fell from 5 to 1 [mu]mol min-1 mg-1 protein and less SYN-Ab cross-reacting protein was detected. The data suggest that in pea leaves, the bulk of one-carbon-substituted tetrahydrofolates and enzymes for the generation of 10-formyltetrahydrofolate are extra-mitochondrial.     

花生种子发育和萌发过程中贮藏蛋白的合成和降解

以花生品种汕油5 2 3种子为材料,分离纯化花生球蛋白的41 kD和38.5 kD两种主要亚基及伴花生球蛋白的6 0.5 KD亚基并制备抗体.We stern blot分析表明,3种亚基在花生胚组织分化期的胚轴和子叶中就开始合成,其中60.5 kD亚基是最先在胚轴和子叶中大量合成和积累的贮藏蛋白,41 kD和38.5 kD亚基在随后的发育中积累量不断增加;种子萌发时这3种亚基的降解进程不一样,胚轴和子叶中41 kD和38.5kD亚基的降解均先于60.5 kD亚基.     

Purification and properties of two enzymes of meta-cleaving the aromatic ring controlled by the biphenyl biodegradation plasmid pBS 241 from Pseudomonas putida

It was shown that two metapyrocatechases (EC 1.13.11.2) function in Pseudomonas putida BS893. Biphenyl degradative plasmid pBS241 carries the genes of these enzymes. The basic properties of the both enzymes, i. e., MPC1 and MPC2, were investigated. It was found that MPC1 is an enzyme with a molecular mass of 135 kD and has a heterotetrameric subunit structure (alpha 2 beta 2), being made up of two non-identical polypeptides with Mr of 34 and 22.5 kD; pI is 5.15, the pH optimum is at 8.0, a temperature optimum is at 54 degrees C. MPC2 has a molecular mass of 154 kD and possesses a homotetrameric subunit structure (alpha 4); it consists of identical polypeptides with Mr of 41 kD and has a pI of 4.95, a pH optimum at 7.5 and a temperature optimum at 60 degrees C. The substrate specificity of the enzymes was studied, and the Km and Vmax values for substituted catechols were determined. MPC1 shows a high affinity for 2.3-dihydroxybiphenyl and hydrolyzes 3-methylcatechol and catechol (but not 4-methylcatechol) at a low rate. MPC2 has a moderate affinity for catechol, 3- and 4-methylcatechols, but is incapable of cleaving 2.3-dihydroxybiphenyl. Both enzymes share in common some typical properties of metapyrocatechases. The different role of MPC1 and MPC2 in biphenyl catabolism is discussed.     

Characterization of a factor inducing differentiation of mouse myeloid leukemic cells purified from conditioned medium of mouse Ehrlich ascites tumor cells

A factor inducing differentiation of mouse myeloid leukemic cells (MI) into macrophages was purified to apparent homogeneity from 168 1 of CM of Ehrlich ascites tumor cells. The purified factor was half-maximally active at 2 X 10(-11) M. The factor was analyzed by radioiodination, SDS-polyacrylamide gel electrophoresis and autoradiography. Its Mr was 40 000-50 000. On reduction, the factor lost activity, but showed no subunit structure. Treatment of the factor with endo-beta-N-acetylglucosaminidase F, but not endo-beta-N-acetylglucosaminidase H, gave rise to a molecule of Mr 20 000-28 000. The activity of the factor from Ehrlich cells was completely neutralized by antiserum to the factor of Mr 50 000-70 000 from mouse fibroblast L929 cells.     

Role of the endoplasmic reticulum in the synthesis of reserve proteins and the kinetics of their transport to protein bodies in developing pea cotyledons

Developing pea (Pisum sativum L.) cotyledons were labeled with radioactive amino acids, glucosamine, and mannose in pulse an pulse- chase experiments to study the synthesis, glycosylation, and transport of the reserve proteins vicilin and legumin to the protein bodies. Tissue extracts were fractionated on sucrose gradients to isolate either the endoplasmic reticulum (ER) or the protein bodies. Immunoaffinity gels were used to determine radioactivity in the reserve proteins (legumin and vicilin). After pulse-labeling for 45 min with amino acids, about half the total incorporated radioactivity coincided closely with the position of the ER marker enzyme NADH-cytochrome c reductase at a density of 1.13 g . cm-3 on the sucrose gradient. Both radioactivity and enzyme activity shifted to a density of 1.18 g . cm-3 in the presence of 3 mM MgCl2 indicating that the radioactive proteins were associated with the rough ER. Approximately half of the incorporated radioactivity associated with the rough ER was in newly synthesized reserve protein and this accounted for 80% of the reserve protein synthesized in 45 min. Trypsin digestion experiments indicated that these proteins were sequestered within the ER. In pulse-chase experiments, the reserve proteins in the ER became radioactive without appreciable lag and radioactivity chased out of the ER with a half-life of 90 min. Radioactive reserve proteins became associated with a protein body-rich fraction 20-30 min after their synthesis and sequestration by the ER. Pulse-chase experiments with radioactive glucosamine and mannose in the presence and absence of tunicamycin indicated that glycosylation of vicilin occurs in the ER. However, glycosylation is not a prerequisite for transport of vicilin from ER to protein bodies. Examination of the reserve protein polypeptides by SDS PAGE followed by fluorography showed that isolated ER contained legumin precursors (Mr 60,000-65,000) but not the polypeptides present in mature legumin (Mr 40,000 and 19,000) as well as the higher molecular weight polypeptides of vicilin (Mr 75,000, 70,000, 50,000, and 49,000). The smaller polypeptides of vicilin present in vicilin extracted from protein bodies (Mr 12,000-34,000) were absent from the ER. The results show that newly synthesized reserve proteins are preferentially and transiently sequestered within the ER before they move to the protein bodies, and that the ER is the site of storage protein glycosylation.