Differential Proteolysis of Glycinin and beta-Conglycinin Polypeptides during Soybean Germination and Seedling Growth

The degradation of the major seed storage globulins of the soybean (Glycine max [L.] Merrill) was examined during the first 12 days of germination and seedling growth. The appearance of glycinin and β-conglycinin degradation products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cotyledon extracts followed by electroblotting to nitrocellulose and immunostaining using glycinin and β-conglycinin specific antibodies. The three subunits of β-conglycinin were preferentially metabolized. Of the three subunits of β-conglycinin, the larger α and α′ subunits are rapidly degraded, generating new β-conglycinin cross-reactive polypeptides of 51,200 molecular weight soon after imbibition of the seed. After 6 days of growth the β-subunit is also hydrolyzed. At least six polypeptides, ranging from 33,100 to 24,000 molecular weight, appear as apparent degradation products of β-conglycinin. The metabolism of the glycinin acidic chains begins early in growth. The glycinin acidic chains present at day 3 have already been altered from the native form in the ungerminated seed, as evidenced by their higher mobility in an alkaline-urea polyacrylamide gel electrophoresis system. However, no change in the molecular weight of these chains is detectable by sodium dodecyl sulfate-polyarylamide gel electrophoresis. Examination of the glycinin polypeptide amino-termini by dansylation suggests that this initial modification of the acidic chains involves limited proteolysis at the carboxyl-termini, deamidation, or both. After 3 days of growth the acidic chains are rapidly hydrolyzed to a smaller (21,900 molecular weight) form. The basic polypeptides of glycinin appear to be unaltered during the first 8 days of growth, but are rapidly degraded thereafter to unidentified products. All of the original glycinin basic chains have been destroyed by day 10 of growth.     

Nonidentical subunits of citrate lyase from Klebsiella aerogenes

Citrate lyase from Klebsiella aerogenes has been shown to contain 3 different subunits by SDS gel electrophoresis. On re-electrophoresis, each of these polypeptides is found to migrate in the same manner as it did in the first electrophoresis. The 3 subunits have also been separated by gel filtration on an agarose column in 6 M urea.     

Subunit glycosylation of Lupinus angustifolius seed globulins

Reduced polypeptide subunits of α-, β- and γ-conglutins from Lupinus angustifolius seeds were resolved by preparative SDS gel electrophoresis of the fluorescent labelled proteins, into four, six and two major components, respectively. All subunits were glycosylated, to varying degrees, containing mannose, galactose and glucosamine. The major glycopeptides released by pronase digestion of each conglutin had similar galactose/mannose ratios; the MW of the glycopeptide released from α- and β-conglutin was ca 5000. Although on average, each molecule of α-conglutin contains one main oligosaccharide chain, and β-conglutin two, the presence of carbohydrate in all polypeptide subunits suggests that some subunits may arise by proteolytic cleavage of a larger polypeptide after glycosylation. The presence of minor glycopeptide components indicates that modification of carbohydrate chains during seed development may also occur.     

Characterization of three neurosecretory proteins from the A median neurosecretory cells of Locusta migratoria by coupled chromatographic,electrophoretic and isoelectrofocusing methods

Proteins from the nervous corpora cardiaca (N-CC) of Locusta migratoria were analysed using a combination of chromatography, electrophoresis and electrofocusing. Three major cysteine or/and cystine-rich proteins were identified as neurosecretory proteins of the median neurosecretory cells (M-NSC) by comparing the pattern of separated proteins of (1) intact N-CC and (2) depleted N-CC after intracerebral axotomy of the M-NSC.These three neurosecretory proteins are: a trimer, a dimer and a monomer. The trimer and the monomer are composed of apparent 18,000 subunits and 9000 polypeptide chains. The dimer is composed of apparent 7000 subunits composed of putative 4000 polypeptide chains. The three neurosecretory proteins are acidic (pH_i approx. 5.6 for the trimer, 5.1 for the monomer and 4.0 for the dimer). Their relationship to the neurophysins and to the neurosecretory protein isolated from brain and corpora cardiaca of the locust by Friedel et al. (1980) (Gen. comp. Endocr.41, 487–498) is discussed. The role of these three neurosecretory proteins (carrier protein, neurohormone or precursor of neurohormone) is as yet unknown.     

The isolation of bovine-heart cytochrome c oxidase subunits Dependence on phospholipid and cholate content

The polypeptide chains of bovine-heart cytochrome c oxidase were preparatively isolated by a simple large-scale procedure based on gel permeation chromatography in the presence of sodium dodecyl sulphate.The resolution of the subunits as a function of the cholate and phospholipid content of the preparation was investigated.Cholate, and to a lesser extent, phospholipids interfere with the separation of the subunits; however, they do not prevent dissociation of the enzyme by SDS.Bovine-heart cytochrome c oxidase consists of six major subunits (estimated molecular weights in thousands: 40, 25, 20, 14, 12 and 10). In addition, the enzyme preparation contains at least five minor constituents, present in less than stoichiometric amounts.The first two of the three large subunits, all of which are hydrophobic, have amino-terminal N-formylmethionine. Subunit III, however, has a free methionine N-terminus.     

Helical arrays of Escherichia coli small ribosomal subunits produced in vitro.

In vitro conditions have been determined for obtaining ordered helical ribbons of small ribosomal subunits from Escherichia coli. These ribbons, suitable for study by three-dimensional reconstruction, are the first ordered arrays of ribosomes or ribosomal subunits to be produced in vitro.Although small ribosomal subunits remain in solution for extended periods (up to 6 months) during this procedure, their structural integrity, as assessed by acrylamide/agarose gel electrophoresis, by sucrose gradients, and by electron microscopy, is not significantly altered.Electron micrographs of ribbons of small subunits diffract to 60 Å resolution. Optical diffraction patterns suggest that adjacent subunits within helical ribbons are related by a 2-fold screw parallel to the long axis of the ribbon and the helical repeat distance measured from electron micrographs is 220 Å.     

Subunit composition of a high molecular weight oligomer: Limulus polyphemus hemocyanin

The hemocyanin of Limulus polyphemus is a 48-subunit aggregate. This 3.3 × 10⁶-dalton oligomer is composed of structurally and functionally heterogeneous subunits. Using polyacrylamide electrophoresis J. Markl, A. Markl, W. Schartau, and B. Linzen (J. Comp. Physiol. Ser. B130,283–292, 1979) observed 12 bands; while using immunoelectrophoresis, M. Hoylaerts, G. Preaux, R. Witters, and R. Lontie (Arch. Int. Physiol. Biochem.87, 417–418, 1979) and J. Lamy, J. Lamy, J. Weill, J. Bonaventura, C. Bonaventura, and M. Brenowitz. (Arch. Biochem. Biophys.196, 324–339, 1979) observed 8 subunits. To proceed with an analysis of subunit roles in assembly it is first necessary to determine the number of distinct subunits. Refinement of the chromatographic separation procedures has led to the isolation of 8 immunologically distinct subunits as well as additional charge isomers which cannot be distinguished immunologically. Alkaline electrophoresis revealed 15 bands and isoelectric focusing up to 17. On the basis of extensive control experiments, including composit acrylamide-agarose immunoelectrophoresis and checks for conformational isomers, aggregation, proteolysis, and other types of degradation, we conclude that the electrophoretic heterogeneity of immunologically identical subunits is not artifactual. We have extended the nomenclature used by Lamy et al. (1979) to include the electrophoretic heterogeneity by using primes (′) to denote electrophoretically distinguishable subunits which are immunologically identical. A number of patterns have become apparent by correlating the results obtained by the different techniques. For example, immunologically pure subunit II, which shows 3 bands on alkaline electrophoresis, is in fact a mixture of electrophoretically distinct subunits II, II′, II″. Except for subunits II, II′, and II″ immunoelectrophoretically identical subunits are typically homogeneous on sodium dodecyl sulfate-gels. However, slight differences in the apparent molecular weight are observed on high-resolution gels between immunologically unrelated subunits. The immunological identity and electrophoretic differences suggest that the charge isomers which are immunologically identical have similar antigenic surfaces. If a charge substitution is not in a critical location, we would expect the electrophoretically distinct but immunologically identical subunits to have identical assembly roles. Comparison of the results for Limulus hemocyanin with the hemocyanin of related species Eurypelma californicum and Androctanus australis, which have 7 and 8 immunologically distinct subunits, respectively, suggests that the calcium-mediated aggregation from 24 to 48 subunits of Limulus does not require more extensive subunit complexity.     

Subunit structure of γ-conglycinin in soybean seeds

Dissociated subunits of purified γ-conglycinin were isolated on a DEAE-Sephadex A-50 column. A single band was seen on two kinds of gel electrophoresis and isoleucine was shown as the only N-terminal amino acid. The isolated subunit reacted with antisera to the native γ-conglycinin. The M_r of the subunit was 51 000–51 500 estimated by urea-acetic acid and SDS-urea gel electrophoresis. A value of 50 000 was obtained by gel filtration with guanidine-hydrochloric acid on Sepharose CL-6B. The γ-conglycinin molecule was found to be made up of three subunits. This was determined by cross-linking the subunits and then submitting them to gel electrophoresis. Differences and similarities of subunit structure among γ-conglycinin, β-conglycinin and glycinin are discussed.     

Subunits of Limnodrilus erythrocruorin.

The dissociation of the erythrocruorin of the oligochaete $\text{Limnodrilus gotoi}$ was investigated using polyacrylamide gel electrophoresis at neutral pH. In the presence of 0.1% SDS, the erythrocruorin dissociated into five subunits possessing molecular weights of 13,000 (1), 20,000 (2), 23,000 (3), 25,000 (4) and 47,000 (5). In the presence of SDS and mercaptoethanol, the erythrocruorin dissociated into two subunits, whose molecular weights were 13,000 (I) and 28,000 (II). Subunit I accounts for 70–80% of the whole molecule. SDS electrophoresis of the isolated subunits 1 through 5 in the presence of mercaptoethanol showed that subunit I was derived from both subunits 1 and 5, while subunit II was derived from subunits 2–4. These results suggest that $\text{Limnodrilus}$ erythrocruorin consists of at least five polypeptide chains: two chains of 13,000 and three chains of 28,000.     

Kinetics of ligand binding to ferrous heme groups of hemoglobin MSaskatoon.

Hemoglobin M_Saskatoon (α₂^Aβ₂^63tyr) has two α chains in the normal ferrous state, while its two β chains are in the ferric state. The reaction of hemoglobin M_Saskatoon with carbon monoxide at pH 7 and 20 °C in the presence and absence of dithionite was studied. In the absence of dithionite only the α chains react and the combination rate is slow and similar to that of normal deoxyhemoglobin. After the addition of dithionite the rate of reaction is greatly increased initially and then decreases to a rate similar to that seen in the absence of dithionite. The dissociation of oxygen from hemoglobin M_Saskatoon at pH 7 and 20 °C was found for the α subunits to be similar to that seen for normal oxyhemoglobin. This similarity in the kinetic properties of normal hemoglobin and the α subunits of hemoglobin M_Saskatoon in both ligand combination and dissociation reactions indicates that the α subunits of hemoglobin M_Saskatoon undergo a structural transition from a low to high affinity form on liganding. Since the β subunits react rapidly with carbon monoxide even when the α subunits are unliganded, it appears that the ligand binding sites of the β chains are uncoupled from the state of liganding of the α subunits.     

A simple electrophoretic procedure for the determination of the polypeptide composition of the subunits of Fraction 1 protein

A rapid and convenient method is described for resolving the polypeptide composition of Fraction 1 protein. Using crude leaf extracts of a number of Lycopersicon species, Fraction 1 protein was first separated by polyacrylamide gel electrophoresis and the gel slices containing the protein were isoelectrofocused in the presence of 8 m urea. Isoelectric focusing was also applied directly on subunits in gel slices obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polypeptide composition produced is in agreement with previous determinations obtained by more elaborated techniques.     

Primary structure of hemoglobin from cobraNaja naja naja

Cobra snakeNaja naja naja hemoglobin shows four bands on Triton electrophoresis. We present the primary structure of oneα and oneβ chain. The separation of polypeptide chains was achieved by ion exchange chromatography on carboxymethyl cellulose column. The amino acid sequence was established by automatic Edman degradation of the native chains and tryptic and hydrolytic peptides in a gas-phase sequencer. The structural data are compared with those of human and other reptile hemoglobins and reveal not only large variations from human but within reptiles. The amino acid exchanges involve several subunit contacts and heme binding sites. This is the first study on the hemoglobin of a land snake. There are only two amino acid sequences of sea snake hemoglobin (Microcephalophis gracilis gracilis andLiophis miliaris) reported in the literature.     

Chromatographic resolution of the subunits of calf brain tubulin

Two procedures are described for column chromatographic separation of the α and β subunits of brain tubulin using hydroxylapatite in the presence of (i) urea or (ii) sodium dodecyl sulfate and β-mercaptoethanol. In the first system the α and β chains are partially resolved, and in the second the subunits are resolved into three peaks which we designate α₁, α₂, and β.     

Controlled proteolysis of nascent polypeptides in rat liver cell fractions. II. Location of the polypeptides in rough microsomes

Rough microsomes were incubated in an in vitro amino acid-incorporating system for labeling the nascent polypeptide chains on the membrane-bound ribosomes. Sucrose density gradient analysis showed that ribosomes did not detach from the membranes during incorporation in vitro. Trypsin and chymotrypsin treatment of microsomes at 0° led to the detachment of ribosomes from the membranes; furthermore, trypsin produced the dissociation of released, messenger RNA-free ribosomes into subunits. Electron microscopic observations indicated that the membranes remained as closed vesicles. In contrast to the situation with free polysomes, nascent chains contained in rough microsomes were extensively protected from proteolytic attach. By separating the microsomal membranes from the released subunits after proteolysis, it was found that nascent chains are split into two size classes of fragments when the ribosomes are detached. These were shown by column chromatography on Sephadex G-50 to be: (a) small (39 amino acid residues) ribosome-associated fragments and (b) a mixture of larger membrane-associated fragments excluded from the column. The small fragments correspond to the carboxy-terminal segments which are protected by the large subunits of free polysomes. The larger fragments associated with the microsomal membranes depend for their protection on membrane integrity. These fragments are completely digested if the microsomes are subjected to proteolysis in the presence of detergents. These results indicate that when the nascent polypeptides growing in the large subunits of membrane-bound ribosomes emerge from the ribosomes they enter directly into a close association with the microsomal membrane.     

Proteomic approach to characterize mitochondrial complex I from plants

Mitochondrial NADH dehydrogenase complex (complex I) is by far the largest protein complex of the respiratory chain. It is best characterized for bovine mitochondria and known to consist of 45 different subunits in this species. Proteomic analyses recently allowed for the first time to systematically explore complex I from plants. The enzyme is especially large and includes numerous extra subunits. Upon subunit separation by various gel electrophoresis procedures and protein identifications by mass spectrometry, overall 47 distinct types of proteins were found to form part of Arabidopsis complex I. An additional subunit, ND4L, is present but could not be detected by the procedures employed due to its extreme biochemical properties. Seven of the 48 subunits occur in pairs of isoforms, six of which were experimentally proven. Fifteen subunits of complex I from Arabidopsis are specific for plants. Some of these resemble enzymes of known functions, e.g. carbonic anhydrases and l-galactono-1,4-lactone dehydrogenase (GLDH), which catalyzes the last step of ascorbate biosynthesis. This article aims to review proteomic data on the protein composition of complex I in plants. Furthermore, a proteomic re-evaluation on its protein constituents is presented.     

The role of O-linked and N-linked oligosaccharides on the structure–function of glycoprotein hormones: Development of agonists and antagonists

Thyrotropin (TSH) and the gonadotropins; follitropin (FSH), lutropin (LH) and human chorionic gonadotropin (hCG) are a family of heterodimeric glycoprotein hormones. These hormones composed of two noncovalently linked subunits; a common α and a hormone specific β subunits. Assembly of the subunits is vital to the function of these hormones. However, genetic fusion of the α and β subunits of hFSH, hCG and hTSH resulted in active polypeptides. The glycoprotein hormone subunits contain one (TSH and LH) or two (α, FSHβ and hCGβ) asparagine-linked (N-linked) oligosaccharides. CGβ subunit is distinguished among the β subunits because of the presence of a carboxyl-terminal peptide (CTP) bearing four O-linked oligosaccharide chains. To examine the role of the oligosaccharide chains on the structure–function of glycoprotein hormones, chemical, enzymatic and site-directed mutagenesis were used. The results indicated that O-linked oligosaccharides play a minor role in receptor binding and signal transduction of the glycoprotein hormones. In contrast, the O-linked oligosaccharides are critical for in vivo half-life and bioactivity. Ligation of the CTP bearing four O-linked oligosaccharide sites to different proteins, resulted in enhancing the in vivo bioactivity and half-life of the proteins. The N-linked oligosaccharide chains have a minor role in receptor binding of glycoprotein hormones, but they are critical for bioactivity. Moreover, glycoprotein hormones lacking N-linked oligosaccharides behave as antagonists. In conclusion, the O-linked oligosaccharides are not important for in vitro bioactivity or receptor binding, but they play an important role in the in vivo bioactivity and half-life of the glycoprotein hormones. Addition of the O-linked oligosaccharide chains to the backbone of glycoprotein hormones could be an interesting strategy for designing long acting agonists of glycoprotein hormones. On the other hand, the N-linked oligosaccharides are not important for receptor binding, but they are critical for bioactivity of glycoprotein hormones. Deletion of the N-linked oligosaccharides resulted in the development of glycoprotein hormone antagonists. In the case of hTSH, development of an antagonist may offer a novel therapeutic strategy in the treatment of thyrotoxicosis caused by Graves' disease and TSH secreting pituitary adenoma.     

Homology between ricin and Ricinus communie agglutinin: Amino terminal sequence analysis and protein synthesis inhibition studies

The existence of three forms of ricin and two forms of the Ricinus communis agglutinin (RCA) was established using cation exchange chromatography, isoelectric focusing, and polyacrylamide gel electrophoresis. The preparation of the RCA we obtained was 60–75 times more potent than ricin in the agglutination of erythrocytes, but was about 4% as effective as an inhibitor of cell-free protein synthesis. When reduced with 2-mercaptoethanol, the RCA was activated 3000-fold as an inhibitor of cell-free protein synthesis, whereas ricin was activated about 600-fold by the same treatment. A mixture of the RCA A chains was about one-fifth as effective as the ricin A chain in the inhibition of cell-free protein synthesis. The purified polypeptide subunits of the castor bean lectins were subjected to automated Edman degradation. The sequence for 17 of the first 19 residues of the agglutinin A chain was determined. The first seven residues of the ricin A chain were determined and they are identical with those of the RCA A chain. Nineteen turns of Edman degradation on the RCA B chain resulted in the identification of 18 amino acids. The sequence determined for the first 17 residues of the ricin B chain was identical with that of the RCA B chain. It is likely that the identity of the ricin/RCA A and B chain sequences extends further along the polypeptide chains than the sequences we have determined. The similar structural and catalytic potentials of the RCA and ricin suggest that they bear a precursor-product relationship.     

Purification and some properties of Bacillus macerans cycloamylose (cyclodextrin) glucanotransferase

Bacillus macerans cycloamylose (cyclodextrin) glucanotransferase (EC 2.4.1.19) was purified by the technique of starch adsorption and DEAE-cellulose column chromatography, and then crystallized from an ammonium sulfate solution containing mM calcium chloride. The crystals of the enzyme were rod-shaped and showed a single band by disc-gel electrophoresis. The purified enzyme was dissociated into two subunits by sodium dodecyl sulfate-disc electrophoresis. The subunits had no enzyme activity. Details of each purification step and some properties of the enzyme are described in this paper.     

Studies on subunit structure and evidence that ligandin is a heterodimer

Several lines of evidence indicate that ligandin consists of two different subunits. The protein dissociates into two components that are detected by electrophoresis in a discontinuous sodium dodecyl sulfate system, or in acid-urea gels, and by isoelectric focusing in the presence of urea. The apparent molecular weights of the two polypeptides are 25,000 and 22,000. Alkylated or succinylated ligandins also exhibit subunit heterogeneity and resolved into two bands in these electrophoretic systems. Cross-linked ligandin showed only one band in sodium dodecyl sulfate-gel electrophoresis indicating that the two subunits are part of a heterodimeric protein rather than monomers of two different proteins. No dansylated terminal amino acids were detected suggesting that the NH2-terminal residues of both chains are blocked. One mole of arginine or phenylalanine was released per mole of ligandin after digestion with carboxypeptidase B or A, respectively. Tryptic maps of succinylated ligandin were consistent with identical disposition of arginine residues in both chains, but several additional tryptic peptides were obtained with native ligandin as compared to the predicted number if both subunits were identical. These observations are consistent with the possibility that both subunits contain common sequences and that a small peptide of about 25 to 30 amino acid residues is cleaved from the COOH-terminal of the larger subunit to produce the smaller subunit.