Partial structural analysis of a highly basic low molecular weight protein from rat testis

Automated Edman degradation of a testis-specific basic protein isolated from the rat gave the following NH₂-terminal sequence of amino acids:

Cleavage of the native protein with cyanogen bromide produced two fragments which were purified by gel filtration. Amino acid analysis of the smaller fragment revealed it to be the NH₂-terminal undecapeptide resulting from cleavage at Met₁₁. The partial sequence analysis of the intact protein coupled with compositional analyses of these cyanogen bromide peptides indicate that the basic testis protein contains 24 basic amino acids and a single methionine in a sequence of 54 amino acids.     

Some factors that influence the nonenzymatic glycation of peptides and polypeptides by glyceraldehyde

The rate of reaction of glyceraldehyde with a series of peptides was found to be dependent on their amino acid composition, sequence, and chain length. The presence of a histidine near the NH₂-terminal increased the rate of glycation, whereas the presence of a carboxyl group near the reaction site led to a decrease in reaction rate. In general, tripeptides reacted faster than dipeptides, and dipeptides reacted faster than amino acids. Sodium phosphate and 2,3-diphosphoglycerate enhanced the rate of reaction of glyceraldehyde with all the dipeptides tested. Sodium chloride inhibited the reaction in phosphate buffer, but not in HEPES buffer. The NH₂-terminal heptapeptide from the -chain of human hemoglobin A (HbA), where histidine is the second residue, reacted with glyceraldehyde faster than the NH₂-terminal hexapeptide from the -chain. The glycation of tetrameric human Hb by glyceraldehyde was found to be dependent on the ligation state of the protein since deoxy-HbA reacted about 50% more with glyceraldehyde than did liganded HbA. The enhanced glycation of deoxy HbA was mainly attributable to the more extensive reaction at the NH₂-terminal of the -chain. The presence of a histidine adjacent to the NH₂-terminal at this site may facilitate the Amadori rearrangement. The glycation of horse Hb in which the second residue is glutamine was not increased under anaerobic conditions.     

Reevaluation of the amino acid sequence of porcine follitropin

We have reevaluated the sequence of porcine follicle-stimulating hormone (pFSH) with more recent protein-sequencing methodology. This has led to revision of the earlier proposed sequence. As with almost all reported gonadotropin -subunits, NH₂-terminal heterogeneity was found in the porcine FSH -subunit (FSH), starting with residue Phe (1), Asp (3), Gly (4), or Thr (7). In the -subunit, there were found to be at least two molecular species, starting with residue Asn (1) (minor 20%) or Cys (3) (major 80%) as NH₂-terminal and ending at residue Glu (108) as COOH-terminal. The net effect of the present revisions is to increase the homology of pFSH with other reported follitropin sequences. Apparent differences in the half-cystine placements in a previous proposal for pFSH compared with other species of FSH are no longer tenable. The half-cystine placements thus remain a constant structural feature throughout the gonadotropin hormones (choriogonadotropin, follitropin, and lutropin).     

Primary structure of zymogen of streptococcal proteinase

The complete amino acid sequence has been derived for the zymogen of streptococcal proteinase. The protein yielded a unique sequence containing 337 amino acids in a single polypeptide chain. The NH₂-terminal residue of the zymogen is aspartic acid and the COOH terminus is proline. The signal peptide commonly associated with the intracellular form of many proteins secreted from eukaryotic cells was absent from the zymogen sequence. The transformation of the zymogen to the enzyme under controlled conditions of proteolysis by trypsin and by streptococcal protease itself involves the removal of 84 amino acid residues from the NH₂ terminus of the zymogen. The zymogen-to-enzyme conversion is accompanied by a change in serological specificity. An intermediate, modified zymogen formed in the transformation process contains only 12 amino acid residues less than the zymogen but shows the serological reactivity of both the zymogen and the enzyme.     

A Gene Encoding a Cytochrome c Oxidase-Like Protein Is Located Closely to the Cytochrome c-553 Gene in the Anaerobic Bacterium,Desulfovibrio vulgaris (Miyazaki F)

The gene encoding cytochrome c-553 from Desulfovibrio vulgaris (Miyazaki F) was cloned using a synthetic oligodeoxyribonucleotide probe. The nucleotide sequence indicated that cytochrome c-553 was synthesized as a precursor protein with an NH₂-terminal signal sequence of 23 residues. In the cloned DNA fragment, there are three other open reading frames whose products have 191, 157, 541 amino acid residues, respectively. The putative ORF-4 product is highly homologous with the cytochrome c oxidase subunit I from various organisms.     

Structure and function relationship of staphylocoagulase

The bacterial protein staphylocoagulase binds stoichiometrically to human prothrombin, resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of -thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by -chymotrypsin. Limited -chymotryptic cleavage of staphylocoagulase yielded three large fragments, of 43, 30, and 20 kD. The 43-kD fragment exhibited a high affinity for human prothrombin (K_d=1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (K_d=0.46 nM). A complex of the 43-kD fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. The 30-kD fragment exhibited weaker affinity for prothrombin (K_d=120 nM.) While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. The 20-kD fragment was found only to bind to prothrombin. The NH₂-terminal sequence analyses of these fragments revealed that the 43-kD fragment constitutes the NH₂-terminal portion of staphylocoagulase, and contains the 30-kD and 20-kD fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH₂-terminal region of the intact protein. The 43-kD fragment contained 324 amino acids with a molecular weight of 38,098. The 43-kD fragment had an unusual amino acid composition based on a sequence in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounted for more than 45% of the total. A comparison of the amino acid sequence of the 43-kD fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with that of trypsin, -chymotrypsin, and elastase.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Chicken smooth muscle myosin light chain kinase is acetylated on its NH2-terminal methionine

The reported cDNA structrre, of chicken smooth muscle myosin light chain kinase (smMLCK) encodes a protein of 972 residues (Olsonet al. Proc. Natl. Acad. Sci USA, 87: 2284–2288, 1990). The calculated Mr is 107, 534 whereas the estimate by SDS-PAGE is approximately 130, 000. Gibson and Higgins (DNA Sequence (in press)) have recently reported the possibility of errors, in the cDNA sequence for non-muscle MLCK and that the NH₂-terminus of both it and smMLCK may extend beyond the reported coding region. The native smMLCK is NH₂-terminally blocked. A CNBr peptide derived from smMLCK contains the NH₂-terminal sequence Asp-Phe-Arg-Ala corresponding to residues 2 to 4 in the smMLCK sequence indicating, that Met-1 is present. Using a limited thermolysin digest we isolated an NH₂-terminally blocked peptide by reversed-phase HPLC. This thermolytic peptide had a mass of approximately 797 by time of flight mass spectrometry. Amino acid analysis and Edman sequencing of a CNBr-subfragment of the thermolytic peptide indicated that it had the composition and sequence, (Met)-Asp-Phe-Arg-Ala-Asn, with a calculated mass of 753. The difference in mass corresponds to the NH₂-terminal Met being blocked by actylation. The results demonstrate that the NH₂-terminal sequence of smMLCK inferred from the reported cDNA sequence is correct and that the proposed initiating, Met is not removed, but modified by -NH₂ acetylation of the translation product.     

Studies on lipase directed export of Escherichia coli beta-lactamase in Staphylococcus carnosus

Summary The lipase (lip) gene of Staphylococcus hyicus was used to study the expression of the Escherichia coli -lactamase (bla) gene in S. carnosus. The bla gene, devoid of its promotor and most of the signal sequence, was fused to the lip structural gene at various positions. A set of 11 secretion vectors (pLL1 to pLL11) was isolated and analysed. All secretion vectors caused -lactamase production and activity in S. carnosus. However, the amount of hybrid proteins secreted was influenced by the length of the NH₂-terminal lipase portion. An increased concentration, comparable to that of the native lipase, of secreted lipase/-lactamase hybrid proteins was only found when the lipase portion of the construct comprised more than 101 amino acids of the NH₂-terminal region of the lipase preprotein; the proposed lipase signal peptide is 36 amino acids long. If the hybrid proteins constructed contained 101 or less amino acids of the NH₂-terminal lipase preprotein, only low amounts of secreted hybrid proteins were detectable and a significant portion of the hybrid proteins and -lactamase activity was found in the cellular fraction. The results indicate that the lipase possesses adjacent to the signal peptide a peptide domain that is essential for the secretion of the lipase/-lactamase hybrid proteins.Abbreviations Cm chloramphenicol - bla gene beta lactamase coding gene of Escherichia coli - lip gene lipase-coding gene of Staphylococcus hyicus - PA polyacrylamide - PAGE PA gel electrophoresis - SDS sodium dodecyl sulphate - [] indicates plasmid-carrier state     

Sequence analysis and structural features of the largest known protamine isolated from the sperm of the archaeogastropod Monodonta turbinata

Protamine of the archaeogastropod mollusc Monodonta turbinata has been isolated and characterized. With a mass of 13,476 Da, it is the largest known prolamine. Amino acid sequence of this protamine (106 residues) was established from data provided by automated sequence analysis and mass spectrometry of the protein and of its fragments. The primary structure of the NH₂-terminal region exhibits repetitive sequence motifs Basic-Ser (mainly R-S) and both central and COOH-terminal regions are composed by arginine clusters. The amino acid sequence of Monodonta turbinata protamine shows structural similarities with other protamines from invertebrates and from birds and mammals.     

Crotalus adamanteus phospholipase A2-α: Subunit structure,NH2-terminal sequence,and homology with other phospholipases

Automated Edman degradation of reduced and carboxymethylated phospholipase A₂-α from Crotalus adamanteus venom revealed a single amino acid sequence extending 30 residues into the protein from the amino terminus. The singularity of the sequence and the yields of the phenylthiohydantoin amino acids thus obtained indicate that the subunits comprising the phospholipase dimer are identical. Further chemical evidence in support of subunit identity was obtained by cleavage of phospholipase A₂-α with cyanogen bromide. Compositional analysis of the protein revealed one residue of methionine per monomer and the sequence determination placed this amino acid at position 10 in the sequence of 133 amino acids. Cyanogen bromide cleavage of the protein, followed by reduction and carboxymethylation afforded the expected 2 fragments: an NH₂-terminal decapeptide (CNBr-1) and a larger COOH-terminal fragment of 123 residues (CNBr-II). Automated Edman degradation of the latter has extended the sequence analysis to 54 residues in the NH₂-terminal segment of the monomer chain. Comparison of this sequence with those derived for phospholipases from other snake venoms, from bee venom, and from porcine pancreas has revealed striking homologies in this region of the molecules. As expected on the basis of their phylogenetic classification, the phospholipases from the pit vipers C. adamanteus and Agkistrodon halys blomhoffii are more similar to one another in sequence than to the enzyme from the more distantly related viper, Bitis gabonica. Furthermore, the very close similarities in sequence observed among all of these phospholipases in regions corresponding to residues 24 through 53 in the C. adamanteus enzyme suggest that this segment of the polypeptide plays an important role in phospholipase function and probably constitutes part of the active site.     

Isolation, Purification, and Subcellular Localization of Isozymes of Superoxide Dismutase from Scots Pine (Pinus sylvestris L.) Needles

Two of four isozymes of superoxide dismutase (SOD) (EC 1.15.1.1) were purified from Scots pine (Pinus sylvestris L.) needles. One form was cytosolic (SOD-1) and the other was associated with chloroplasts (SOD-3). The holoenzyme molecular masses was estimated at approximately 35 kilodaltons by gel filtration. The subunit molecular weight of the dimeric enzymes was estimated to 16.5 kilodaltons (SOD-1) and 20.4 kilodaltons (SOD-3) on sodium dodecyl sulfatepolyacrylamide gels. The NH₂-terminal sequence of the pine enzymes showed similarities to other purified superoxide dismutases located in the corresponding compartment. The cytosolic form revealed two additional amino acids at position 1 and 2 at the NH₂-terminal. Both forms were cyanide- and hydrogenperoxide-sensitive and SOD-3 was found to contain approximately one copper atom per subunit, indicating that they belong to the cupro-zinc SODs. The isoelectric point was 4.9 and 4.5 for SOD-1 and SOD-3, respectively.     

The primary structure of a cDNA for PsaN,encoding an extrinsic lumenal polypeptide of barley photosystem I

A cDNA clone encoding a 15.501 Da photosystem I (PSI) subunit of barley was isolated using an oligonucleotide based on the NH₂-terminal amino acid sequence of the isolated protein. The polypeptide, which migrates with an apparent molecular mass of 9.5 kDa on denaturing SDS-PAGE, has been designated PSI-N, and the corresponding gene is PsaN. Analysis of the deduced protein sequence indicates a mature protein of 85 amino acid residues and a molecular mass of 9818 Da. PSI-N is a hydrophilic, extrinsic protein with no predicted membrane-spanning regions. The transit peptide of 60 residues (5683 Da) contains a predicted hydrophobic -helix, suggesting that the protein is routed into the thylakoid lumen. Thus, PSI-N is the second known lumenal protein component associated with PSI, together with PSI-F.     

Biogenesis of the Saccharomyces cerevisiae Mating Pheromone a-Factor

The Saccharomyces cerevisiae mating pheromone a-factor is a prenylated and carboxyl methylated extracellular peptide signaling molecule. Biogenesis of the a-factor precursor proceeds via a distinctive multistep pathway that involves COOH-terminal modification, NH₂-terminal proteolysis, and a nonclassical export mechanism. In this study, we examine the formation and fate of a-factor biosynthetic intermediates to more precisely define the events that occur during a-factor biogenesis. We have identified four distinct a-factor biosynthetic intermediates (P0, P1, P2, and M) by metabolic labeling, immunoprecipitation, and SDSPAGE. We determined the biochemical composition of each by defining their NH₂-terminal amino acid and COOH-terminal modification status. Unexpectedly, we discovered that not one, but two NH₂-terminal cleavage steps occur during the biogenesis of a-factor. In addition, we have shown that COOH-terminal prenylation is required for the NH₂-terminal processing of a-factor and that all the prenylated a-factor intermediates (P1, P2, and M) are membrane bound, suggesting that many steps of a-factor biogenesis occur in association with membranes. We also observed that although the biogenesis of a-factor is a rapid process, it is inherently inefficient, perhaps reflecting the potential for regulation. Previous studies have identified gene products that participate in the COOH-terminal modification (Ram1p, Ram2p, Ste14p), NH₂-terminal processing (Ste24p, Axl1p), and export (Ste6p) of a-factor. The intermediates defined in the present study are discussed in the context of these biogenesis components to formulate an overall model for the pathway of a-factor biogenesis.In Saccharomyces cerevisiae, the peptide mating pheromones a-factor and α-factor function to promote conjugation between cells of the opposite mating type, MATa and MATα (Marsh et al., 1991; Sprague and Thorner, 1992). Like the peptide hormones secreted by higher eukaryotes, the yeast mating pheromones are initially synthesized as larger precursors that undergo posttranslational modification and proteolytic processing before their export from the cell. Despite their functional equivalence as signaling molecules, the a-factor and α-factor pheromones are structurally quite dissimilar and exemplify distinct paradigms for biogenesis. The maturation of α-factor is well characterized and involves the “classical” secretory pathway (ER→ Golgi→ secretory vesicles; Julius et al., 1984). Subsequent to its translocation across the ER membrane, the α-factor precursor undergoes signal sequence cleavage, glycosylation, a series of proteolytic processing steps in the lumenal compartments of the secretory pathway, and then exits the cell via exocytosis (Fuller et al., 1986; Sprague and Thorner, 1992). In contrast to our extensive understanding of α-factor maturation, our view of the events involved in a-factor biogenesis is still incomplete. An important difference between the two pheromones is that secretion of a-factor is mediated by a “nonclassical” export mechanism (Kuchler et al., 1989; McGrath and Varshavsky, 1989; Michaelis, 1993). The purpose of the present study is to delineate the steps of a-factor biogenesis that occur before its export, by the identification and characterization of a-factor biosynthetic intermediates.Mature bioactive a-factor is a prenylated and methylated dodecapeptide, derived by the posttranslational maturation of a precursor encoded by the similar and functionally redundant genes MFA1 and MFA2 (Brake et al., 1985; Michaelis and Herskowitz, 1988). The structures of the precursor and mature forms of a-factor derived from MFA1 are shown in Fig. ​Fig.1.1. The a-factor precursor can be subdivided into three functional segments: (a) the mature portion (shaded in Fig. ​Fig.1),1), which is ultimately secreted; (b) the NH₂-terminal extension; and (c) the COOH-terminal CAAX motif (C is cysteine, A is aliphatic, and X is one of many residues). As shown here, and also suggested by our previous studies, the biogenesis of a-factor occurs by an ordered series of events involving first COOH-terminal CAAX modification, then NH₂-terminal processing, and finally export from the cell (He et al., 1991; Michaelis, 1993; Sapperstein et al., 1994). Open in a separate windowFigure 1Structure of precursor and mature forms of a-factor encoded by MFA1. The a-factor precursor encoded by MFA1 is shown with the NH₂-terminal extension, COOH-terminal CAAX motif, and mature portion (shaded gray) indicated. Every fifth residue is numbered. Mature a-factor derived from this precursor is modified on its COOH-terminal cysteine residue by a farnesyl moiety and a carboxyl methyl group, as indicated.The COOH-terminal maturation of the a-factor precursor is directed by its CAAX sequence. The CAAX motif is present at the COOH terminus of numerous eukaryotic proteins, most notably the Ras proteins, and is known to signal a triplet of posttranslational modifications. These include prenylation of the cysteine residue, proteolysis of the COOH terminal AAX residues (VIA for a-factor), and methylation of the newly exposed cysteine carboxyl group (Clarke, 1992; Zhang and Casey, 1996). The yeast enzymes that mediate the modification of CAAX-terminating proteins are known from genetic and biochemical studies. RAM1 and RAM2 encode the subunits of the cytosolic farnesyltransferase enzyme (Fujiyama et al., 1987; He et al., 1991; Powers et al., 1986; Schafer et al., 1990). An “AAX” endoprotease has been detected as a membrane-associated activity in yeast extracts, although the corresponding gene(s) remains elusive (Ashby et al., 1992; Hrycyna and Clarke, 1992). STE14 encodes the prenylcysteine-dependent carboxyl methyltransferase that mediates methylation, the final step in modification of CAAX proteins; Ste14p is also membrane associated (Hrycyna and Clarke, 1990; Hrycyna et al., 1991; Marr et al., 1990; Sapperstein et al., 1994). In mutants (ram1, ram2, and ste14) defective in CAAX modification, biologically active a-factor is not produced.The events involved in the NH₂-terminal proteolytic processing of the a-factor precursor are less well-defined than those of COOH-terminal maturation. It was recently shown that a protease encoded by the AXL1 gene is required for one step of the NH₂-terminal processing of a-factor (Adames et al., 1995). Axl1p belongs to the insulin-degrading enzyme (IDE)¹ subfamily of proteases; an AXL1 homologue, Ste23p, was also found to perform a role at least partially redundant to that of Axl1p in a-factor processing (Adames et al., 1995). Recently, we have identified another gene, STE24, whose product participates in the NH₂-terminal processing of the a-factor precursor in a manner distinct from Axl1p and Ste23p (Fujimura-Kamada and Michaelis, 1997). Based on a priori inspection of the precursor and mature forms of a-factor (Fig. ​(Fig.1),1), a single NH₂-terminal proteolytic cleavage event (between residues N21 and Y22) might have been predicted; however, we provide evidence in the present study that the proteolytic processing of the NH₂terminal extension of the a-factor precursor occurs in two distinct steps.The final event in a-factor biogenesis is the export of the fully matured pheromone from the cell. The absence of a canonical NH₂-terminal signal sequence in the MFA1 and MFA2 sequences, as well as the lack of effect upon a-factor secretion of sec mutants blocked at various steps in the classical secretory pathway, led to the suggestion of a nonclassical export mechanism for a-factor export (McGrath and Varshavsky, 1989; Sterne, 1989). Indeed, a-factor export is now known to be mediated by Ste6p, a member of the ATP-binding cassette (ABC) superfamily of proteins (Kuchler et al., 1989; McGrath and Varshavsky, 1989). ABC proteins carry out the ATP-dependent membrane translocation of a variety of compounds, including small peptides, hydrophobic drugs, and even prenylcysteine derivatives, by an uncharacterized mechanism (Gottesman and Pastan, 1993; Zhang et al., 1994). It is notable that a-factor undergoes COOH-terminal modification and NH₂-terminal proteolytic maturation before Ste6p-mediated membrane translocation. This order of events contrasts with those of the biogenesis of the α-factor precursor and other classical secretory substrates, which undergo ER membrane translocation first and are matured only subsequently.In the present study, we aimed to elucidate the events that occur during a-factor biogenesis, before its export from the cell. Our approach was to identify a-factor biosynthetic intermediates, determine their chemical composition and localization properties, and examine the efficiency of their formation and the effects of an a-factor CAAX mutation on their formation. In addition to identifying the biosynthetic intermediates we expected, which include the unmodified a-factor precursor (P0), the COOHterminally modified a-factor precursor (P1), and mature a-factor (M), we unexpectedly uncovered a novel and unanticipated intermediate. This species, designated P2, is fully COOH-terminally modified and has had only a segment of its NH₂-terminal extension proteolytically removed. The existence of the P2 intermediate provides evidence that an additional unpredicted step occurs during the NH₂-terminal processing of the a-factor precursor. The biosynthetic intermediates we identify here, considered together with known a-factor biogenesis components, are presented in terms of a comprehensive model for the a-factor biogenesis pathway.     

The occurrence of a premature form of egg-specific protein in vitellogenic follicles ofBombyx mori

Summary Analysis of yolk proteins of the silkworm,Bombyx mori, by SDS-polyacrylamide gel electrophoresis and immunoblotting showed that there was a developmental change in subunit composition of egg-specific protein; egg-specific protein consisting of 72 kDa subunits alone (premature form) was found in vitellogenic follicles, whereas the protein in mature eggs was composed of 72 kDa and 64 kDa subunits (mature form). The premature form of egg-specific protein was purified from young ovaries to homogeneity using a high performance liquid chromatography system. The purified protein had an apparent molecular mass of 225 kDa which could not be distinguished from that of the mature form. By circular dichroism analysis, both egg-specific proteins were estimated to have about 30% -helix and 20% -sheet, but the mature form showed a relatively rigid conformation in the aromatic region. The premature egg-specific protein purified from vitellogenic ovaries, consisted of three 72 kDa subunits, whereas mature egg-specific protein was composed of two 72 kDa subunits and one 64 kDa subunit. All of these subunits showed the same immunoreactivity towards antiserum raised against the mature form. An identical NH₂-terminal amino acid sequence was found in both 72 kDa polypeptides and 64 kDa polypeptide for the initial 10 amino acids.Abbreviations SDS sodium dodecyl sulfate - PMSF phenylmethylsulfonyl fluoride - PAGE polyacrylamide gel electrophoresis - HPLC high performance liquid chromatography - ESP egg-specific protein - Vtn vitellin     

The Role of the Supernumerary Subunit of Rhodobactersphaeroides Cytochrome bc 1 Complex

The smallest molecular weight subunit (subunit IV), which contains no redox prosthetic group,is the only supernumerary subunit in the four-subunit Rhodobacter sphaeroides bc ₁ complex.This subunit is involved in Q binding and the structural integrity of the complex. When thecytochrome bc ₁ complex is photoaffinity labeled with [³H]azido-Q derivative, radioactivity isfound in subunits IV and I (cytochrome b), indicating that these two subunits are responsiblefor Q binding in the complex. When the subunit IV gene (fbcQ) is deleted from the R.sphaeroides chromosome, the resulting strain (RSIV) requires a period of adaptation beforethe start of photosynthetic growth. The cytochrome bc ₁ complex in adapted RSIVchromatophores is labile to detergent treatment (60–75% inactivation), and shows a four-fold increasein the K _m for Q₂H₂. The first two changes indicate a structural role of subunit IV; the thirdchange supports its Q-binding function. Tryptophan-79 is important for structural andQ-binding functions of subunit IV. Subunit IV is overexpressed in Escherichia coli as a GSTfusion protein using the constructed expression vector, pGEX/IV. Purified recombinant subunitIV is functionally active as it can restore the bc ₁ complex activity from the three-subunit corecomplex to the same level as that of wild-type or complement complex. Three regions in thesubunit IV sequence, residues 86–109, 77–85, and 41–55, are essential for interaction withthe core complex because deleting one of these regions yields a subunit completely or partiallyunable to restore cytochrome bc ₁ from the core complex.     

Glycinin composition of several perennial species related to soybean

The 7S and 11S seed storage proteins from four perennials related to soybean (Glycine canescens, G. tomentella, G. tabacina, and G. clandestina) were analyzed by sodium dodecyl sulfate-gel electrophoresis. Each species yielded a unique electrophoretic pattern that varied in the total number of bands and their relative mobilities. In every case, the electrophoretic patterns were substantially different from CX635-1-1-1, the strain of G. max used in this study for comparison. Size heterogeneities among both the 7S and 11S polypeptides of the perennials were evident.

Abundant proteins in the 11S fraction from G. tomentella (CSIRO No. 1133) were separated by chromatography on DEAE-Sephadex and then their apparent molecular weights, amino acid compositions, and NH₂-terminal amino acid sequences were determined. A group of proteins were obtained which resembled the A_1b-polypeptide components of glycinin from G. max. They had the same size (M_r 37,000), identical NH₂-terminal sequences, and similar amino acid compositions to A_1b. A second group of acidic proteins (M_r 50,000) in G. tomentella had NH₂-terminal sequences homologous to the A₅ component (M_r 10,000) of glycinin. The latter group of polypeptides had a substantially higher apparent molecular weight than any acidic polypeptide components of glycinin analyzed previously. A third group of polypeptides purified from G. tomentella were the same size as basic polypeptides of glycinin and had homologus NH₂-terminal sequences. The results indicated that the perennials exhibit variability in their seed proteins at a level not found among the cultivars of G. max and G. soja and may be useful in studies concerning the origin and organization of genes involved in the synthesis of storage proteins in cultivated soybeans.