Protection of specific sites in 23 S and 5 S RNA from chemical modification by association of 30 S and 50 S ribosomes.

The effect of 30S subunit attachment on the accessibility of specific sites in 5 S and 23 S RNA in 50 S ribosomal subunits was studied by means of the guanine-specific reagent kethoxal. Oligonucleotides surrounding the sites of kethoxal substitution were resolved and quantitated by diagonal electrophoresis. In contrast to the extensive protection of sites in 16 S RNA in 70 S ribosomes (Chapman &; Noller, 1977), only two strongly (approx. 90%) protected sites were detected in 23 S RNA. The nucleotide sequences at these sites are

in which the indicated kethoxal-reactive guanines (with K above them) are strongly protected by association of 30 S and 50 S subunits. The latter sequence has the potential to base-pair with nucleotides 816 to 821 of the 16 S RNA, a site which has been shown to be protected from kethoxal by 50 S subunits and essential for subunit association. Six additional sites in 23 S RNA are partially (30 to 50%) protected by 30 S subunits. One of these sequences,

is complementary to nucleotides 787 to 792 of 16 S RNA. a site which is also 50 S-protected and essential for association. Of the two kethoxal-reactive 5 S RNA sites in 50 S subunits, G₁₃ is partially protected in 70 S ribosomes. while G₄₁ remains unaffected by subunit association.The relatively small number of kethoxal-reactive sites in 23 S RNA that is strongly protected in 70 S ribosomes suggests that subunit association may involve contacts between single-stranded sites in 16 S RNA and 50 S subunit proteins or non-Watson-Crick interactions with 23 S RNA. in addition to the two suggested base-paired contacts.     

Alpha-thrombin-catalyzed activation of human platelet factor XIII: relationship between proteolysis and factor XIIIa activity

The kinetics of activation of platelet factor XIII, an a-subunit dimer, were characterized by determining rate constants for activation peptide (AP) release, generation of activity, and exposure of the active-site thiol group. The specificity constant (kappacat/Km) for alpha-thrombin-catalyzed AP release, 1.2 x 10(5) M-1s-1, was found to be similar to that for AP release from the tetramer plasma factor XIII (a2b2) [Janus, T.J., Lewis, S. D., Lorand, L., & Shafer, J. A. (1983) Biochemistry 22, 6269-6272], implying that the b subunits of plasma factor XIII do not hinder alpha-thrombin-catalyzed cleavage of AP from the a subunit. Platelet factor XIIIa activity was generated at a rate approximately twice the rate of AP release. This difference in rates was shown to be consistent with a reaction pathway for activation of platelet factor XIII wherein full factor XIIIa activity is generated when one AP is removed from the dimeric zymogen so that removal of the second AP has no detectable effect on catalytic activity. In accord with this conclusion, the rate constant for exposure of the active-site thiol group, as measured by the incorporation of [1-14C]-iodoacetamide, was about twice that observed for the removal of AP.(ABSTRACT TRUNCATED AT 250 WORDS)     

The origins of modern proteomes

Distributions of phylogenetically related protein domains (fold superfamilies), or FSFs, among the three Superkingdoms (trichotomy) are assessed. Very nearly 900 of the 1200 FSFs of the trichotomy are shared by two or three Superkingdoms. Parsimony analysis of FSF distributions suggests that the FSF complement of the last common ancestor to the trichotomy was more like that of modern eukaryotes than that of archaea and bacteria. Studies of length distributions among members of orthologous families of proteins present in all three Superkingdoms reveal that such lengths are significantly longer among eukaryotes than among bacteria and archaea. The data also reveal that proteins lengths of eukaryotes are more broadly distributed than they are within archaeal and bacterial members of the same orthologous families. Accordingly, selective pressure for a minimal size is significantly greater for orthologous protein lengths in archaea and bacteria than in eukaryotes. Alignments of orthologous proteins of archaea, bacteria and eukaryotes are characterized by greater sequence variation at their N-terminal and C-terminal domains, than in their central cores. Length variations tend to be localized in the terminal sequences; the conserved sequences of orthologous families are localized in a central core. These data are consistent with the interpretation that the genomes of the last common ancestor (LUCA) encoded a cohort of FSFs not very different from that of modern eukaryotes. Divergence of bacterial and archaeal genomes from that common ancestor may have been accompanied by more intensive reductive evolution of proteomes than that expressed in eukaryotes. Dollo's Law suggests that the evolution of novel FSFs is a very slow process, while laboratory experiments suggests that novel protein genesis from preexisting FSFs can be relatively rapid. Reassortment of FSFs to create novel proteins may have been mediated by genetic recombination before the advent of more efficient splicing mechanisms.     

Resistance of factor XIII to degradation or activation by plasmin

The effect of plasmin on the subunit polypeptides of factor XIII has been investigated. purified human plasma (a2b2) and platelet (a2) zymogens and the enzyme (a2) were incubated with plasmin at plasmin: factor XIII ratios of 0.03-0.5 casein units per mg protein. Under conditions in which plasmin readily digested fibrinogen and casein, it had no effect on either a2b2 or a2. There was no evidence for cleavage of peptide bonds in the zymogens, and all the potential catalytic activity was retained after prolonged incubation. Similarly a2*, either in the presence or absence of b subunit, was also unaffected by plasmin incubation. 90% of the activity was recovered after incubation of factor XIII with plasmin. b subunit was also not degraded. Additionally, no evidence was obtained that plasmin could activate factor Xiii. These results indicate that in purified systems there is no significant interaction between plasmin and factor XIII.     

Platelet factor XIII. The collagen receptor?

We have studied the binding of collagen fibers with platelet proteins using affinity chromatography on collagen-Sepharose. Only a few proteins from a platelet lysate were trapped by this column. When denatured collagen (gelatin) was used as the affinity ligand, the major protein did not bind and was identified as platelet Factor XIII by polyacrylamide gel electrophoresis, immunoprecipitation, and enzymic activity. This is a zymogen form of transglutaminase, which corresponds to the "a" subunit of the coagulation factor in plasma. Immunoglobulins specific for platelet Factor XIII obtained from antiserum raised against plasma Factor XIII were able to initiate platelet aggregation by themselves, in strong contrast to nonspecific antibodies. This specific immunoglobulin-mediated platelet aggregation required the presence of Ca2+. It was inhibited by aspirin and prostacyclin, but not by specific inhibitors for other agonists. These data suggest the possibility that the zymogen form of Factor XIII is located on the surface of platelets and may play a key role as the receptor for collagen-induced platelet aggregation.     

Differences in the Subunit Structure of Human Fibrin formed by the Action of Arvin,Reptilase and Thrombin

INTEREST has focused recently on the clinical use of proteolytic enzymes similar in properties to thrombin which can directly cleave fibrinogen. Potentially the most important are arvin, derived from the venom of Agkistrodon rhodostoma and reptilase, isolated from the venom of Bothrops atrox. These only release fibrinopeptide A from fibrinogen^1–3, whereas thrombin cleaves fibrinopeptides A and B from fibrinogen to form fibrin. Thrombin also activates fibrin stabilizing factor (FSF) which introduces amide bonds between the subunits of soluble fibrin⁴. FSF rapidly forms covalent links between pairs of γ(C)-chains giving γ(C)-dimers and in a slower reaction α(A)-chains are linked to produce high molecular weight polymers⁵. Although reptilase, like thrombin, activates FSF⁶, arvin apparently does not, which would explain why the fibrin formed by arvin seems to be more friable than that produced by thrombin or reptilase⁷.     

Studies on factor XIII antigen in congenital factor XIII deficiency. A tentative classification of the disease in two groups.

Immunological and immunofluorescent studies carried out on plasma and platelets of three cases of congenital factor XIII deficiency are reported. Two of these patients were originally thought to have normal factor XIII subunit S and no subunit A. However, repeated assays carried out using different lots of antiserum showed that in reality the patients lacked both subunit S and subunit A. The false positive finding was due to the presence of a anti-factor VIII contaminant in the antiserum originally used. The third patient had a normal subunit S and no subunit A. No factor XIII antigen was found by the indirect immunofluorescent technique in normal, factor XIII deficiency and von Willebrand's disease platelets. On the contrary, by using the non-monospecific antiserum a fluorescent pattern similar to that observed by using an anti-factor VIII antiserum, had been noted. On the basis of the data presented in this paper a tentative classification of factor XIII deficiency in two groups is proposed: Type I is characterized by the lack of both factor XIII subunit S and A. Type II is characterized by a normal subunit S and no subunit A. The need for a re-evaluation of published case of factor XIII deficiency by means of monospecific antisera is indicated.     

Intermediate structure of normal human haemoglobin: methaemoglobin in the deoxy quaternary conformation

This paper describes a new method of producing a crystalline intermediate between the unligated and ligated states of haemoglobin, suitable for X-ray analysis, by the use of a lattice strengthening reagent. Acrylamide is polymerized in the liquid of crystallization after the crystal has grown, forming a stiff supporting gel between the haemoglobin molecules, but not covalent bonds with them. The structure of human haemoglobin A crystallized in the deoxy quaternary structure (T-state²) and then oxidized by air after lattice strengthening (tertiary structure made met, or r-state) was determined to 3.5 Å resolution by the difference Fourier technique. Marked changes in tertiary structure in the region of the haem pockets and the contacts between the subunits (α₁β₂) are observed. The iron is seen to move towards the plane of the porphyrin, causing a change of tilt of the haem. This appears to act as a lever setting in train stereochemical changes that loosen several hydrogen bonds within and between subunits, on which the stability of the tertiary and quaternary deoxy structures depend. The liganding water molecule itself causes a slight opening of the haem pocket in the α subunit, and a substantial one in the β subunit. The structural changes seen here in going from the tertiary deoxy to the aquomet state within the quaternary T-structure are similar, but opposite, to those seen earlier in going from aquomet to deoxy in the quaternary R-structure of BME-haemoglobin. Changes in tertiary structure associated with addition of ligand to the T-structure or the removal of ligand from the R-structure are thus seen to be complementary. Electron density maps show the α haems to undergo autoxidation more readily than the β haems, just as the β haems were reduced more easily than the α haems in BME-haemoglobin.     

The ecto-5'-nucleotidase subunits in dimers are not linked by disulfide bridges but by non-covalent bonds

It has long been considered that ecto-5'-nucleotidase (eNT) dimers consist of subunits linked by disulfide bonds. Hydrophilic (6.7S) and amphiphilic (4.0S) dimers were separated by sedimentation analysis of eNT purified from bull seminal plasma. Hydrophilic (4. 2S) and amphiphilic (2.6S) eNT monomers were obtained after reduction of disulfide bonds in dimers. The amphiphilic eNT dimers or monomers were converted into their hydrophilic variants with phosphatidylinositol-specific phospholipase C. SDS-PAGE plus Western blot showed 68 kDa subunits, regardless of the addition of beta-mercaptoethanol to the SDS mixture. Active eNT monomers were obtained by addition of 1 M guanidinium chloride (Gdn) to dimers, and unfolded subunits by addition of 4 M Gdn. The results unambiguously demonstrate that the subunits in eNT dimers are not linked by disulfide bridges, but by non-covalent bonds, and that dissociation precedes inactivation and unfolding.     

Genetic mapping and validation of the loci controlling 7S α′ and 11S A-type storage protein subunits in soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.]

Key message

Four soybean storage protein subunit QTLs were mapped using bulked segregant analysis and an F₂ population, which were validated with an F₅ RIL population.

Abstract

The storage protein globulins β-conglycinin (7S subunit) and glycinin (11S subunits) can affect the quantity and quality of proteins found in soybean seeds and account for more than 70% of the total soybean protein. Manipulating the storage protein subunits to enhance soymeal nutrition and for desirable tofu manufacturing characteristics are two end-use quality goals in soybean breeding programs. To aid in developing soybean cultivars with desired seed composition, an F₂ mapping population (n = 448) and an F₅ RIL population (n = 180) were developed by crossing high protein cultivar ‘Harovinton’ with the breeding line SQ97-0263_3-1a, which lacks the 7S α′, 11S A₁, 11S A₂, 11S A₃ and 11S A₄ subunits. The storage protein composition of each individual in the F₂ and F₅ populations were profiled using SDS-PAGE. Based on the presence/absence of the subunits, genomic DNA bulks were formed among the F₂ plants to identify genomic regions controlling the 7S α′ and 11S protein subunits. By utilizing polymorphic SNPs between the bulks characterized with Illumina SoySNP50K iSelect BeadChips at targeted genomic regions, KASP assays were designed and used to map QTLs causing the loss of the subunits. Soybean storage protein QTLs were identified on Chromosome 3 (11S A₁), Chromosome 10 (7S α′ and 11S A₄), and Chromosome 13 (11S A₃), which were also validated in the F₅ RIL population. The results of this research could allow for the deployment of marker-assisted selection for desired storage protein subunits by screening breeding populations using the SNPs linked with the subunits of interest.

     

Molecular subunits and transamidase activity of factor XIII in patients with deep vein thrombosis

Thrombin formed during the clotting process leads to the activation of factor XIII and then to its consumption. Transamidase activity of factor XIII and its concentrations of subunits "a" and "b" were measured in plasmas of patients with deep vein thrombosis of the calves. The patients revealed a significant decrease of both factor XIII activity and its concentration of subunit "a". A subsequent rise of transamidase activity and concentration of subunit "a" of factor XIII to the normal values was observed in two weeks' time. It is assumed that estimation of factor XIII activity and/or its concentration of subunit "a" may be an additional method for detecting venous thrombosis and monitoring its therapy.     

The vacuolar ATPase ofNeurospora crassa

The filamentous fungusNeurospora crassa has many small vacuoles which, like mammalian lysosomes, contain hydrolytic enzymes. They also store large amounts of phosphate and basic amino acids. To generate an acidic interior and to drive the transport of small molecules, the vacuolar membranes are densely studded with a proton-pumping ATPase. The vacuolar ATPase is a large enzyme, composed of 8–10 subunits. These subunits are arranged into two sectors, a complex of peripheral subunits called V₁ and an integral membrane complex called V₀. Genes encoding three of the subunits have been isolated.vma-1 andvma-2 encode polypeptides homologous to the and subunits of F-type ATPases. These subunits appear to contain the sites of ATP binding and hydrolysis.vma-3 encodes a highly hydrophobic polypeptide homologous to the proteolipid subunit of vacuolar ATPases from other organisms. This subunit may form part of the proton-containing pathway through the membrane. We have examined the structures of the genes and attempted to inactivate them.     

Characterization of N-polyhedrin of two baculovirus strains pathogenic for Orgyia pseudotsugata.

N-polyhedrin of inclusion bodies of two nucleopolyhedrosis viruses of Orgyia pseudotsugata was characterized. Alkali-dissolved N-polyhedrin from both virus strains was of similar size and consisted of 12S molecule of 209 000 daltons. Eight subunits of approximately 26 000 daltons were found to form the 12S molecules. N-polyhedrin from both viruses showed two main antigens by immunodiffusion. The subunits appear to possess one antigen and, upon formation of the 12S molecule, a new antigen is created. Both the subunit and 12S antigens from the two virus strains were shown to be antigenically related. The 12S molecule of both viruses also appears to possess a minor antigen unique to each virus.     

Cross-linking of a major fibroblast surface-associated glycoprotein (fibronectin) catalyzed by blood coagulation factor XIII

The surface proteins of cultured human skin fibroblasts were iodinated and then exposed to one or more of the following blood coagulation proteins: thrombin, fibrinogen, and factor XIII (plasma protransglutaminase). Radiolabeled polypeptides were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. After exposure to physiological concentrations of activated factor XIII (XIII_a), the band of radioactivity corresponding to the major labeled surface protein (fibronectin, molecular weight = 2.2 × 10⁵ daltons) was cross-linked to a very high molecular weight complex. The cross-linking reaction was inhibited by fibrin (which is known to bind the catalytic subunit of XIII_a). Cross-linking of labeled cell surface fibronectin to fibrin could not be demonstrated. The fibrillar pattern of surface fibronectin appeared unaffected by cross-linking when studied by immunofluorescence. Cross-linking of cell surface fibronectin by XIII_a requires highly specific enzyme-substrate and protein-protein interactions, and may be an important physiological reaction.     

A Relaxed Specificity in Interchain Disulfide Bond Formation Characterizes the Assembly of a Low-Molecular-Weight Glutenin Subunit in the Endoplasmic Reticulum

Wheat (Triticum spp.) grains contain large protein polymers constituted by two main classes of polypeptides: the high-molecular-weight glutenin subunits and the low-molecular-weight glutenin subunits (LMW-GS). These polymers are among the largest protein molecules known in nature and are the main determinants of the superior technological properties of wheat flours. However, little is known about the mechanisms controlling the assembly of the different subunits and the way they are arranged in the final polymer. Here, we have addressed these issues by analyzing the formation of interchain disulfide bonds between identical and different LMW-GS and by studying the assembly of mutants lacking individual intrachain disulfides. Our results indicate that individual cysteine residues that remain available for disulfide bond formation in the folded monomer can form interchain disulfide bonds with a variety of different cysteine residues present in a companion subunit. These results imply that the coordinated expression of many different LMW-GS in wheat endosperm cells can potentially lead to the formation of a large set of distinct polymeric structures, in which subunits can be arranged in different configurations. In addition, we show that not all intrachain disulfide bonds are necessary for the generation of an assembly-competent structure and that the retention of a LMW-GS in the early secretory pathway is not dependent on polymer formation.The unique ability of wheat (Triticum spp.) flour to form a dough that has the rheological properties required for the production of leavened bread and other foods is largely due to the characteristics of the proteins that accumulate in wheat endosperm cells during seed development (Gianibelli et al., 2001). Among these endosperm proteins, a major role is played by prolamines, a large group of structurally different proteins sharing the characteristic of being particularly high in Pro and Gln.On the basis of their polymerization status, wheat prolamines can be subdivided into two groups, the gliadins and the glutenins. While gliadins are monomeric, glutenins are heterogeneous mixtures of polymers where individual subunits are held together by interchain disulfide bonds (Galili et al., 1996; Tatham and Shewry, 1998). The subunits participating to the formation of these large polymers have been classified into four groups according to their electrophoretic mobility (Gianibelli et al., 2001). The A group is constituted by the so-called high-molecular-weight glutenin subunits (HMW-GS), while polypeptides in groups B, C, and D are collectively termed low-molecular-weight glutenin subunits (LMW-GS). While only three to five HMW-GS are expressed in common wheat endosperm, LMW-GS include a very large number of different polypeptides.Different models of glutenin assembly have been proposed (see Gianibelli et al., 2001 for a review), but the determination of their precise structure and M_r distribution has been hampered by their large size and complex subunit composition. Crucially, because disulfide bonds appear to be the major factor affecting polymer stability, it would be very useful to know whether the pairing between specific Cys residues, rather than random assembly, controls glutenin polymer formation. Indeed, data obtained with HMW-GS indicate that the formation of certain types of intermolecular disulfide bonds is particularly favored (Tao et al., 1992; Shimoni et al., 1997). In the case of LMW-GS, at least two functionally distinct types of subunits can be distinguished. Subunits of the first type, to which the majority of B-type subunits belong, would act as chain extenders, because they contain two Cys residues that remain available for the formation of interchain disulfide bonds. Subunits of the second type, containing a single Cys residue able to form an interchain disulfide bond, would instead act as chain terminators (Kasarda, 1989). Most of the members of this second group are indeed modified gliadins that participate to polymer formation thanks to the presence of extra Cys residues (D''Ovidio and Masci, 2004). Given the complexity of the situation found in wheat endosperm, where many different subunits are synthesized at the same time and can participate in the formation of complex high-M_r polymers, the study of glutenin polymer formation can take advantage of the use of heterologous expression systems where the behavior of individual subunits can be more easily monitored. For instance, the expression of HMW-GS in transgenic tobacco (Nicotiana tabacum) has provided insights into the rules governing the assembly of some of the subunits belonging to this class (Shani et al., 1994; Shimoni et al., 1997). In this work, we have used heterologous expression of wild-type and modified LMW-GS in tobacco protoplasts to study the assembly of this class of gluten polypeptides. Our results confirm that disulfide bonds are crucial for the assembly of these proteins and indicate that a relaxed specificity in Cys pairing from different subunits can drive the formation of complex glutenin polymers.     

Mitoribosomes from Candida utilis. Morphological, physical, and chemical characterization of the monomer form and of its subunits

Highly purified mitochondrial ribosomes (mitoribosomes) have been obtained from the yeast Candida utilis. Sedimentation analysis in sucrose gradients made in 5 mM MgCl₂, 1 mM Tris, pH 7.4 and 50 mM KCl clearly distinguishes mitoribosomes (72S) from cytoplasmic ribosomes (cytoribosomes) (78S). Mitoribosomes are completely dissociated into 50S and 36S subunits at 10^-4 M MgCl₂ whereas complete dissociation of cytoribosomes into 61S and 37S subunits occurs only at 10^-6 M MgCl₂ Electron microscopy of negatively stained mitoribosomes (72S peak) shows bipartite profiles, about 265 x 210 x 200 A Characteristic views are interpreted as frontal, dorsal, and lateral projections of the particles, the latter is observed in two enantiomorphic forms Mitoribosome 50S subunits display rounded profiles bearing a conspicuous knoblike projection, reminiscent of the large bacterial subunit. The 36S subunits show a variety of angular profiles. Mitoribosomal subunits are subject to artifactual dimerization at high Mg²⁺ concentration Under these conditions, a supplementary 80S peak arises. Electron microscopic observation of the 80S peak reveals closely paired particles of the 50S type Buoyant density determinations after glutaraldehyde fixation show a single peak at ρ = 1.48 for mitoribosomes and 1.53 for cytoribosomes In the presence of ethylenediaminetetraacetate (EDTA), two species of RNA, 21S and 16S, are obtained from mitoribosomes, while 25S and 17S RNA are obtained from cytoribosomes It is established that the small and large RNA species are derived from the 36S and 50S subunits, respectively, by extraction of the RNA from each subunit The G + C content of the RNA is lower for mitoribosomes (33%) than for cytoribosomes (50%). Incubation of C utilis mitochondria with leucine-¹⁴C results in the labeling of 72S mitoribosomes. The leucine-¹⁴C incorporation is inhibited by chloramphenicol and resistant to cycloheximide Puromycin strips the incorporated radioactivity from the 72S mitoribosomes, which is consistent with the view that leucine-¹⁴C is incorporated into nascent polypeptide chains at the level of mitoribosomes     

Protein involved in the binding of dihydrostreptomycin to ribosomes of Escherichia coli

At increasing ammonium chloride concentrations, 30 S subunits on one hand, and 50 S subunits, 16 S BNA and 23 S RNA on the other hand, show a different behaviour with respect to dihydrostreptomycin binding. Within a wide range (10 to 250 mm) binding to 30 S subunits is not affected by NH₄Cl, whereas binding to 50 S and the RNAs decreases by increasing NH₄Cl concentrations. 30 S subunits lose more than 90% of their binding capacity by washing with 1.15 m-LiCl (SP_1.5³).The split proteins SP_1.15 were analysed by DEAE-cellulose chromatography and Sephadex G100 gel filtration. After reconstitution with the non-binding 2.0 core the proteins S3 and S5 can bind dihydrostreptomycin independently of each other; the S5-dependent binding is stimulated by S9 and S14 (S10). The Scatchard plot revealed 0.8 binding sites per 30 S subunit. We conclude that S3 and S5 are part of one binding site of dihydrostreptomycin.     

Interaction Involving Disulfide Bridges between Subunits of Soybean Seed Globulin and between Subunits of Soybean and Sesame Seed Globulins

Native subunit proteins of glycinin, the acidic and the basic subunits designated as AS₁₊₂, AS₂₊₃, AS₄, AS₅, and AS₆ and BS, respectively, were isolated by DEAE-Sephadex A-50 column chromatography in the presence of 6 m urea and 0.2 m 2-mercaptoethanol.

Reconstitution of intermediary subunits involving a disulfide bridge from native acidic and basic subunits was investigated. Formation of the intermediary subunit was observed in combinations between BS and each acidic subunit except AS₆. The yields of the reconstituted intermediary subunits differed from one another.

Further, formation of the intermediary complexes was observed when native acidic and basic subunits of soybean glycinin and sesame 13 S globulin, respectively (or reverse combinations), were mixed under reductively denatured condition and subjected to the reconstitution procedure. Considerring the overall evidence, we may conclude that the complexes are probably a hybrid intermediary subunit.