Boar proacrosin is a single-chain molecule which has the N-terminus of the acrosin A-chain (light chain)

Boar proacrosin was isolated from spermatozoa by a novel procedure under conditions preventing proenzyme activation. The spermatozoal extract was fractionated by gel filtration and reversed-phase FPLC, all in acidic solutions. Isolated proacrosin had a molecular mass of 55/53 kDa (doublet) and was devoid of amidolytic activity. Its single N-terminal sequence corresponded to that of the 23-residue acrosin A-chain and continued with that of the acrosin B-chain. Autoactivation at pH 7.8 did not influence the molecular mass. However, activated material contained two parallel N-terminal sequences, those of the A- and B-chain. Thus, activation of proacrosin is analogous to that of other serine proteinase proenzymes.     

Human low-molecular-mass kininogen. Amino-acid sequence of the light chain; homology with other protein sequences

The chemical structure of the polysaccharide moiety of the lipopolysaccharide Rhodopseudomonas sphaeroides ATCC 17023 was established. Mild acetic acid hydrolysis of isolated lipopolysaccharide, followed by preparative high-voltage paper electrophoresis afforded three oligosaccharides. They were characterized by chemical and physicochemical studies to be: GlcA(alpha 1----4)dOclA8P, Thr(6') GlcA(alpha 1----4)GlcA and GlcA(alpha 1----4)dOclA, where GlcA is D-glucuronic acid and dOc1A is 3-deoxy-D-manno-octulosonic acid. Carboxyl-reduction of the lipopolysaccharide followed by acid hydrolysis gave a trisaccharide: GlcA(alpha 1----4)Glc(alpha 1----4)Glc, showing the presence of three residues of glucuronic acids in the O-specific chain and indicating that only two of them are reducible by NaBH4. The linkage between the polysaccharide and lipid A was shown to be through a single 1,4-linked residue of dOc1A attached by a 2,6'-linkage to the lipid A moiety.     

Glutamyl-tRNA synthetase of Escherichia coli. Isolation and primary structure of the gltX gene and homology with other aminoacyl-tRNA synthetases

The gltX gene encoding the glutamyl-tRNA synthetase of Escherichia coli and adjacent regulatory regions was isolated and sequenced. The structural gene encodes a protein of 471 amino acids whose molecular weight is 53,810. The codon usage is that of genes highly expressed in E. coli. The amino acid sequence deduced from the nucleotide sequence of the gltX gene was confirmed by mass spectrometry of large peptides derived from the glutamyl-tRNA synthetase. The observed peptides confirm 73% of the predicted sequence, including the NH2-terminal and the COOH-terminal segments. Sequence homology between the glutamyl-tRNA synthetase and other aminoacyl-tRNA synthetases of E. coli was found in four segments. Three of them are aligned in the same order in all the synthetases where they are present, but the intersegment spacings are not constant; these ordered segments may come from a progenitor to which other domains were added. Starting from the NH2-end, the first two segments are part of a longer region of homology with the glutaminyl-tRNA synthetase, without need for gaps; its size, about 100 amino acids, is typical of a single folding domain. In the first segment, containing sequences homologous to the HIGH consensus, the homology is consistent with the following evolutionary linkage: gltX----glnS----metS----ileS and tyrS.     

The primary structure of crotalase,a thrombin-like venom enzyme,exhibits closer homology to kallikrein than to other serine proteases

Amino acid sequences of crotalase, totaling 98 residues or about 37% of the molecule, were determined by Edman degradation and were compared with the published sequences of nine serine proteases. Homologous alignment could be found for all crotalase sequences except one decapeptide. Comparison between crotalase and porcine pancreatic kallikrein showed the largest number of identical amino acids (36%). This finding has led to experiments which demonstrate that crotalase has specific enzymatic properties resembling kallikrein.     

Isolation and primary structure of the CCI papain-like cysteine proteinases from the latex of Carica candamarcensis hook.

The dried latex of the mountain papaya, Carica candamarcensis, was chromatographed on CM-Sephadex C50, giving rise to three peaks (CCI, CCII and CCIII) with amidase activity on N-alpha-benzoyl-DL-arginine-4-nitroanilide. The less basic, most active, peak, CCI, was separated into two components, CCIa and CCIb, by reverse-phase HPLC under denaturing conditions. The primary structures of CCIa and CCIb are presented. They were deduced from sequence analysis of the whole proteins and peptides resulting from enzymatic digestions. Both proteinases are made of 213 amino acid residues, CCIb sharing 88-89% similarity with the three subvariants (G90/R212, E90/R212, E90/K212) of CCIa. 139-140 amino acid residues (65.8%) of CCIa and 141 residues (66.5%) of CCIb are common to papain. The seven cysteine residues are aligned with those of papain and the catalytic triad (Cys25, His159, Asn175) of all cysteine peptidases of the papain family is conserved. The similarity with the other cysteine proteases from Carica papaya is discussed.     

Phosphorylation and dephosphorylation of a light chain of the chicken gizzard myosin molecule.

Chicken gizzard myosin was incubated with ATP and/or "native" tropomyosin (NTM) of gizzard muscle in the presence or absence of calcium ions. One of the two light chains of the myosin molecule was phosphorylated in the presence of Ca, but not in its absence. The phosphorylated gizzard myosin was dephosphorylated by a crude preparation of myosin light-chain phosphatase obtained from gizzard muscle. In a superprecipitation test in the presence of EGTA, actomyosin reconstituted from dephosphorylated gizzard myosin did not superprecipitate, whereas actomyosin reconstituted from phosphorylated gizzard myosin showed superprecipitation activity which was inhibited by skeletal NTM and reactivated by Ca.     

Variability of the canonical loop conformations in serine proteinases inhibitors and other proteins

Canonical loops of protein inhibitors of serine proteinases occur in proteins having completely different folds. In this article, conformations of the loops have been analyzed for inhibitors belonging to 10 structurally different families. Using deviation in C_α-C_α distances as a criterion for loop similarity, we found that the P3-P3′ segment defines most properly the length of the loop. When conformational differences among loops of individual inhibitors were compared using root mean square deviation (rmsd) in atomic coordinates for all main chain atoms (Δr method) and rmsd operating in main chain torsion angles (Δt method), differences of up to 2.1 Å and 72.3°, respectively, were observed. Such large values indicate significant conformational differences among individual loops. Nevertheless, the overall geometry of the inhibitor-proteinase interaction is very well preserved, as judged from the similarity of C_α-C_α distances between C_α of catalytic Ser and C_α of P3-P3′ residues in various enzyme-inhibitor complexes. The mode of interaction is very well preserved both in the chymotrypsin and subtilisin families, as distances calculated for subtilisin-inhibitor complexes are almost always within the range of those for chymotrypsin-inhibitor complexes. Complex formation leads to conformational changes of up to 160° for χ₁ angle. Side chains of residue P1 and P2′ adopt in different complexes a similar orientation (χ₁ angle = −60° and −180°, respectively). To check whether the canonical conformation can be found among non–proteinase-inhibitor Brookhaven Protein Data Bank structures, two selection criteria—the allowed main chain dihedral angles and C_α-C_α distances for the P3-P3′ segment—were applied to all these structures. This procedure detected 132 unique hexapeptide segments in 121 structurally and functionally unrelated proteins. Partial preferences for certain amino acids occurring at particular positions in these hexapeptides could be noted. Further restriction of this set to hexapeptides with a highly exposed P1 residue side chain resulted in 40 segments. The possibility of complexes formation between these segments and serine proteinases was ruled out in molecular modeling due to steric clashes. Several structural features that determine the canonical conformation of the loop both in inhibitors and in other proteins can be distinguished. They include main chain hydrogen bonds both within the P3-P3′ segment and with the scaffold region, P3-P4 and P3′-P4′ hydrophobic interactions, and finally either hydrophobic or polar interactions involving the P1′ residue. Proteins 32:459–474, 1998. © 1998 Wiley-Liss, Inc.     

The complete primary structure of three isoforms of a boar sperm-associated acrosin inhibitor.

Acrosin inhibitors of seminal vesicle origin, after binding to their acceptor molecules on the anterior part of ejaculated sperm, are thought to be important capacitation factors, protecting zona binding sites during sperm uterine passage, and then dissociating to allow sperm binding to the zona pellucida of the oocyte. Each species so far tested possess an heterogeneous population of isoinhibitors which may display overlapping but not identical biological functions. Here we report the complete primary structure of three isoforms of a boar sperm-associated acrosin inhibitor, whose sequences are 90% identical to the seminal plasma counterpart. Despite this high analogy, the differences between the sperm-associated and the seminal plasma inhibitors may confer to them different physico-chemical properties which are postulated to be of functional importance.     

Benzamidine as a spectroscopic probe for the primary specificity subsite of trypsin-like serine proteinases

Summary Formation and dissociation of the benzamidine: -trypsin adduct is accompanied by reversible spectral changes in the ultraviolet region (between 230 and 300 nm). The pH-independent difference extinction coefficient of the adduct (benzamidine: -trypsin complex minus the free proteinase) is 1.75 mM^–1 cm^–1 at 248 nm. This signal can be used in studies of inhibitor and substrate binding by rapid kinetic techniques. Therefore, following the spectral changes associated with the displacement of benzamidine from the primary specificity subsite, the kinetics of the -trypsin: BPTI complex formation were investigated between pH 2.9 and 7.6 (I = 0.1 M) at 21 ± 0.5 °C. Under all the experimental conditions the -trypsin: BPTI complex formation, examined by benzamidine displacement experiments, may be described in terms of a simple competition event. On the other hand, the very same reaction followed by displacement of another spectroscopic probe, proflavine, appears to involve the ternary proflavine: -trypsin:BPTI adduct (7). The difference between the kinetic processes of -trypsin: BPTI complex formation, observed by using benzamidine and proflavine as reaction indicators, suggests that the two dye molecules bind at non-coincident regions of the proteinase active center. The advantages in using benzamidine as a sensitive probe specific for the S₁ subsite of the recognition center of trypsin-like proteinases, as compared to proflavine, are emphasized.Abbreviations BPTI bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor) - pNGB p-nitrophenyl-p-guanidinobenzoate - NaDodSO₄ sodium dodecyl sulfate     

The two-chain coiled-coil molecule of native epidermal keratin intermediate filaments is a type I-type II heterodimer.

The composition of the two-chain coiled-coil molecule of murine epidermal keratin intermediate filaments (KIF) containing keratins 1 (type II) and 10 (type I) has been explored using native-type KIF as well as KIF reassembled in vitro from protein dissolved in urea solutions or from mixtures of 3H-labeled and unlabeled purified chains. By use of cross-linking, high resolution polyacrylamide gel electrophoresis and blotting for 3H-labeled keratins or with an anti-mouse keratin 10 antibody, it was found that individual keratin chains form type I or type II homodimers and homotetramers in solution that do not assemble into KIF in vitro. When mixed in urea solutions of 5 M or greater, such homo-oligomers rapidly rearrange into mostly heterodimers and heterotetramers that support filament assembly. On the other hand, prekeratin, isolated from newborn mouse epidermis with 0.1 M sodium citrate buffer, pH 2.6, under conditions that do not dissociate the native coiled-coil molecule, consists exclusively of type I-II heterodimers and heterotetramers. It is necessary to dissolve prekeratin in 8-9.5 M urea for several hours in order to dissociate the native heterodimer molecule and incorporate tracer amounts of a single 3H-labeled keratin chain. These data establish that native KIF consist of heterodimer coiled-coil molecules. Furthermore, heterodimers are much more stable than homodimers and are the favored form of association in solution. However, some homodimers (10-30% of total) always form after dissolution in concentrated urea and can be assimilated into KIF during reassembly in vitro. The isolation of alpha-helix-enriched dimer particles from the 2B region of the rod domains upon limited proteolysis confirmed the presence of mostly heterodimer and some homodimer molecules in reassembled KIF. These properties of keratin chains in urea solutions hereby clarify a number of conflicting reports in the literature concerning the composition of the coiled-coil molecule. The presence of some homo-oligomeric species in reassembled KIF correlates with earlier observations of polymorphism as well as stoichiometry.     

Isolation and thermal stability studies of two novel serine proteinases from the fungus Tritirachium album Limber.

A number of serine proteinases are secreted into the culture medium when Tritirachium album Limber is supplied with protein as the only nitrogen source. From this population of proteinases, we have isolated two novel proteolytic enzymes, designated as proteinase R and T. We have compared the thermal stability of these two proteinases with that of subtilisin BPN' and proteinase K. Both of these proteinases were thermally stable in the absence of detergents in buffers of low (4.0) and high (10.0) pH. The thermal stability of proteinase T was not affected by the presence of 1.0% SDS either at pH 8.0 or 10.0 in contrast to proteinase R which became heat labile. At low pH, the presence of SDS was detrimental to the stability of all the proteinases.     

Three-dimensional structure of a light chain dimer crystallized in water. Conformational flexibility of a molecule in two crystal forms

The three-dimensional structure of an immunoglobulin light chain dimer (Mcg) crystallized in deionized water (orthorhombic form) was determined at 2.0 A resolution by phase extension and crystallographic refinement. This structure was refined side-by-side with that of the same molecule crystallized in ammonium sulfate (trigonal form). The dimer adopted markedly different structures in the two solvents. "Elbow bend" angles between pseudo 2-fold axes of rotation relating pairs of "variable" (V) and "constant" (C) domains were found to be 132 degrees in the orthorhombic form and 115 degrees in the trigonal form. Modes of association of the V domains and, to a lesser extent, the pairing interactions of the C domains were different in the two structures. Alterations in the V domain pairing were reflected in the shapes of the binding regions and in the orientations of the side-chains lining the walls of the binding sites. In the trigonal form, for instance, the V domain interface was compartmentalized into a main binding cavity and a deep pocket, whereas these spaces were continuous in the orthorhombic structure. Patterns of ordered water molecules were quite distinct in the two crystal types. In some cases, the solvent structures could be correlated with conformational changes in the proteins. For example, close contacts between V and C domains of monomer 1 of the trigonal form were not retained in orthorhombic crystals. Ordered water molecules filled the space created when the two domains moved apart.     

The serpin SQN-5 is a dual mechanistic-class inhibitor of serine and cysteine proteinases

SQN-5 is a mouse serpin that is highly similar to the human serpins SCCA1 (SERPINB3) and SCCA2 (SERPINB4). Previous studies characterizing the biochemical activity of SQN-5 showed that this serpin, like SCCA2, inhibited the chymotrypsin-like enzymes mast cell chymase and cathepsin G. Using an expanded panel of papain-like cysteine proteinases, we now show that SQN-5, like SCCA1, inhibited cathepsins K, L, S, and V but not cathepsin B or H. These interactions were characterized by stoichiometries of inhibition that were nearly 1:1 and second-order rate constants of >10(4) M(-1) s(-1). Reactive site loop (RSL) cleavage analysis showed that SQN-5 employed different reactive centers to neutralize the serine and cysteine proteinases. To our knowledge, this is the first serpin that serves as a dual inhibitor of both chymotrypsin-like serine and the papain-like cysteine proteinases by employing an RSL-dependent inhibitory mechanism. The ability of serpins to inhibit both serine and/or papain-like cysteine proteinases may not be a recent event in mammalian evolution. Phylogenetic studies suggested that the SCCA and SQN genes evolved from a common ancestor approximately 250-280 million years ago. When the fact that mammals and birds diverged approximately 310 million years ago is considered, an ancestral SCCA/SQN-like serpin with dual inhibitory activity may be present in many mammalian genomes.     

蛇毒丝氨酸蛋白酶结构与功能关系

蛇毒丝氨酸蛋白酶(snake venome serine proteinases,SVSP)是分布于多种蛇毒中的一类蛋白质水解酶,它们与凝血及纤溶系统关系密切,具有一定的临床应用价值。本文主要介绍了几种空间结构已知的SVSP在结构上的特征,以及由此而产生的底物特异性及生化特性的差异,显示了天然SVSP中存在着与功能相关的结构变异多样性,这为进一步开发及医学利用SVSP提供了有益的信息。     

Molecular reconstruction and homology modelling of the catalytic domain of the common ancestor of the haemostatic vitamin-K-dependent serine proteinases

The vitamin-K-dependent serine proteinases of coagulation have evolved by a process of gene duplication and divergence, acquiring along the way a considerable degree of functional diversity that has equipped them for their different roles in haemostasis. The cDNA sequences encoding the catalytic domains of the early mammalian ancestors of five vitamin-K-dependent factors (factors VII, IX and X, protein C and prothrombin) were reconstructed by employing cDNA sequence data from a range of extant mammals and by using established phylogenies. The cDNA sequence of the putative common ancestor of these early mammalian proteins was then reconstructed from the five sequences by using a deduced phylogeny that was different in a number of respects from those previously proposed. Factor IX is proposed to have branched off early on, followed by protein C and prothrombin and finally factors VII and X. Significant differences in mutation rates were observed between proteins within a species; factor IX exhibited a lower mutation rate than the other proteins, consistent with its early emergence. Differences in mutation rates were also observed between species for a given protein and these exhibited an inverse correlation with generation time. A biophysically plausible structure for the ancestral vitamin-K-dependent factor protein was constructed by comparative methods. Studies of the functional architecture of this model provide new insights into the evolution of protein-binding specificity in this family of proteins. Received: 3 April 1996 / Revised: 24 April 1996