Activation of hepatocyte growth factor by proteolytic conversion of a single chain form to a heterodimer.

Hepatocyte growth factor (HGF) is a heterodimeric protein consisting of a heavy chain and a light chain held by a disulfide bond. These chains are produced by endoproteolytic processing from a single chain precursor. In this study, we examined whether the processing is a prerequisite for the mitogenic activity of HGF on hepatocytes in primary culture. Single chain HGF was proteolytically converted to the heterodimeric form during incubation with hepatocytes and was as mitogenic as the heterodimeric form. When the conversion was inhibited by serine-protease inhibitors, the mitogenic activity of single chain HGF was markedly reduced. Furthermore, a mutant resistant to the proteolytic processing, which was prepared by in vitro mutagenesis, completely lost the mitogenic activity. From these results, we concluded that the single chain form of HGF is endoproteolytically processed by a serine-protease and that this processing is a prerequisite for the mitogenic activity of HGF.     

The amino acid sequence of a lambda light chain presenting abnormal physicochemical and antigenic features

The amino acid sequence of the light chain of a human monoclonal IgA1 (Mem) was established, in part by analogy with already known sequences. By homology its variable part was shown to belong to the V lambda I subgroup while the isotype-associated amino acid residues characterized it as Mcg+, Kern+ and Oz-. The normal primary structure of this chain was in contrast to its abnormal physical and antigenic properties: (a) its apparent molecular mass estimated by SDS/polyacrylamide gel electrophoresis, by gel filtration chromatography and by gradient ultracentrifugation was found to be lower by approximately equal to 10% than the values (23.5 kDa) of 'normal' light chain used as controls; (b) the lambda I chain Mem, when tested in native state was not antigenically reactive. These abnormalities were reverted when the chain was treated with 8 M urea. These data suggest that the abnormal behaviour of lambda I chain Mem is at a conformational level.     

具有谷胱甘肽过氧化物酶活性的含硒单链抗体酶制备

 利用RT PCR从分泌有谷胱甘肽结合部位的单克隆抗体杂交瘤细胞株 2F3中 ,扩增出单抗重链可变区和轻链可变区基因 .经DNA测序后 ,用Linker(Gly4 Ser1) 3 构建成单链抗体 (scFv)表达载体pTMF scFv ,将重组质粒pTMF scFv转化到大肠杆菌BL2 1(DE3) ,实现了单链抗体的高效表达 .表达的单链抗体占菌体总蛋白 2 5%～ 30 % .该重组蛋白以包涵体形式存在 ,分子量为 30kD .经过金属螯合亲和层析纯化、复性和凝胶过滤纯化 ,得到电泳均一的单链抗体 .再经化学诱变 ,得到含硒单链抗体酶 ,其谷胱甘肽过氧化物酶活性为 330 0U μmol.采用荧光滴定法测定了单链抗体对谷胱甘肽的结合常数     

An autoantibody to single-stranded DNA: comparison of the three-dimensional structures of the unliganded Fab and a deoxynucleotide-Fab complex.

Crystal structures of the Fabs from an autoantibody (BV04-01) with specificity for single-stranded DNA have been determined in the presence and absence of a trinucleotide of deoxythymidylic acid, d(pT)3. Formation of the ligand-protein complex was accompanied by small adjustments in the orientations of the variable (VL and VH) domains. In addition, there were local conformational changes in the first hypervariable loop of the light chain and the third hypervariable loop of the heavy chain, which together with the domain shifts led to an improvement in the complementarity of nucleotide and Fab. The sugar-phosphate chain adopted an extended and "open" conformation, with the base, sugar, and phosphate components available for interactions with the protein. Nucleotide 1 (5'-end) was associated exclusively with the heavy chain, nucleotide 2 was shared by both heavy and light chains, and nucleotide 3 was bound by the light chain. The orientation of phosphate 1 was stabilized by hydrogen bonds with serine H52a and asparagine H53. Phosphate 2 formed an ion pair with arginine H52, but no other charge-charge interactions were observed. Insertion of the side chain of histidine L27d between nucleotides 2 and 3 resulted in a bend in the sugar-phosphate chain. The most dominant contacts with the protein involved the central thymine base, which was immobilized by cooperative stacking and hydrogen bonding interactions. This base was intercalated between a tryptophan ring (no. H100a) from the heavy chain and a tyrosine ring (no. L32) from the light chain. The resulting orientation of thymine was favorable for the simultaneous formation of two hydrogen bonds with the backbone carbonyl oxygen and the side chain hydroxyl group of serine L91 (the thymine atoms were the hydrogen on nitrogen 3 and keto oxygen 4).     

Monte Carlo simulations of a polymer chain conformation. The effectiveness of local moves algorithms and estimation of entropy

A linear chain on a simple cubic lattice was simulated by the Metropolis Monte Carlo method using a combination of local and non-local chain modifications. Kink-jump, crankshaft, reptation and end-segment moves were used for local changes of the chain conformation, while for non-local chain rearrangements the "cut-and-paste" algorithm was employed. The statistics of local micromodifications was examined. An approximate method for estimating the conformational entropy of a polymer chain, based on the efficiency of the kink-jump motion respecting chain continuity and excluded volume constraints, was proposed. The method was tested by calculating the conformational entropy of the undisturbed chain, the chain under tension and in different solvent conditions (athermal, theta and poor) and also of the chain confined in a slit. The results of these test calculations are qualitatively consistent with expectations. Moreover, the obtained values of the conformational entropy of self avoiding chain with ends fixed over different separations, agree very well with the available literature data.

Phosphorylation of myosin light chain by a protease-activated kinase from rabbit skeletal muscle

A protease-activated protein kinase that phosphorylates the P light chain of myosin in the absence of Ca2+ and calmodulin has been isolated from rabbit skeletal muscle. The enzyme has properties similar to protease-activated kinase I from rabbit reticulocytes [S. M. Tahara and J. A. Traugh (1981) J. Biol. Chem. 256, 11588-11564], which has been shown to phosphorylate the P light chain of myosin [P. T. Tuazon, J. T. Stull, and J. A. Traugh (1982) Biochem. Biophys. Res. Commun. 108, 910-917]. The protease-activated kinase from skeletal muscle has been partially purified by chromatography on DEAE-cellulose, phosphocellulose and hydroxyapatite. The enzyme phosphorylates histone as well as the P light chain of myosin following activation by proteolysis. Stoichiometric phosphorylation of myosin light chain was observed with the protease-activated kinase and myosin light chain kinase. The sites phosphorylated by the protease-activated kinase and myosin light chain kinase were examined by two-dimensional peptide mapping following chymotryptic digestion. The phosphopeptides observed with the protease-activated kinase were different from those obtained with the Ca2+-dependent myosin light chain kinase, indicating that the two enzymes phosphorylated different sites on the P light chain of skeletal muscle myosin. When actomyosin from skeletal muscle was examined as substrate, the P light chain was phosphorylated following activation of the protease-activated kinase by limited proteolysis.     

Purification and characterization of a novel intracellular acid proteinase from the plasmodia of a true slime mold, Physarum polycephalum

An acid proteinase was purified to apparent homogeneity from the plasmodia of a slime mold, Physarum polycephalum, by a combination of detergent extraction, acid precipitation, and column chromatographies on DEAE-Sephadex, hydroxylapatite, CM-Sephadex, and Sephadex G-100. The enzyme was shown to be composed of two polypeptide chains (a 31-kDa heavy chain and a 23-kDa light chain) cross-linked by disulfide bond(s). The NH2-terminal amino acid sequence of the heavy chain was determined to be Ala-Gly-Val- Asp-Gly-Tyr-Ile-Val-Pro-Tyr-Val-Ile-Phe-Asp-Leu-Tyr-Gly-Ile-Pro-Tyr and that of the light chain to be Ala-Glu-Pro-Pro-Ile. The heavy chain contained carbohydrate moiety composed of mannose, glucosamine, fucose, and glucose. The enzyme was optimally active at pH 1.7 toward hemoglobin as a substrate. Among the proteinase inhibitors tested only diazoacetyl-D,L-norleucine methyl ester, a typical aspartic proteinase inhibitor, inhibited the acid proteinase in the presence of cupric ions. It was insensitive to the other typical aspartic proteinase inhibitors, pepstatin A and 1,2-epoxy-3-(p-nitrophenoxy)propane. The enzyme hydrolyzed Lys-Pro-Ile-Glu-Phe(4-NO2)-Arg-Leu at the Phe-Phe(4-NO2) bond, but could not hydrolyze another synthetic pepsin-substrate, N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine. The enzyme showed a unique substrate specificity toward oxidized insulin B chain. The major cleavage sites were the bonds Gly8-Ser9, Leu11-Val12, Cya19-Gly20, and Phe24-Phe25, and the Gly8-Ser9 bond was most susceptible. These results indicate that the enzyme is a novel type of intracellular acid proteinase with a unique substrate specificity.     

Isolation and characterization of a cDNA coding for human myeloperoxidase

A cDNA encoding the carboxyl-terminal fragment of the human myeloperoxidase heavy chain was isolated and characterized. It was then used to determine the locations of the myeloperoxidase light and heavy chains in the polypeptide precursor. A cDNA library from poly(A)+ RNA from human leukemia HL-60 cells was constructed in pBR322 and screened by differential hybridization with enriched and depleted cDNA probes and then by hybridization with an oligonucleotide probe. A cDNA clone containing 1278 bp with an open reading frame of 474 bp and a 3' noncoding region of 804 bp was isolated. The amino acid sequence deduced from the nucleotide sequence consisted of 158 residues including a sequence of 14 amino acids known to be present in the heavy chain of the molecule. The cDNA also included a stop codon of TAG followed by a noncoding sequence that included a potential recognition site for polyadenylylation and a poly(A) tail. RNA transfer blot analysis with the cDNA probe indicated that myeloperoxidase mRNA was approximately 3.3 kb in length. In vitro translation of the mRNA selected by cDNA hybridization revealed preferential synthesis of a 74,000-Da polypeptide precursor that could be precipitated with anti-myeloperoxidase IgG. Antibodies specific for the heavy and light chains of myeloperoxidase were isolated from antiserum by affinity chromatography employing Sepharose columns covalently bound to the heavy or light chains. Antibodies specific for the light chain or the heavy chain readily precipitated the 74,000-Da precursor polypeptide. These results indicated that myeloperoxidase is synthesized as a single chain which undergoes processing into a light and heavy chain. Furthermore, the heavy chain of myeloperoxidase originates from the carboxyl terminus of the precursor polypeptide.     

Isolation and cell-free translation of a human immunoglobulin light chain messenger RNA.

Human myeloma cell line RPMI 8226 synthesizes and secretes a lambda immunoglobulin light chain. Total cellular RNA, obtained from cells grown in culture, was used for the isolation of poly(A)-containing mRNA by oligo(dT)-cellulose chromatography. The poly(A)-containing mRNA was translated in an Ehrlich ascites extract. Immunoprecipitation of the cell-free products showed that a polypeptide chain antigenically related to human lambda chain was synthesized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the cell-free product was larger than the secreted light chain. On tryptic digestion the cell-free product and the secreted light chain exhibited essentially identical peptide patterns except for an additional peptide derived from the cell-free product. We conclude that the lambda mRNA from this human myeloma cell line codes for a precursor to the secreted lambda chain as described for immunoglobulin mRNAs from murine plasmacytomas.     

Proteolytic dissection of a hapten binding site

IgG Gar, a human myeloma protein that binds riboflavin with a high affinity, was used to derive variable region fragments from the heavy chain and the light chain. Riboflavin binding ability of the active site generated by V(H) and light chain and the active site generated by V(H) and V(L) was compared to riboflavin binding by the F(ab) fragment. The riboflavin binding ability of the F(ab) fragment is the same as the intact molecule, while the binding ability of the active site formed by V(H) and light chain is lowered by two to three orders of magnitude, indicating that the removal of C(H1) domain decreases the interaction between riboflavin and the amino acids that is important in tight binding of riboflavin. Removal of the third hypervariable region and the constant region domain from the light chain further lowers the binding constant by one order of magnitude. The results indicate that the V(H) and V(L) segments of IgG Gar can reconstitute a riboflavin binding site. The decrease in affinity probably reflects a decrease in the rigidity with which the hypervariable loops are held together to place the contact amino acid residues in optimal contact with the hapten.     

Expression of a recombinant murine IgE in transfected myeloma cells

We constructed a recombinant gene encoding an immunoglobulin (Ig) heavy chain consisting of the variable region from the phosphorylcholine (PC)-specific secreting myeloma MOPC167 and the epsilon constant region from SJL mice. This gene, cloned into the shuttle vector pSV2gpt, was transfected into J558L myeloma cells, and stable transformants that expressed the epsilon gene were cloned. The IgE heavy chain in these transformants is associated with the endogenous lambda light chain and is secreted as an intact IgE molecule. However, the secreted IgE does not bind to PC conjugated to bovine serum albumin (PC-BSA). The MOPC167 kappa chain gene was cloned into the shuttle vector pSV2neo and was transfected into the epsilon heavy-chain transformant. Stable transformants were cloned that expressed both the epsilon heavy chain and the kappa light chain. IgE secreted from such a transformant was shown to bind to PC-BSA. Both types of secreted recombinant IgE bound to rat basophilic leukemia (RBL) cells, but only the IgE produced by the cell line transformed with the MOPC167 kappa gene could be cross-linked with PC-BSA to cause serotonin release.     

Novel structure of the N-acetylgalactosamine containing N-glycosidic carbohydrate chain of batroxobin, a thrombin-like snake venom enzyme.

The structure of the Asn-linked carbohydrate chain of batroxobin, a thrombin-like enzyme from Bothrops atrox moojeni snake venom, has been determined. The sugar chain was isolated from batroxobin by hydrazinolysis followed by pyridylamination (PA). The PA-oligosaccharide chain was purified by HPLC on an anion exchange or reverse phase columns, and its structure was examined by sequential exoglycosidase digestion, 600 MHZ 1H NMR spectroscopy and methylation analysis. The results indicate that the oligosaccharide chain has the following structure involving a novel linkage, NeuAc alpha 2----3GalNAc.     

Production of a non‐triple helical collagen α chain in transgenic silkworms and its evaluation as a gelatin substitute for cell culture

We generated transgenic silkworms that synthesized human type I collagen α1 chain [α1(I) chain] in the middle silk glands and secreted it into cocoons. The initial content of the recombinant α1(I) chain in the cocoons of the transgenic silkworms was 0.8%. The IE1 gene, a trans‐activator from the baculovirus, was introduced into the transgenic silkworm to increase the content of the chain. We also generated silkworms homozygous for the transgenes. These manipulations increased the α1(I) chain content to 8.0% (4.24 mg per cocoon). The α1(I) chain was extracted and purified from the cocoons using a very simple method. The α1(I) chain contained no hydroxyprolines due to the absence of prolyl‐hydroxylase activity in the silk glands. Circular dichroism analysis showed that the secondary structure of the α1(I) chain is similar to that of denatured type I collagen, demonstrating the absence of the triple helical structure. Human skin fibroblasts were seeded on the α1(I) chain‐coated dishes. The cells attached and spread, although at decreased chain concentrations the spreading rate was lower than that of the collagen and gelatin. Cynomolgus monkey embryonic stem cells cultured on the α1(I) chain‐coated dishes maintained an undifferentiated state after 30 passages, and their pluripotency was confirmed by teratoma formation in severe combined immunodeficient mice. These results show that the recombinant human α1(I) chain is a promising candidate biomaterial as a high‐quality and safe gelatin substitute for cell culture. Biotechnol. Bioeng. 2010;106: 860–870. © 2010 Wiley Periodicals, Inc.     

Phosphorylation of a clathrin light chain of coated vesicles in the presence of histones

Coated vesicles isolated from bovine brain contained a protein kinase(s) which phosphorylated phosvitin and an endogenous protein with a molecular weight (Mr) of 48,000. A clathrin light chain (Mr 33,000), a constituent of the coat structure of the coated vesicles, was also phosphorylated when histone was added to the incubation medium. The clathrin light chain was phosphorylated with GTP as well as ATP as the phosphoryl donor. The phosphorylation reaction was inhibited by heparin. An additional 1.35 mol of PO4/mol was incorporated into the clathrin light chain which had contained approximately 1.5 mol of PO4/mol when the coated vesicles were incubated with ATP, Mg2+, and histone. Phosphoamino acid determination revealed the presence of 32P-phosphorylated threonine and serine in phosvitin, threonine in the endogenous protein (Mr 48,000) and serine in the clathrin light chain (Mr 33,000).     

Facile synthesis of a D-galactono-1,6-lactone derivative, a precursor of a copolyester

2,3,4,6-Tetra-O-methyl-d-galactonic acid (5) was readily prepared from d-galactono-1,4-lactone (1) in 47% yield. The sequence involves tritylation of HO-6 of 1, followed by O-permethylation and deprotection. Lactonization of 5 led to the per-O-methyl-d-galactono-1,6-lactone, which was copolymerized with epsilon-caprolactone by ring-opening polymerization catalyzed by scandium triflate. The incorporation of the sugar comonomer into the polyester chain was about 10%.     

Isolation of an active variable-domain fragment from a homogeneous rabbit antibody heavy chain. Physiochemical and immunological properties.

A fragment corresponding to most of the variable domain of the rabbit heavy chain (VH) was obtained by tryptic digestion of the midly reduced and aminoethylated heavy chain from rabbit antibody 3T72. The domain size peptide was purified by gel filtration and shown to extend between residues 11(Leu) and 122(Lys) of the heavy chain by sequence analysis. The molecular size of the fragment (approximately 11 000) was determined by gel filtration under denaturing conditions. Under nondenaturing conditions (20 mM sodium acetate, pH 5.5, 0.1 M NaCl), however, the fragment exists as a mixture of monomeric and dimeric species. The varable-domain fragment retains the allotypic determinants of the heavy chain (a1), as shown by double diffusion on agar plates and radioimmunoassay. Upon recombination of the heavy-chain variable-domain fragment with its homologous light chain, partial recovery of specific binding activity toward the SIII polysaccharide antigen was demonstrated. The method reported here is reproducible (with yields varying between 40 and 60%) and may provide a general method for obtaining the variable region of the heavy chain for antigen binding and allotypic and amino acid sequence studies.     

Evidence for a nucleation step in a lipid-protein interaction-kinetics of the incorporation of polymyxin into phosphatidic acid bilayer vesicles

Incubation of 8-anilino-1-naphthalene sulfonic acid with ricin and its isolated A and B polypeptide chains showed an increase in fluorescence at 470 nm. The A chain induced more fluorescence enhancement than either ricin or ricin B chain. The addition of B chain to A chain resulted in decreased fluorescence enhancement which was pH dependent. Sephadex gel filtration showed that A and B chain efficiently reassociated and the reassociation was not dependent on formation of the interchain disulfide bond and could not be prevented by high salt concentration.     

Identification of a soluble GM-CSF binding protein in the supernatant of a human choriocarcinoma cell line.

We identified two forms of the receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) made by the human choriocarcinoma cell line JEG-3 using an affinity-labeling technique. The protein was identified in the detergent-extract was 78 kDa, very similar to that of the membrane-bound GM-CSF receptor alpha chain expressed in a wide variety of hematopoietic and nonhematopoietic cells, including JEG-3. In contrast, a 62-kDa GM-CSF binding protein, or the soluble GM-CSF receptor, was identified in the supernatant of JEG-3 cells. Utilizing the same affinity labeling technique, we did not detect the soluble GM-CSF binding protein in the supernatant of several hematopoietic cell lines, such as U-937 and KG-1, which express membrane bound alpha chain as well as beta chain. The 62-kDa soluble GM-CSF receptor is produced in abundant amounts by JEG-3, but in very small amounts, if any, by hematopoietic cell lines.     

Metabolism of a novel side chain modified Delta8(14)-15-ketosterol, a potential cholesterol lowering drug: 28-hydroxylation by CYP27A1

The synthetic inhibitors of sterol biosynthesis, 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one and 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one, are of interest as potential cholesterol lowering drugs. Rapid metabolism of synthetic 15-ketosterols may lead to a decrease, or loss, of their potency to affect lipid metabolism. 3beta-Hydroxy-5alpha-cholest-8(14)-en-15-one is reported to be rapidly side chain oxygenated by rat liver mitochondria. In an attempt to reduce this metabolism, the novel side chain modified 15-ketosterol 3beta-Hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one was synthesized. We have examined the metabolism by recombinant human CYP27A1 of this novel side chain modified 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one and compared the rate of metabolism with that of the previously described 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one. Both sterols were found to be efficiently metabolized by recombinant human CYP27A1. None of the two 15-ketosterols was significantly metabolized by microsomal 7alpha-hydroxylation. Interestingly, CYP27A1-mediated product formation was much lower with the side chain modified 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one than with the previously described 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one. A surprising finding was that this novel side chain modified sterol was metabolized mainly in the C-28 position by CYP27A1. The data on 28-hydroxylation by human CYP27A1 provide new insights on the catalytic properties and substrate specificity of this enzyme. The finding that 3beta-hydroxy-24S-methyl-5alpha-cholesta-8(14),22-dien-15-one with a modified side chain is metabolized at a dramatically slower rate than the previously described 15-ketosterol with unmodified side chain may be important for future development of synthetic cholesterol lowering sterols.