Non-specific depolymerization of chitosan by pronase and characterization of the resultant products.

Pronase (type XXV serine protease from Streptomyces griseus) efficiently depolymerizes chitosan, a linear beta-->1,4-linked polysaccharide of 2-amino-deoxyglucose and 2-amino-2-N-acetylamino-D-glucose, to low-molecular weight chitosans (LMWC), chito-oligomers (degree of polymerization, 2-6) and monomer. The maximum depolymerization occurred at pH 3.5 and 37 degrees C, and the reaction obeyed Michaelis-Menten kinetics with a Km of 5.21 mg.mL(-1) and Vmax of 138.55 nmoles.min(-1).mg(-1). The molecular mass of the major product, LMWC, varied between 9.0 +/- 0.5 kDa depending on the reaction time. Scanning electron microscopy of LMWC showed an approximately eightfold decrease in particle size and characterization by infrared spectroscopy, circular dichroism, X-ray diffractometry and 13C-NMR revealed them to possess a lower degree of acetylation, hydration and crystallinity compared to chitosan. Chitosanolysis by pronase is an alternative and inexpensive method to produce a variety of chitosan degradation products that have wide and varied biofunctionalities.     

Sites of cleavage of glucagon by insulin-glucagon protease.

To study the mechanism of degradation of glucagon with purified insulin-glucagon protease, glucagon was reacted with the enzyme at various times of incubation. The proteolysis was followed by the production of flourescamine-reacting material as well as reaction with dansyl chloride, cleavage by acid hydrolysis, and identification by thin layer chromatography. For quantitative measurement of the degradation products, [¹⁴C] dansyl derivatives were produced, identified by autoradiography, and counted. In the degradation products in addition to histidine, the dansyl derivatives of tyrosine, phenylalanine, two leucines, alanine and lysine were identified. For comparison, glucagon was also reacted with chymotrypsin and the degradation products consisted of threonine, serine, two leucines and valine. Thus, insulin-glucagon protease degrades glucagon in a manner distinct from that of chymotrypsin.     

微生物降解4-羟基苯甲酸的研究进展

4-羟基苯甲酸(4HBA)是在自然界中广泛存在的芳香族化合物,也是很多天然产物和人工合成化合物的中间代谢产物。4HBA的代谢途径有原儿茶酸开环途径、脱碳酸途径和厌氧微生物的苯甲酰-CoA还原途径,以及尚未完全阐明的龙胆酸开环途径。从4HBA转化为龙胆酸的过程包含NIH重排反应步骤,本综述重点介绍NIH重排反应的研究进展并初步介绍了涉及4HBA降解过程中的酶。在本综述中,结合我们的研究工作介绍了一个嗜热Bacillus sp.B1菌株降解4HBA等芳香族化合物的代谢途径,最后对4HBA降解过程中的NIH重排反应研究进行了展望。     

Total amino acid stabilization during cell-free protein synthesis reactions

Limitations in amino acid supply have been recognized as a substantial problem in cell-free protein synthesis reactions. Although enzymatic inhibitors and fed-batch techniques have been beneficial, the most robust way to stabilize amino acids is to remove the responsible enzymatic activities by genetically modifying the source strain used for cell extract preparation. Previous work showed this was possible for arginine, serine, and tryptophan, but cysteine degradation remained a major limitation in obtaining high protein synthesis yields. Through radiolabel techniques, we confirmed that cysteine degradation was caused by the activity of glutamate-cysteine ligase (gene gshA) in the cell extract. Next, we created Escherichia coli strain KC6 that combines a gshA deletion with previously described deletions for arginine, serine, and tryptophan stabilization. Strain KC6 grows well, and active cell extract can be produced from it for cell-free protein synthesis reactions. The extract from strain KC6 maintains stable amino acid concentrations of all 20 amino acids in a 3-h batch reaction. Yields for three different proteins improved 75-250% relative to cell-free expression using the control extract.     

Sequence of the sites phosphorylated by protein kinase C in the smooth muscle myosin light chain

We have determined the sequence of the sites phosphorylated by protein kinase C in the turkey gizzard smooth muscle myosin light chain. In contrast to previous work (Nishikawa, M., Hidaka, H., and Adelstein, R. S. (1983) J. Biol. Chem. 258, 14069-14072), two-dimensional tryptic peptide maps of both heavy meromyosin and the isolated myosin light chain showed two major phosphopeptides, one containing phosphoserine and the other phosphothreonine. We have purified the succinylated tryptic phosphopeptides using reverse phase and DEAE high pressure liquid chromatography. The serine-containing peptide, residues 1-4 (Ac-SSKR), is the NH2-terminal peptide. The phosphorylated serine residue may be either serine 1 or serine 2. The threonine-containing peptide, residues 5-16, yielded the sequence AKAKTTKKRPQR. Analysis of the yields and radioactivity of the products from automated Edman degradation showed that threonine 9 is the phosphorylation site.     

Chemical mechanism of the serine acetyltransferase from Haemophilus influenzae

The pH dependence of kinetic parameters was determined in both reaction directions to obtain information about the acid-base chemical mechanism of serine acetyltransferase from Haemophilus influenzae (HiSAT). The maximum rates in both reaction directions, as well as the V/K(serine) and V/K(OAS), decrease at low pH, exhibiting a pK of approximately 7 for a single enzyme residue that must be unprotonated for optimum activity. The pH-independent values of V(1)/E(t), V(1)/K(serine)E(t), V/K(AcCoA)E(t), V(2)/E(t), V(2)/K(OAS)E(t), and V/K(CoA)E(t) are 3300 +/- 180 s(-1), (9.6 +/- 0.4) x 10(5) M(-1) s(-1), 3.3 x 10(6) M(-1) s(-1), 420 +/- 50 s(-1), (2.1 +/- 0.5) x 10(4) M(-1) s(-1), and (4.2 +/- 0.7) x 10(5) M(-1) s(-1), respectively. The K(i) values for the competitive inhibitors glycine and l-cysteine are pH-independent. The solvent deuterium kinetic isotope effects on V and V/K in the direction of serine acetylation are 1.9 +/- 0.2 and 2.5 +/- 0.4, respectively, and the proton inventories are linear for both parameters. Data are consistent with a single proton in flight in the rate-limiting transition state. A general base catalytic mechanism is proposed for the serine acetyltransferase. Once acetyl-CoA and l-serine are bound, an enzymic general base accepts a proton from the l-serine side chain hydroxyl as it undergoes a nucleophilic attack on the carbonyl of acetyl-CoA. The same enzyme residue then functions as a general acid, donating a proton to the sulfur atom of CoASH as the tetrahedral intermediate collapses, generating the products OAS and CoASH. The rate-limiting step in the reaction at limiting l-serine levels is likely formation of the tetrahedral intermediate between serine and acetyl-CoA.     

Structure and proteolysis of the growth hormone receptor on rat hepatocytes

125I-Labeled human growth hormone is isolated in high molecular weight (Mr) (300,000, 220,000, and 130,000) and low molecular weight complexes on rat hepatocytes after affinity labeling. The time-dependent formation of low molecular weight complexes occurred at the expense of the higher molecular weight species and was inhibited by low temperature or inhibitors of serine proteinases. Exposure to reducing conditions induced loss of Mr 300,000 and 220,000 species and augmented the amount of Mr 130,000 complexes. The molecular weight of growth hormone (22,000) suggests that binding had occurred with species of Mr 280,000, 200,000, and 100,000. Two-dimensional gel electrophoresis demonstrated that the 100,000-dalton receptor subunit is contained in both the 280,000- and 200,000-dalton species. Reduction of interchain disulfide bonds in the growth hormone receptor did not alter its elution from gel filtration columns, but intact, high molecular weight receptor constituents were separated from lower molecular weight degradation products. Digestion of affinity-labeled growth hormone-receptor complexes with neuraminidase increased the mobility of receptor constituents on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These observations show that the growth hormone receptor is degraded by hepatic serine proteinases to low molecular weight degradation products which can be separated from intact receptor by gel filtration. Intact hormone-receptor complexes are aggregates of 100,000-dalton sialoglycoprotein subunits held together by interchain disulfide bonds and by noncovalent forces.     

Molecular cloning and fibrin(ogen)olytic activity of a bumblebee (Bombus hypocrita sapporoensis) venom serine protease

Although several bee venom serine protease genes have been previously described, fibrin(ogen)olytic activity of these serine proteases has been reported for only two bumblebees to date, Bombus ignitus and B. terrestris. Here, we cloned venom serine proteases from the other bumblebee species, B. hypocrita sapporoensis and B. ardens ardens. The venom serine protease genes of B. h. sapporoensis and B. a. ardens consist of 358 amino acids and 357 amino acids, respectively. We compared the predicted mature protein sequences of these serine protease genes to those previously reported for other bees. A phylogenetic analysis shows that B. h. sapporoensis venom serine protease is further immediately close to B. ignitus and B. terrestris venom serine proteases, excluding the venom serine protease of B. a. ardens. Using B. h. sapporoensis venom serine protease (Bs-VSP), we identified that Bs-VSP acts as a fibrin(ogen)olytic enzyme. We also found that Bs-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. Our results further define roles for bumblebee venom serine proteases as fibrin(ogen)olytic agents.     

Comb-like derivatives of amylose having (1-6)-linked malto-oligosaccharide side-chains.

The preparation of acetylated glycosyl bromide derivatives of the higher malto-oligosaccharides was studied by using beta-cyclodextrin or linear malto-oligosaccharides of d.p. 6 and 7. The products were treated with 2,3-di-O-phenylcarbamoyl-6O-tritylamylose in the presence of silver perchlorate (reaction A), and with 2,3-di-O-phenylcarbamoylamylose in the presence of mercuric cyanide and mercuric bromide (reaction B). After removal of the substituents, the branched molecules were characterized by their iodine-binding properties, beta-amylolytic degradation, and their priming activity in phosphorolytic synthesis. The distance between the branch points of the backbone chain was 25-55 and 100-150 units in the products from reactions A and B, respectively. Thus, the frequency of branching was considerably lower than in the comb-like molecules having D-glucose side-chains previously described.     

Analysis of fluorogenic Smith degradation products of 7-(1,3-disulfonaphtyl)amino-disaccharides for linkage position analysis of carbohydrates

The linkage position of a glycosidic bond to the reducing-end residue of a pyridylamino (PA-) sugar can be determined sensitively by Smith degradation and HPLC [K. Omichi and S. Hase, (1994) J. Biochem. 115, 429-434]. With the aim of enhancing the sensitivity of this method of linkage position analysis to the fmol-level, use of the 7-(1,3-disulfonaphtyl)amino (DSNA-) group instead of the PA-group as a fluorescent tag was examined. Smith degradation of DSNA-disaccharides with a DSNA-hexose, DSNA-N-acetylglucosamine, or DSNA-N-acetylgalactosamine reducing-end residue was carried out. HPLC and FAB-MS of the fluorogenic Smith degradation products showed that the DSNA-group was stable under the Smith degradation reaction conditions, and that the reaction proceeded in a manner similar to that using PA-disaccharides to give the predicted products. Fluorogenic Smith degradation products specific to the glycosidic linkage position were well separated by reversed-phase HPLC, and were easily assignable by comparing the HPLC elution positions with those of standard compounds. The method was successfully applied to analyzing the structure of an N-linked sugar chain.     

Secondary substrate-binding exosite in the serine protease domain of activated protein C important for cleavage at Arg-506 but not at Arg-306 in factor Va

Proteolytic inactivation of activated factor V (FVa) by activated protein C (APC) is a key reaction in the regulation of hemostasis. We now demonstrate the importance of a positive cluster in loop 37 of the serine protease (SP) domain of APC for the degradation of FVa. Lysine residues in APC at positions 37, 38, and 39 form a secondary binding site for FVa, which is important for cleavage of FVa at Arg-506 while having no effect on Arg-306 cleavage. In contrast, topological neighbors Lys-62, Lys-63, and Arg-74 in APC appear of minor importance in FVa degradation. This demonstrates that secondary binding exosites of APC specifically guide the proteolytic action of APC, resulting in a more favorable degradation of the 506-507 peptide bond as compared with the 306-307 bond.     

14-3-3 binding to the IGF-1 receptor is mediated by serine autophosphorylation

The phosphoserine-binding 14-3-3 proteins have been implicated in playing a role in mitogenic and apoptotic signaling pathways. Binding of 14-3-3 proteins to phosphoserine residues in the C-terminus of the insulin-like growth factor-1 receptor (IGF-1R) has been described to occur in a variety of cell systems, but the kinase responsible for this serine phosphorylation has not been identified yet. Here we present evidence that the isolated dimeric insulin-like growth factor-1 receptor kinase domain (IGFKD) contains a dual specific (i.e. tyrosine/serine) kinase activity that mediates autophosphorylation of C-terminal serine residues in the enzyme. From the total phosphate incorporation of approximately 4 mol per mol kinase subunit, 1 mol accounts for serine phosphate. However, tyrosine autophosphorylation proceeds more rapidly than autophosphorylation of serine residues (t(1/2) approximately 1 min vs. t(1/2) approximately 5 min). Moreover, dot-blot and far-Western analyses reveal that serine autophosphorylation of IGFKD is sufficient to promote binding of 14-3-3 proteins in vitro. The proof that dual kinase activity of IGFKD is necessary and sufficient for 14-3-3 binding was obtained with an inactive kinase mutant that was phosphorylated on serine residues in a stoichiometric reaction with the catalytically active enzyme. Thus, the IGF-1R itself might be responsible for the serine autophosphorylation which leads to recognition of 14-3-3 proteins in vivo.     

The effect of the typical peptidases (proteases) inhibitors on the degradation of Glp6 (125I) Tyr8SP6-11 hexapeptide in the nuclear and synaptosomal fraction of the cortex and hippocampus of rat brain.

The main peptidase PN/cutting Tyr8-Gly9 or Gly9-Leu10 bond (of sequence Glp6-Phe7-Tyr8-Gly9-Leu10-MetNH2) seems to be, at least in part, cysteine type enzyme. Cutting of Phe7-Tyr8 bond with PC enzyme is apparently negligible. Further degradation of labelled PN products seems to be accomplished with PI, being serine enzyme at least in part. Metalloenzymes, including "enkephalinase", seem to be of minor importance in hexapeptide degradation, at least in its very low concentration. Some typical inhibitors enhance the degradation what might be explained assuming that products of action of one peptidase strongly inhibit the other peptidase's action. Namely, products of PN and PI seem to inhibit PC except the hippocampal synaptosomes where the opposite is true.     

Ethylene production from methionine

1. A new reaction is described in which ethylene is formed from the Cu(+)-catalysed breakdown of methionine in phosphate buffer at 30 degrees in air. Some of the other products of the reaction are methionine sulphone, methionine sulphoxide, homocysteic acid, homocystine, acrolein, dimethyl disulphide, methanethiol, ethyl methyl sulphide, methane and ethane. These are considered to be produced in different reaction pathways. 2. Hydrogen peroxide is an intermediate in this reaction and can support ethylene production in the model system in anaerobic atmospheres. Cuprous copper is the active form that catalyses the formation of ethylene from an oxidized intermediate. The initial reaction is probably a Strecker degradation, but the aldehyde product is further degraded to ethylene and other products. 3. Methional (CH(3).S.CH(2).CH(2).CHO) is the most effective producer of ethylene in the model system and appears to be an intermediate in the reaction. 4. The evidence, from both tracer studies and from other precursors of ethylene in the reaction, indicates that ethylene is derived from the -CH(2).CH(2)- group of methionine.     

Hydrogen exchange kinetics and the mechanism of reaction B of yeast tryptophan synthase

It has been shown that yeast tryptophan synthase (L-serine hydro-lyase (adding indoleglycerol-phosphate) EC 4.2.1.20) catalyses tritium exchange reactions between protons on the alpha-carbon of L-serine of L-tryptophan, and water. The absolute rates of these reactions and indole-serine condensation (reaction B), all of which are pyridoxal phosphate-dependent, were measured. L-Serine exchange was resolved into two components, a high-affinity, slow, Michaelian reaction (KmS,H = 0.06 mM, kcats,H 3 X 10(-3) s-1) and a faster reaction (kcat greater than 2.5 S-1) which was not saturated even at 100 mM L-serine. Hydrogen exchange by tryptophan was a Michaelian process (KmT,H = 2.9 mM; kcatT,H = 0.6 s-1). Indole did not inhibit either exchange reaction. A plausible explanation of the results, that reaction B has a ping-pong mechanism with serine as first substrate and water and L-tryptophan as first and second products, respectively, was inadequate because of the observations that L-tryptophan is as first and second products, respectively, was inadequate because of the observations that L-tryptophan is synthesised with less than 1 mol of exchanged proton per mol amino acid, and that the ratio kcat/Km for serine changes between enzyme reactions. A branched modification with two enzyme-serine complexes, only one of which will exchange protons with water, will fit all the results.     

Reaction of hydrogen peroxide with ferrylhemoglobin: superoxide production and heme degradation

The reaction of Fe(II) hemoglobin (Hb) but not Fe(III) hemoglobin (metHb) with hydrogen peroxide results in degradation of the heme moiety. The observation that heme degradation was inhibited by compounds, which react with ferrylHb such as sodium sulfide, and peroxidase substrates (ABTS and o-dianisidine), demonstrates that ferrylHb formation is required for heme degradation. A reaction involving hydrogen peroxide and ferrylHb was demonstrated by the finding that heme degradation was inihibited by the addition of catalase which removed hydrogen peroxide even after the maximal level of ferrylHb was reached. The reaction of hydrogen peroxide with ferrylHb to produce heme degradation products was shown by electron paramagnetic resonance to involve the one-electron oxidation of hydrogen peroxide to the oxygen free radical, superoxide. The inhibition by sodium sulfide of both superoxide production and the formation of fluorescent heme degradation products links superoxide production with heme degradation. The inability to produce heme degradation products by the reaction of metHb with hydrogen peroxide was explained by the fact that hydrogen peroxide reacting with oxoferrylHb undergoes a two-electron oxidation, producing oxygen instead of superoxide. This reaction does not produce heme degradation, but is responsible for the catalytic removal of hydrogen peroxide. The rapid consumption of hydrogen peroxide as a result of the metHb formed as an intermediate during the reaction of reduced hemoglobin with hydrogen peroxide was shown to limit the extent of heme degradation.     

A potential processing enzyme in prokaryotes: Oligopeptidase B,a new type of serine peptidase

Basic amino acid pairs in polypeptides represent important markers for processing enzymes to produce biologically active products. Such enzymes related to the serine peptidase subtilisin have recently been identified in eukaryotes. Herein is described and kinetically characterized a new type of processing enzyme, oligopeptidase B, which is encountered in the prokaryote Escherichia coli, and belongs to the prolyl oligopeptidase family of serine peptidases. The enzyme hydrolyzes the peptides at the carboxy end of dibasic sites by two orders of magnitude faster with respect to monobasic substrates. The k_cat/K_m is extremely high, 63 μM⁻¹ s⁻¹, for the substrate benzyloxycarbonyl-L-arginyl-L-arginyl-7-(4- methylcoumaryl)amide. The bell-shaped pH dependence of the rate constant is perturbed by some ionizing group(s). This effect is abolished at 1 M NaCl. In addition, high ionic strength inhibits the reaction considerably by increasing K_m, which is indicative of an electrostatic interaction between the arginyl residues and the enzymatic carboxy groups. In distinction from that found with most serine endopeptidases, kinetic deuterium isotope measurements with oligopeptidase B indicate that the rate-limiting step of the reaction is a physical step rather than a chemical one characterized by general acid/base catalysis. The present result will contribute to our understanding of the processing phenomena in prokaryotes, as well as in higher organisms. Proteins 28:375–379, 1997. © 1997 Wiley-Liss, Inc.     

Immunochemical studies on blood groups. 53. A study of various conditions of alkaline borohydride degradation on human and hog blood group substances and on known oligosaccharides

Blood group A, B, H, Le^a, Le^b, and I substances, their products of periodate oxidation and Smith degradation, and disaccharides containing 3-O-substituted reducing N-acetylhexosamines were treated with base-borohydride under three defined sets of conditions. Procedures for the assay and quantitation of the possible reduced base-degradation products, including hexenetetrol(s), 3-deoxygalactitol, galactitol, reduced chromogens, N-acetylglucosaminitol, and N-acetylgalactosaminitol are described. Extensive degradation occurred by two methods. 1 m NaBH₄ in 0.05 n NaOH at 50 ° cleaves the glycosidic linkage of the oligosaccharide chains from serine and threonine with reduction of the terminal-reducing N-acetylgalactosamine with minimal base degradation. The method is useful for isolation of complete reduced oligosaccharides from blood group substances; the structural implications of the free and oligosaccharide-bound N-acetylgalactosaminitol released are discussed.     

A 3,4-dichloroisocoumarin-resistant component of the multicatalytic proteinase complex.

The multicatalytic proteinase complex (MPC) exhibits three proteolytic activities designated as trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing (PGPHA). Evidence based on inhibitor and specificity studies indicates that each of the three activities is associated with a different component of the complex. Inactivation of the three activities by the serine proteinase inhibitor, 3,4-dichloroisocoumarin (DCI), reveals the presence of an additional DCI-resistant component that cleaves natural peptides including neurotensin, dynorphin, angiotensin II, the oxidized B-chain of insulin, and also proinsulin at a rate greater than that of the native uninhibited complex. Examination of the reaction products of neurotensin (NT) and proinsulin degradation showed cleavage of the Ile12-Leu13 bond in NT and cleavage of the Leu44-Ala45 and Val39-Gly40 bonds within the connecting peptide (C-chain) of bovine proinsulin, suggesting preferential cleavage of bonds on the carboxyl side of branched chain amino acids. Although resistant to inhibition by DCI, the component was sensitive to inhibition by the isocoumarin derivatives, 7-amino-4-chloro-3-[3-(isothioureido)propoxy]isocoumarin and 4-chloro-7-guanidino-3-(2-phenylethoxy)isocoumarin. Degradation of NT was activated by leupeptin, chymostatin, and antipain indicating that binding of these aldehyde inhibitors at one site can stimulate proteolytic activity at a different site of the complex. The DCI-resistant component seems to constitute a major component of the complex active in degradation of natural peptides and proteins.     

Liquid chromatographic determination of penicillins by postcolumn alkaline degradation using a hollow-fiber membrane reactor

A high-performance liquid chromatographic method using a hollow-fiber membrane reactor is described for the determination of penicillins. This method involves separation of penicillins on a C18 column, postcolumn reaction with sodium hydroxide and mercury (II) chloride introduced into the main flow stream using sulfonated hollow-fiber membrane reactors immersed in each solution (4 M sodium hydroxide and 3 X 10(-2) M mercury (II) chloride plus 10(-2) M nitric acid), and detection at 290 nm based on the uv absorbance of the degradation products. At penicillin concentrations of 5 micrograms/ml, within- and between-run precisions (relative standard deviation) were 0.24-2.39 and 1.19-4.13%, respectively. The detection limits of the proposed method were 1-5 ng at a signal-to-noise ratio of 3. The method was applied to assays of ampicillin and its metabolites in human serum and urine.