Purification and biochemical characterization of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione reductase from rat lung and inhibition effects of some antibiotics

G6PD, 6PGD and GR have been purified separately in the single step from rat lung using 2′, 5′-ADP Sepharose 4B affinity chromatography. The purified enzymes showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of the enzymes were estimated to be 134?kDa for G6PD, 107?kDa for 6PGD and 121?kDa for GR by Sephadex G-150 gel filtration chromatography, and the subunit molecular weights was respectively found to be 66, 52 and 63?kDa by SDS-PAGE. Optimum pH, stable pH, optimum ionic strength, optimum temperature, K_M and V_max values for substrates were determined. Product inhibition studies were also performed. The enzymes were inhibited by levofloxacin, furosemide, ceftazidime, cefuroxime and gentamicin as in vitro with IC₅₀ values in the range of 0.07–30.13?mM. In vivo studies demonstrated that lung GR was inhibited by furosemide and lung 6PGD was inhibited by levofloxacin.     

Direct Resolution of dl-Lysine-3, 5-Dimtrobenzoate

The inhibitory activity of an angiotensin I-converting enzyme (ACE) detected in soy sauce was fractionated into two major fractions of high molecular weight (Hw) and low molecular weight (Lw) by gel filtration chromatography on Bio-gel P-2 after treating with ethanol. The Hw fraction reduced the blood pressure in hypertensive rats after orally administering, while the Lw fraction did not. The ACE inhibitor in the Hw fraction was further purified by Dowex 50W ion-exchange chromatography and four subsequent steps of HPLC. On the basis of the SIMS-mass spectrum, NMR spectrum and other characteristics, the purified ACE inhibitor was identified as nicotianamine (N-[N-(3-amino-3-carboxypropyl)-3-amino-3- carboxypropyl]azetidine-2-carboxylic acid). The IC₅₀ value for this ACE was 0.26 µM.     

Enzyme antigens associated with pigeon breeder's disease. I. Isolation and characterization of basic hydrolases

A survey of the hydrolytic enzymes present in pigeon dropping extracts (PDE) has shown that this material contains a variety of proteolytic and nonproteolytic activities. These enzymes were separated into their basic and acidic components by chromatography on DEAE-cellulose. Staining of immunoprecipitates with specific chromogenic substrates demonstrated the presence of antibodies in symptomatic breeders to several of the basic enzymes in PDE. Five distinct hydrolytic activities were isolated from the basic group of enzymes. Trypsin, elastase, and two forms of collagenase were the specific proteolytic activities isolated. A phospholipase was also purified from these preparations. The purified elastase consisted of a single polypeptide chain (M _r =22,000). The purified trypsin had a molecular weight (M _r =25,000) and charge similar to those reported for elastase and, like elastase, the trypsin from PDE appeared to be composed of a single polypeptide chain. Two molecular weight forms of collagenase were found; both hydrolyzed bovine collagen. The high-molecular-weight collagenase (M _r =51,000) was shown to be a glycoprotein consisting of two polypeptides (M _r =24,000). It was readily separated from the low-molecular-weight collagenase (M _r =15,000) by gel filtration. The phospholipase (M _r =99,000) appeared to be a dimer. The relevance of these enzymes to the development of pigeon breeder's disease is discussed.     

Enzyme antigens associated with pigeon breeder's disease. II. Isolation and characterization of acidic hydrolases

The hydrolytic enzymes in pigeon dropping extracts (PDE) were separated into basic and acidic components by DEAE-cellulose chromatography. Six distinct hydrolytic activities were isolated from the acidic group of enzymes. Elastase, trypsin, and two forms of collagenase were the proteases identified. An esterase and a phospholipase were also detected. The sera of symptomatic pigeon breeders, analyzed by crossed immunoelectrofocusing techniques, were shown to contain antibodies to the enzymes trypsin, collagenase, and esterase in the acidic fractions of PDE by staining the immune precipitates with specific chromogenic substrates. These enzymes were purified by preparative isoelectric focusing, affinity chromatography, and gel filtration. The purified elastase hydrolyzed elastin and consisted of a single polypeptide chain (M _r =23,100). Trypsin hydrolyzed a synthetic arginine substrate, but not elastin or collagen. Its size (M _r =27,500) and subunit structure were similar to those reported here for elastase. Both enzymes were inhibited by -1-antitrypsin,N-p-tosyl-L-lysine chloromethylketone, and phenylmethylsulfonyl fluoride. Two distinct collagenases were found; both cleaved bovine collagen. The high-molecular-weight (HMW) collagenase was a glycoprotein (M _r =117,500) and consisted of three polypeptide chains (M _r =37,100); a low-molecular-weight (LMW) collagenase (M _r =12,500) was also isolated. The HMW collagenase was inhibited byEDTA,p-chloromercuribenzoic acid, and phenylmethylsulfonyl fluoride, but not by -1-antitrypsin. The source of these enzymes as well as their relationship to the basic hydrolytic enzymes of PDE are discussed.     

SYNTHESIS OF GLYCOPROTEINS IN BRAIN: IDENTIFICATION, PURIFICATION AND PROPERTIES OF GLYCOSYL TRANSFERASES FROM PURIFIED SYNAPTOSOMES OF GUINEA PIG CEREBRAL CORTEX

Abstract— Four glycoprotein:glycosyl transferases (a fetuin:N-acetylglucosaminyl transferase; a bovine submaxillary mucin: N-acetylgalactosaminyl transferase; a collagen: glucosyl transferase and an orosomucoid: galactosyl transferase) were purified 34-, 45-, 37- and 47-fold, respectively, from synaptosomes prepared from guinea pig cerebral cortex. Purifications were achieved by centrifugation and by column chromatography on Sephadex G-100 and G-150 of 0 , 1% (w/v) Triton X-100 extractsof the purified cerebral cortical synaptosomes. The enzymes were separated from endogenous acceptors and were highly specific for specific macromolecular acceptors; small molecules were ineffective as acceptors. The fetuin: N-acetylglucosaminyl transferase functioned only with fetuin minus N-acetylneuraminic acid, galactose and N-acetylglucosamine; the bovine submaxillary mucin: N- acetylgalactosaminyl transferase with bovine submaxillary much minus N-acetylneuraminic acid and N-acetylgalactosamine; the collagen: glucosyl transferase with collagen minus glucose; and the orosomucoid: galactosyl transferase with either orosomucoid minus N-acetylneuraminic acid and galactose or fetuin minus N-acetylneuraminic acid and galactose. Each transferase required a specific (XDP)-monosaccharide for transfer. The transferases were entirely dependent on either Mn²⁺ or Mg²⁺ for activation and Fe²⁺ and Hg²⁺ inhibited each of the four enzymes. The optimum pH's for the enzymes were: for fetuin: N-acetylglucosaminyl transferase, 7 , 4–8.0; for bovine submaxillary mucin: N-acetylgalactosaminyl transferase, 7 , 7; for collagen: glucosyl transferase, 7 , 7 and for orosomucoid: galactosyl transferase, 6 , 6. The enzymes were distributed subsynaptosomally primarily in the synaptosomal plasma membrane and in the mitochondria of the synaptosome. The respective values for K_m (μM) and V_mex (pmoles/h/mg of protein) for the transferases were: fetuin: N-acetylglucosaminyl transferase, 12 and 143; for bovine submaxillary mucin: N-acetylgalactosaminyl transferase, 25 and 166; for collagen: glucosyl transferase, 4 and 10 and for orosomucoid:galactosyl transferase, 8 and 111.     

Substitution of the amino acid at position 102 with polar and aromatic residues influences substrate specificity of lactate dehydrogenase

The Gin residue at amino acid position 102 ofBacillus stearothermophilus lactate dehydrogenase was replaced with Ser, Thr, Tyr, or Phe to investigate the effect on substrate recognition. The Q102S and Q102T mutant enzymes were found to have a broader range of substrate specificity (measured byk _cat/K _m) than the wild-type enzyme. However, it is evident that either Ser or Thr at position 102 are of a size able to accommodate a wide variety of substrates in the active site and substrate specificity appears to rely largely on size discrimination in these mutants. The Q102F and Q102Y mutant enzymes have low catalytic efficiency and do not show this relaxed substrate specificity. However, their activities are restored by the presence of an aromatic substrate. All of the enzymes have a very low catalytic efficiency with branched chain aliphatic substrates.Abbreviations used BSLDH Bacillus stearothermophilus lactate dehydrogenase - FBP fructose-1,6-bisphosphate - HP hydroxypyruvate - KB ketobutyrate - KC ketocaproate - KV ketovalerate - MDH malate dehydrogenase - PP phenylpyruvate - PYR pyruvate - RBE relative binding energy     

Metabolism of prostaglandin endoperoxide by microsomes from human lung parenchyma and comparison with metabolites produced by pig, bovine, rat, mouse and guinea-pig

The metabolism of PGH₂ by human lung parenchymal microsomes was characterized by radiometric high performance liquid chromatography and compared with metabolism by pig, bovine, rat, mouse, and guinea pig lung microsomes. Microsomes from human lung synthesized 0.74 nmoles/mg protein and 0.72 nmoles/mg protein, PGI₂ (6-Keto-PGF_1α) and T×A₂ (T×B₂) respectively, upon incubation with 4.0 nmoles of PGH₂. Pig, bovine, rat, mouse, and guinea pig microsomes respectively synthesized 0.1, 1.0, 0.9, 0.4, and 0.1 nmoles of PGI₂/mg protein, and 0.9, 1.0, 0.7, 0.3, 1.8 nmoles of T×A₂/mg protein, and preparations formed some PGE₂, PGF_2α, and PGD₂. Mouse lung microsomes were unique in synthesizing PGE₂ as the major prostaglandin. The thromboxane synthetase inhibitor 1-benzylimidazole was a specific inhibitor in these six species.     

Isolation and characterization of a bacterial dipeptidyl carboxypeptidase inhibitor from Bacillus subtilis 3-16-20

A bacterial dipeptidyl carboxypeptidase inhibitor was isolated from the culture broth of a bacterium identified as Bacillus subtilis. The inhibitor was purified 33-fold from the culture supernatant of B. subtillis 3-16-20 strain by Q-, and S-Sepharose fast flow, C₁₈ column chromatography, ethanol treatment, and ODS column chromatography. The purified inhibitor has an amino acid sequence of glycyl-prolyl-phenylalanyl-prolylisoleucine. IC₅₀ values of the inhibitor were 177 μM (rabbit lung ACE) and 35 μM (bacterial DCP).     

Inhibitor analysis of angiotensin I-converting and kinin-degrading activities of bovine lung and testicular angiotensin-converting enzyme.

Inhibition of bovine lung and testicular angiotensin-converting enzyme (ACE) by some well-known ACE inhibitors (lisinopril, captopril, enalapril), new substances (Nalpha-carboxyalkyl dipeptides PP-09, PP-35, and PP-36), and phosphoramidon was investigated using Cbz-Phe-His-Leu and FA-Phe-Phe-Arg (C-terminal analogs of angiotensin I and bradykinin, respectively) as the substrates. The somatic (two domains) and testicular (single domain) isoenzymes demonstrated different kinetic parameters for hydrolysis of these substrates. All of the inhibitors were competitive inhibitors of both ACE isoforms, and the Ki values were substrate-independent. The relative potencies of the inhibitors for both enzymes were: lisinopril > captopril > PP-09 > enalapril > PP-36 > PP-35 > phosphoramidon. The inhibition efficiency of PP-09 was comparable with those of the well-known ACE inhibitors. Captopril was more effectively bound to the somatic ACE (Ki = 0.5 nM) than to the testicular isoform (Ki = 6.5 nM).     

茶树CsPLK基因的鉴定与表达分析

茶树中富含茶氨酸、儿茶素和咖啡碱等重要功能成分,具有较高的价值功效,茶树在生命周期中经常遭受逆境胁迫,维生素B6(VB6)在植物体内参与逆境应答,吡哆醛激酶(pyridoxal kinase,PLK)是VB6补救途径中的关键酶。为进一步了解PLK在茶树生物合成中的功能和作用机理,该研究基于茶树基因组数据库,以龙井43为材料,采用逆转录PCR(RT-PCR)的方法从茶树中克隆出CsPLK的基因。结果表明:该基因序列长为1 179 bp,编码393个氨基酸; CsPLK蛋白和已知物种中PLK蛋白具有较高的同源性,都是核糖激酶超家族成员;通过构建pET-CsPLK载体进行原核表达,并鉴定出重组蛋白有很强的催化活性;组织表达特异性分析表明,叶中的表达量比茎、根的高,在根中最低;荧光定量PCR表示,低温诱导CsPLK上调表达,干旱诱导CsPLK下调表达,发现该基因在茶树中有明显的逆境应答,推测CsPLK在茶树的生长发育、逆境胁迫发挥重要作用。     

Purification and characterization of milk-clotting enzymes from oyster mushroom (<Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> (Fr.) Kumm)

Three enzymes with milk-clotting activity have been isolated from the fruiting bodies of Pleurotus ostreatus (Fr.) Kumm) by (NH₄)₂SO₄ precipitation, gel chromatography on Sephadex G75, and ion exchange chromatography on carboxymethylcellulose (CMC). Isoelectric points of the enzymes, as determined by isoelectrofocusing, equaled 4.2, 6.7, and 8.8. Inhibition analysis showed that the enzymes with isoelectric points of 4.2 and 6.7 belong to the class of metal-dependent proteinases, while the enzyme with the isoelectric point of 8.8 belongs to the serine protease class.     

Purification and characterization of sulfatases from Haliotis rufescens: evidence for changes in synthesis and heterogeneity during development

Summary The digestive glands of many marine molluscs are rich sources of arylsufatase enzymes which may function in the catabolism of sulfated polysaccharides in the diets of herbivorous species. Arylsulfatases, partially purified from the hepatopancreas of the red abalone, Haliotis rufescens, were investigated with respect to heterogeneity, catalytic requirements, and timing of induction during development. Four hepatopancreatic enzymes were purified from adult animals using a combination of hydrophobic interaction and anion-exchange chromatography. Zymograms of the four partially-purified enzymes produced by electrophoresis under nondenaturing conditions revealed a fifth, relatively more basic isozyme. All four partially-purified enzymes appear to be monomeric, with molecular weights of approximately 43 000 Da each, as measured by gel filtration. The affinities for p-nitrocatechol sulfate, pH optima, and strengths of inhibition by anions displayed by these enzymes are similar to the values reported for other molluscan arylsulfatases. Three of the four enzymes have K _m values between 0.8 and 2.0 mM for p-nitrocatechol sulfate; the remaining enzyme (A₂) has a K _m of 6.7 mM. All four enzymes have pH and temperature optima of 5.5 and 45°C, respectively. Three of the four enzymes have-t_1/2(50°C) values of 3.5 min; the enzyme A₄ has a t_1/2 has a t_1/2(50°C) of 8.5 min. A monoclonal antibody directed against form A_1b does not cross react with any of the other hepatopancreatic arylsulfatases when assayed by Western blot, confirming the structural heterogeneity of the adult enzymes.Total arylsulfatase activity increases in a biphasic manner during early abalone development, with the first increase occurring early in larval maturation. The secoad phase of enzyme expression is dependent upon the induction of settlement and metamorphosis of the competent veliger larvae, strongly suggesting that the expression of arylsulfatase synthesis (and the maturation of the digestive gland, the hepatopancreas) is controlled by genetic events which occur as a result of metamorphosis. Competent veliger larvae express only two arylsulfatase forms, which share many physicochemical and kinetic characteristics with the adult hepatopancreatic enzymes. However, neither of the larval arylsulfatases is recognized by the monoclonal antibody to form A_1b from adult hepatopancreas. Endogenous enzyme inhibitor levels in larvae remain constant throughout the period of arylsulfatase induction, and therefore do not contribute to the control of arylsulfatase activity levels during development.These results are the first documentation of the developmental induction of a specific protein(s) in abalone as a result of metamorphosis. The significance of the timing of arylsulfatase expression is discussed in relation to potential physiological substrates and the dietary switching which occurs at metamorphosis. Possible genetic events which are consistent with the observed patterns of expression of these enzymes also are considered.Abbreviations BSA bovine serum albumin - C centigrade - Da daltons - DEAE diethylaminoethyl - ELISA enzyme-linked immunosorbent assay - FPLC fast protein and polynucleotide liquid chromatography - GABA -aminobutyric acid - HAT hypoxanthine, aminopterin, thymidine - Hepes N-(2-Hydroxythyl)piperazine-N'-(2-ethanesulfonic acid) - HPLC high pressure liquid chromatography - KBS Kantor's balanced salt solution - K _m Michaelis constant - PBS phosphate buffered saline - R _m relative mobility - RPMI Roswell Park Memorial Institute - SDS sodium dodecyl sulfate - T time - TBS TRIS buffered saline - V _max maximal velocity     

Angiotensin-converting enzyme insertion/deletion (rs106180) and angiotensin type 1 receptor A1166C (rs106165) genotypes and psoriasis: Correlation with cellular immunity,lipid profile,and oxidative stress markers

Psoriasis is a chronic inflammatory skin condition and angiotensin-converting enzyme (ACE) is a key circulating enzyme converting angiotensin (Ang) I to the vasoactive peptide Ang II. The exact role of ACE insertion (I)/deletion (D) polymorphism (rs106180) in psoriasis is not clear. We aimed to examine whether the ACE I/D and Ang II type 1 receptor (AT1R) A₁₁₆₆C-polymorphisms (rs106165), lipid profile, and stress oxidative are associated with susceptibility to psoriasis. One hundred patients with psoriasis and 100 sex- and age-matched unrelated healthy controls were recruited for this case-control study. ACE I/D and AT1R A₁₁₆₆C polymorphisms were identified by the polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism, respectively, malondialdehyde (MDA) was detected by the high-performance liquid chromatography, serum arylesterase (ARE) activity of paraoxonase and catalase activities were detected by the spectrophotometry, superoxide dismutase (SOD) activity and vascular adhesion protein (VAP)-1 were measured by ELISA. The presence of C allele of AT1R A₁₁₆₆C and I allele of ACE considerably increased the risk of psoriasis by 6.42-fold (P < 0.001). The distribution of II-genotype of ACE was significantly higher in psoriasis patients than in control group and increased the risk of disease by 3.11-times (P = 0.023). The higher levels of MDA in patients and the higher activity of SOD, ARE, and CAT was observed in healthy controls with I/D+I/I-genotype of ACE I/D. This study for the first time demonstrated that the ACE I/D and AT1R A ₁₁₆₆C genes polymorphisms robustly increases the risk of developing psoriasis in population from west of Iran. In addition, these individuals had significantly higher VAP-1 and MDA concentration and lower enzymatic and nonenzymatic antioxidant-status, suggesting that psoriatic patients carrying C allele of AT1R₁₁₆₆ polymorphism may be more susceptible to cardiovascular disease and myocardial infarction compared with A allele.     

Mutagenicity of Isothiocyanates,Isocyanates and Thioureas on Salmonella typhimurium

During the screening program for atrial natriuretic peptide (ANP) receptor ligands of microbial origin, we isolated a novel nonpeptide ANP antagonist, HS-142-1, from a culture broth of Aureo-basidium pullulans var. melanigenum. Structural analysis showed that HS-142-1 was composed of 20–30 kinds of β-1,6-glucan esterified by caproyl groups; each component had an almost equal potency. HS-142-1 inhibited [¹²⁵I]-rANP binding to its receptor in rabbit kidney cortex membranes with an IC₅₀ of 0.3μg/ml and antagonized ANP-induced cGMP production by bovine lung membranes in a dose-dependent fashion. The discovery of this nonpeptide ANP antagonist, HS-142-1, will provide a useful tool to study the physiological significance of natriuretic peptide system.     

Kinetics of hydrolysis of type I, II, and III collagens by the class I and II Clostridium histolyticum collagenases.

The kinetics of hydrolysis of rat tendon type I, bovine nasal septum type II, and human placental type III collagens by class I and class IIClostridium histolyticum collagenases (CHC) have been investigated. To facilitate this study, radioassays developed previously for the hydrolysis of these [³H]acetylated collagens by tissue collagenases have been adapted for use with the CHC. While the CHC are known to make multiple scissions in these collagens, the assays are shown to monitor the initial proteolytic events. The individual kinetic parametersk _cat andK _M have been determined for the hydrolysis of all three collagens by both class I and class II CHC. The specific activities of these CHC toward fibrillar type I and III collagens have also been measured. In contrast to human tissue collagenases, neither class of CHC exhibits a marked specificity toward any collagen type either in solution or in fibrillar form. The values of the kinetic parametersk _cat andK _M for the CHC are similar in magnitude to those of the human enzymes acting on their preferred substrates. Thus, the widely held view that the CHC are more potent collagenases is not strictly correct. As with the tissue collagenases, the local collagen structure at the cleavage sites is believed to play an important role in determining the rates of the reactions studied.     

Properties of the soluble arachidonic acid 15-lipoxygenase and 15-hydroperoxide isomerase from the oomycete Saprolegnia parasitica

The soluble hydroperoxide isomerase and 15-lipoxygenase activities were partially purified from the oomycete Saprolegnia parasitica and some of their properties characterized. Both enzymes co-eluted with a molecular weight of 145,000–150,000 on Sephacryl S-300 chromatography. The enzyme activities also co-eluted on DEAE Sephadex ion exchange chromatography and hydroxylapatite chromatography. Both activities showed similar responses to pH and temperature. Both enzymes showed parallel inhibition by p-hydroxymercuribenzoate and eicosatetraynoic acid. The partially purified hydroperoxide isomerase showed an apparent k_m of 166 μM and a V_max of 5.3 μmol/min/mg protein for exogenous 15-HPETE. It was not stimulated by calcium. These results suggest that the soluble hydroperoxide isomerase and 15-lipoxygenase activities from S. parasitica are both contained on the same protein or protein complex.     

The Preparation of Trypsins and Chymotrypsins From Bovine and Porcine Residues After Insulin Extraction

Bovine and porcine pancreatic residue, remaining after the extraction of insulin, has been used to prepare a proteinase powder. This powder was used as a source of trypsin and chymo-trypsin. The individual enzymes were isolated and purified by chromatography on sulfopropyl (SP)-Sephadex C-25 and affinity chromatography on soybean trypsin inhibitor (STI)-Sepharose. The bovine proteinase powder contained a-chymotrypsin, trypsin and chymotrypsin B in the ratio 5:2:1. The porcine powder contained cationic trypsin, anionic trypsin and cationic chymotrypsin in the ratio 5 : 1. 4 : 3. The isolated enzymes were characterized and found to be identical with enzymes isolated from fresh tissue with the exception of porcine chymotrypsin. Porcine cationic chymotrypsin was isolated as two distinct forms, A-l and A-2, which appear to be different activation products of porcine chymotrypsinogen A. Both forms resemble bovine a-chymotrypsin, a three chain structure, rather than porcine chymo-trypsin A, a two chain structure. Furthermore, the B-chain appears to be cleaved, possibly at residues Phe₈₉-Lys₉₀.     

The in vitro cross-effects of inhibitors of renin-angiotensin and fibrinolytic systems on the key enzymes of these systems

The effects of hypotensive agents (captopril, enalaprilat, and lisinopril) on the activities of components of the fibrinolytic system (FS) and the effects of antifibrinolytic agents (6-aminohexanoic acid (6-AHA) and tranexamic acid (t-AMCHA)) on the activities of angiotensin converting enzyme (ACE) were studied in vitro. Enalaprilat did not affect the FS activity. Captopril considerably inhibited the amidase activities of urokinase (u-PA), tissue plasminogen activator (t-PA), and plasmin ([I]₅₀ (2.0?2.6) ± 0.1 mM), and the activation of Glu-plasminogen by t-PA and u-PA ([I]₅₀ (1.50?1.80) ± 0.06 mM), which may be due to the presence of a mercapto group in the inhibitor molecule. Lisinopril did not affect the amidase activities of FS enzymes, but stimulated Glu-plasminogen activation by u-PA and inhibited activation fibrin-bound Glu-plasminogen by t-PA ([I]₅₀ (12.0 ± 0.5) mM). Presumably, these effects can be explained by the presence in lisinopril of a Lys side residue, whose binding to lysine-binding Glu-plasminogen centers resulted, on the one hand, in the transformation from its closed conformation to a semi-open one and, on the other hand, in its desorption from fibrin. Unspecific inhibition of the activity of ACE, a key enzyme of the renin-angiotensin system, in the presence of 6-AHA and t-AMCHA ([I]₅₀ 10.0 ± 0.5 and 7.5 ± 0.4 mM, respectively) was found. A decrease in the ACE activity along with the growth of the fibrin monomer concentration was revealed. The data demonstrate that, along with endogenous mediated interaction between FS and RAS, relations based on the direct interactions of exogenous inhibitors of one system affecting the activities of components of another system can take place.     

Purifications and Enzymatic Properties of β-Amylase and Pullulanase from Bacillus cereus var. mycoides

A β-amylase and a pullulanase produced by Bacillus cereus var. mycoides were purified by means of ammonium sulfate fractionation, adsorption on starch and celite and Sephadex G–100 column chromatography. The purified enzymes were homogeneous in disc electrophoresis.

The β-amylase released only maltose from amylose, amylopectin, starch and glycogen, and the released maltose was in β-form. The pullulanase released maltose, maltotriose and maltotetraose from β-limit dextrin and maltotriose from pullulan, but not amylose-like substance from amylopectin.

The optimum pHs of β-amylase and pullulanase were about 7 and 6~6.5, respectively. The optimum temperatures of the enzymes were about 50°C. The enzymes were inhibited by the sulfhydryl reagents such as mercuric chloride and p-chloromercuribenzoate, and the inhibitions with p-chloromercuribenzoate were restored by the addition of cysteine. The molecular weights of β-amylase and pullulanase were estimated to be 35,000±5,000 and 110,000±20,000, respectively.     

Comparative Study of the Action of Bovine Duodenal Proteinases (Duodenases) on Polypeptide Substrates

A comparative study of substrate specificity of bovine duodenal proteinases—chymotrypsin-like duodenase (ChlD) and dual-specificity duodenase (dsD)—was carried out using oligopeptide substrates (human proinsulin, glucagon, melittin, angiotensinogen fragment 1-14). ChlD displayed mainly chymotrypsin-like properties towards these substrates, hydrolyzing peptide bonds carboxy-terminally to bulky aliphatic or aromatic residues. In melittin, ChlD additionally cleaved peptide bonds after Thr and Ser residues. Dual-specificity duodenase (dsD) significantly restricted its specificity to only trypsin-like or only chymotrypsin-like or displayed full activity, combining both specificities, depending on substrate. Both ChlD and dsD efficiently hydrolyzed a single peptide bond (Phe8–His9) in angiotensinogen fragment 1-14. The kinetic parameters of angiotensinogen fragment 1-14 cleavage by ChlD and dsD were determined (k _cat/K _m = 80,500 M^-1·sec^-1 for ChlD and 103,000 M^-1·sec^-1 for dsD).