State and reactivity of tryptophyl residues in two bacterial proteases from Sorangium sp.

The state and reactivity of tryptophyl residues in two proteolytic enzymes from Sorangium sp. were investigated by means of the following methods: spectrophotometric oxidation of tryptophans with N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide, and H2O2 in dioxane, optical rotatory dispersion, ultraviolet difference spectrophotometry, solvent perturbation and viscosity measurements. Out of two tryptophyl residues/molecule of alpha-lytic protease, one appears to be completely buried, while the other seems to be exposed. None of these two residues seem to be responsible for the activity of the enzyme. The beta-lytic protease undergoes an irreversible conformational transition between pH 5.0 and 3.5. Out of total four tryptophyl residues/molecule, only one is fully exposed at neutral pH. The other three are gradually exposed in the pH transition region. The degree of exposure and the dimensions of "cavities" shielding tryptophyl residues were estimated. The tryptophyl residues of of beta-lytic protease do not seem to participate in substrate binding or the active site; they are rather one of the determinants of the conformational state of the enzyme.     

生淀粉糖化酶产生菌Cellulosimicrobium sp.SDE的分离鉴定及酶学性质研究

从淀粉制品厂周围土壤中分离到一株高产生淀粉糖化酶的菌株SDE,经形态、生理生化及16S rDNA序列分析将其鉴定为Cellulosimicrobium sp..SDE菌株的最适产酶条件为pH7.0,培养温度为30℃.培养42h粗酶液的酶活达175.3U/mL.该酶以生玉米淀粉为底物时,最适作用pH6.0,最适作用温度40℃,pH6.0-7.0范围内酶活力稳定.在Ca2 存在下,酶的热稳定性很高,80℃保温1 h后,酶活力仍保持50%.Ba2 、Cu2 对酶活有强烈的抑制作用,Ca2 、Zn2 有很强的激活作用.     

Multiple proteases from Streptomyces moderatus. II. Physicochemical and enzymatic properties of the extracellular proteases

The physicochemical and enzymatic properties of five different extracellular proteases of Streptomyces moderatus were studied. The first protease was found to be a metal chelator sensitive protease with a Mr of 21,000 +/- 1000 a and a pI of 4.6. The second enzyme was an anionic trypsin-like protease (Mr 19,000 +/- 1000; pI 3.8) with a Km value of 4.76 X 10(-4) M on N-benzoyl-L-arginine-p-nitroanilide. A Km value of 1.52 X 10(-4) M was obtained when N-benzoyl-L-arginine ethyl ester was used as the substrate. The other three enzymes were found to be serine alkaline proteases with Mr's of 22,000, 29,000, and 23,000 +/- 1000 and with respective pI's of 7.8, 8.4, and 9.2. All the proteases showed optimum activity in the alkaline pH range. One of the three proteases was found to possess chymotrypsin and elastase-like properties. All five proteases were found to be unstable at temperatures above 60 degrees C. Except the trypsin-like protease, which was stable only in acidic pH, all other enzymes were found to be stable over a wide range of pH.     

Gastric proteases of the Greenland cod Gadus ogac. I. Isolation and kinetic properties

The zymogens of three gastric proteases of the Greenland cod (Gadus ogac) were isolated by exclusion chromatography and chromatofocusing. The cod zymogens were activated more rapidly at lower temperatures than porcine pepsinogen and, after activation, were further purified by exclusion chromatography. The cod proteases had more alkaline pH optima and were active over a wider range of pH than porcine pepsin. The specific activity of porcine pepsin on protein substrates was greater than that of the individual cod proteases. However, the cod proteases had cumulative activity on protein substrates that was greater than the sum of their individual activities. Cod protease 1 was active on pepsin-specific substrates, and cod proteases 2 and 3 were active as gastricsin-specific substrates. All three cod proteases had greater milk-clotting activity and hydrolysed hemoglobin to a greater extent than porcine pepsin. The Vmax and Km,app of the cod proteases were dependent upon the substrate, and Vmax/Km,app values of the cod proteases were generally lower than porcine pepsin. It is suggested that the cod proteases together exhibit broad substrate specificity and maintain activity over a wide range of conditions to enhance protein digestion in the cod stomach.     

Isolation, chemical, and physical properties of alpha-1-antitrypsin.

A method of isolation of alpha-1-antitrypsin (alpha-1-AT) in good yield from normal human plasma is described. A key step was affinity chromatography employing an antiserum which had been depleted of alpha-1-AT antibodies. The final preparations were homogeneous by immunological and physicochemical criteria. The specific activity of the purified alpha-1-AT was 0.363 mg of active bovine trypsin inhibited per 1.0 mg of inhibitor. Polyacrylamide gel patterns at both alkaline and acid pH of highly pure preparations frequently, but not invariably, showed multiple hands. Molecular weight studies by sedimentation equilibrium ultracentrifugation in aqueous buffer and in 6 M guanidine as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis suggest that alpha-1-AT is a single polypeptide chain having a molecular weight of 49,500. Other physical and chemical properties of the inhibitor are described. A limited N-terminal sequence (Glu-Asp-Pro-Gln-Gly-Asx-Ala-Ala) was obtained. It was found that alpha-1-AT easily forms polymers and higher aggregates when exposed to denaturing agents such as 8 M urea and 6 M guanidine. The results suggest that aggregation is determined by both covalent and noncovalent forces.     

Isolation and properties of enzymes involved in prostaglandin biosynthesis.

Prostaglandin (PG) endoperoxide synthetase was purified until homogeneity had been attained. The pure enzyme displays both cyclooxygenase and peroxidase activity, in accordance with the work of MIYAMOTO et al. (J. biol. Chem. 252, 2629--2636 (1976)). This enzyme therefore converts arachidonic acid into PGH2. Glutathione S-transferases, in the presence of glutathione, convert PGH2 into a mixture of PGF2alpha, PGE2 and PGD2. A new transferase in sheep lung gives mainly PGF2alpha and PGD2. Isolation and properties of these enzymes will be discussed. Finally, progress will be reported on the isolation of a soluble enzyme from various rat organs such as lung and spleen, which forms almost exclusively prostaglandin D.     

Methylamine dehydrogenase of Pseudomonase sp. J Isolation and properties of the subunits.

Two kinds of subunits, light subunit (Mr =1300) and heavy subunit (Mr=40 000), were isolated from a methylamine dehydrogenase (Mr=105 000) of Pseudomonas sp. J. The isolation of the subunits was carried out by gel chromatography after the enzyme had been treated with 3M guanidine-HCl. Coexistence of both of the subunit exhibited an absorption maximum only at 278 nm but in addition to the peak at 278 nm. The results indicate that the prosthetic group, assumed to be a derivative of pyridoxal, was bound to the light subunit. The spectral changes of the light subunit were observed by addition of methylamine. Various physical and biochemical parameters of the subunits are reported.