Kinin-converting aminopeptidase from human urine partial purification and properties

• 1.1. An aminopeptidase from human urine, which hydrolyses dipeptides and β-naphthylamides of neutral and basic amino acids and which converts the peptides lysylbradykinin and methionyllysyl-bradykinin into bradykinin, was highly purified by a four-step procedure.
• 2.2. The enzyme (mol. wt 100,000) has several similarities with kinin-converting aminopeptidases found in human serum and liver, and is inhibited by 1,10-phenanthroline and puromycin.

     

Stimulants of prostaglandin biosynthesis in human fetal membranes, uterine decidua vera and placenta

Modulation of prostaglandin (PG) biosynthesis by cytosolic fractions derived from homogenates of human amnion, chorion laeve, decidua vera and placenta was examined. PGF_2α and 6-oxo-PGF_1α synthesis by bovine seminal vesicle (BSV) PG synthase was stimulated by the cytosolic fractions of each tissue in a dose-dependent manner. The cytosols from decidua vera and placenta were the most effective in stimulating synthesis and also stimulated PGE₂ biosynthesis. Reduced glutathione (GSH) acted to increase the biosynthesis of PGE₂ at the expense of other PGs both in the presence and absence of various cytosols. These data are indicative that the mode of action of cytosolic fractions on the stimulation of PG biosynthesis is unlike that of GSH. Indomethacin and aspirin, inhibitors of fatty acid cyclooxygenase activity, strongly inhibited the cytosol-induced stimulation of BSV PG synthase.The cytosolic factors that stimulated PG biosynthesis exhibited differential behavior towards boiling and dialysis. The stimulatory effect of all cytosolic fractions was sensitive to boiling except in the case of chorion leave effects toward 6-oxo-PGF_1α production. In dialysis studies we found that the cytosolic components that stimulated the production of PGF_2α were not removed by dialysis except in the case of cytosol of placenta whereas the stimulatory effects of various cytosols toward the biosynthesis of PGE₂ and 6-oxo-PGF_1α were removed by dialysis. These results are indicative of the presence of endogenous factors in human intrauterine tissues that preferentially stimulate the biosynthesis of PGF_2α and 6-oxo-PGF_1α and are further suggestive that PC biosynthesis in intrauterine tissues is, at least in part, regulated by cytosolic factors.     

Arachidonate 15-lipoxygenase from human eosinophil-enriched leukocytes: partial purification and properties

Arachidonate 15-lipoxygenase was purified from human eosinophil-enriched leukocytes after showing that 15-lipoxygenase activity was 100-fold greater in eosinophils than in neutrophils. Partial purification was achieved using ammonium sulfate precipitation, cation-exchange and hydrophobic-interaction chromatography. New evidence is presented suggesting that 15-lipoxygenase has electrostatic and hydrophobic properties distinct from 5-lipoxygenase. In addition, ATP is shown to inhibit, and phosphatidylcholine is shown to stimulate, 15-lipoxygenase, suggesting a regulatory role for these compounds in the lipoxygenation of arachidonic acid.     

Metalloenzyme inhibitor from kidney beans: partial purification and characterization

Inhibitory activity directed against metalloenzymes has been highly purified from extracts of red kidney beans (Phaseolus vulgaris). The inhibitor is a substance of small molecular weight and appears to be a chelator of Zn²⁺. One milligram of the preparation inhibited 23 milligrams carboxypeptidase A. The inhibitor also strongly inhibited carboxypeptidase B and alkaline phosphatase and could activate phosphoglucomutase that had previously been inactivated with Zn²⁺. The isoelectric point of the inhibitor is 4.7. The inhibitor activity was abolished by preincubation with Zn²⁺, Ni²⁺, Co²⁺, or Cu²⁺. The mechanism of inhibition of carboxypeptidases and alkaline phosphatase by the bean inhibitor is apparently due to the complexing and complete removal of Zn²⁺ from the enzymes.     

Cytokinin oxidase from wheat: partial purification and general properties

As part of the study of the possible role(s) of CBF-1, a cytokinin-binding protein abundant in wheat embryo, a cytokinin oxidase was found in wheat (Triticum aestivum L.) germ and partially purified by conventional purification techniques and high performance chromatofocusing. This preparation catalyzes conversion of N⁶-(Δ²-isopentenyl)adenosine to adenosine at a V_max of 0.4 nanomol per milligram protein per minute at 30°C and pH 7.5, the K_m being 0.3 micromolar. This high affinity and the apparent molecular weight of 40,000 estimated by high performance gel permeation on a Spherogel TSK-3000 SW column indicate that this enzyme is different from other cytokinin oxidases previously reported. Oxygen is required for the reaction, as for other cytokinin oxidases already described. N⁶-(Δ²-isopentenyl)adenine and zeatin riboside are also degraded, but N⁶-(Δ²-isopentenyl)adenosine-5′-monophosphate is apparently not a substrate. Benzyladenine is degraded, but to a small extent, and it inhibits slightly the degradation of N⁶-(Δ²-isopentenyl)adenosine. The degradation of N⁶-(Δ²-isopentenyl)adenosine is strongly inhibited by diphenylurea and its highly active derivative N-(2-chloro-4-pyridyl)-N′-phenylurea.     

Purification of prostaglandin E2-9-oxoreductase from human decidua vera

Prostaglandin E2-9- oxoreductase (PGE2-9-OR), the enzyme which converts prostaglandin E2 (PGE2) to prostaglandin F2 alpha (PGF2 alpha), has been detected in human decidua vera. A 105-fold purification was achieved when the centrifuged homogenate was fractionated sequentially by DEAE-Trisacryl, hydroxyapatite-agarose gel, ultrogel AcA 44 and Matrex gel blue A gel chromatographies. The following kinetic constants for PGE2-9-OR have been obtained. The equilibrium constant with respect to PGE2 is 83 microM, the Michaelis constant, Km, for PGE2 is 80 microM, for NADPH 1.6 microM. The maximal velocity for the forward reaction is V1 = .203 pmol/min. The enzyme was inhibited by progesterone, oestradiol-17 beta, cortisol and pharmaceutical drugs. An activating effect could be demonstrated with Ca2+ and oxytocin. The occurrence of PGE2-9-OR in the decidua vera suggests that this enzyme may be responsible for the transformation of PGE2 to PGF2 alpha in these tissues. This may be an important mechanism for the initiation and maintenance of uterine contractions.     

Trypsin inhibitor from Dimorphandra mollis seeds: purification and properties

A trypsin inhibitor from Dimorphandra mollis seeds was isolated to apparent homogeneity by a combination of ammonium sulfate precipitation, gel filtration, ion-exchange and affinity chromatographic techniques. SDS-PAGE analysis gave an apparent molecular weight of 20 kDa, and isoelectric focusing analysis demonstrated the presence of three isoforms. The partial N-terminal amino acid sequence of the purified protein showed a high degree of homology with various members of the Kunitz family of inhibitors. This inhibitor, which inhibited trypsin activity with a Ki of 5.3 x 10(-10) M, is formed by a single polypeptide chain with an arginine residue in the reactive site.     

Mouse bone collagenase inhibitor: purification and partial characterization of the inhibitor from mouse calvaria cultures

The organ culture of neonatal mouse calvaria produced both collagenase and collagenase inhibitor. The inhibitor was purified by a series of column chromatographies: DEAE-cellulose and CM-cellulose ion-exchange chromatography, concanavalin A-Sepharose and heparin-Sepharose affinity chromatography, and finally by Sephacryl S-200 gel filtration. The purified inhibitor migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular mass of 28,000. The inhibitor was purified 140-fold to a specific activity of 163 units/mg with a yield of 18% over the first step of the purification by DEAE-cellulose chromatography. The inhibitor stained positively for carbohydrate with periodic acid-Schiff's reagent indicating, in conjunction with its affinity to concanavalin A, that the inhibitor is a glycoprotein. In addition to mouse bone collagenase, this inhibitor also inhibited chick bone, rat bone, rabbit corneal, and human gingival collagenase, but did not inhibit bacterial collagenase.     

Hypoxanthine phosphoribosyltransferase from human brain: purification and partial characterization

A facile and rapid purification procedure, based upon the heat denaturation of extraneous proteins and GMP-Sepharose affinity chromatography, has been used to purify hypoxanthine phosphoribosyltransferase from human brain. A homogeneous enzyme preparation, as judged by sodium dodecyl sulfate and gradient polyacrylamide gel electrophoresis, was obtained. The subunit molecular weight of the enzyme was estimated as 24,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native molecular weight, determined by gradient gel electrophoresis, was approximately 100,000. These results suggest human brain hypoxanthine phosphoribosyltransferase is a tetramer, consistent with recent results reported for the human erythrocyte enzyme. At least three charge variant forms of the human brain enzyme were distinguished by nondenaturing polyacrylamide gel electrophoresis, electrofocusing, and chromatofocusing. Acidic pI values of approximately 5.7, 5.5, and 5.0 were estimated for the three major species.     

Uracil phosphoribosyltransferase from Acholeplasma laidlawii: partial purification and kinetic properties

Uracil phosphoribosyltransferase was purified 34-fold from sonicated extracts of Acholeplasma laidlawii by ammonium sulfate precipitation, binding to DEAE-Sephadex, Sephadex G-200 chromatography, and hydroxylapatite chromatography. The molecular weight of the enzyme by gel filtration was approximately 80,000. The pH optimum for phosphoribosylation was around 7.5, and the optimum MgCl2 concentration was 5 mM. Initial velocity studies were conducted over a wide range of both uracil and 5-phosphoribosyl-1-pyrophosphate (P-Rib-PP) concentrations, and various equations for biomolecular reaction mechanisms were fitted to the data by nonlinear regression. When the equation for an ordered sequential mechanism was fitted to the data, the Kia thus obtained was not statistically different from zero. This is interpreted as evidence for a nonsequential ("ping-pong") reaction. Graphic analysis of the data by the Hanes-Woolf linear transform supported this conclusion. The enzyme has high affinity for uracil (KmUra = 4.2 microM; KmP-Rib-PP = 66 microM), which provides supporting evidence that this activity is responsible for the incorporation of uracil and uridine into nucleotides.     

Demonstration and partial purification of lactoperoxidase from human colostrum

A peroxidase with stability, chromatographic and immunoreactive properties similar to that of bovine lactoperoxidase has been partly purified from human colostrum. Hydrophobic interaction chromatography on Phenyl-Sepharose C1-4B gave a 10-fold purification with an apparent recovery of about 45%. The enzyme was quantitatively and specifically adsorbed to beads of anti-lactoperoxidase (bovine)-Protein A-Sepharose. No adsorption of the enzyme was observed on immunoadsorbent columns prepared with high-titre polyclonal antibodies raised against human myeloperoxidase and human eosinophile peroxidase.     

An elastolytic proteinase from rabbit leukocytes: purification and partial characterization

A proteinase with elastolytic activity was isolated from granules of rabbit bloodstream leukocytes, and purified to apparent homogeneity by a multi-step procedure consisting of ammonium sulfate precipitation, batch fractionation on DEAE-Sephadex A-50, and finally by preparative isoelectric focusing (IEF) on Sephadex G-75 Superfine. The molecule weight of the enzyme, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was 28,500. This enzyme shows an isoelectric point at pH 9.0. The proteinase is active against natural elastins as well as toward Suc-(Ala)3-NA, Methoxy-Suc-(Ala)2-Pro-Val-NA, and (to a lesser extent) against Suc-(Ala)2-Pro-Leu-NA and Boc-Ala-ONp. The inhibition profile of the isolated enzyme indicates that rabbit granulocyte elastase belongs to the group of serine proteinases. Inhibition by some natural proteinase inhibitors is also observed. Unlike other mammalian elastases, it is insensitive to elastatinal.     

Ribonuclease inhibitor from human placenta: rapid purification and assay.

The ribonuclease inhibitor from human placenta may be isolated in 65% yield (2.5 mg per placenta) in 2 days. The performance of the affinity chromatography on Sepharose-RNase A has been expedited through adaption of the spectrophotometric assay of ribonuclease toward 2',3'-cyclic cytidine monophosphate to determination of the inhibitor activity. The result of these improvements in procedure is increased availability of the ribonuclease inhibitor for study of its chemical properties (Blackburn, P., and Jailkhani, B.L. (1979) J. Biol. Chem. 254, 12488-12493) and for its inclusion into in vitro systems in which inhibition of mammalian neutral ribonucleases is desired.