Donald P. Wallach

Properties of a novel preparation of prostaglandin synthetase from sheep seminal vesicles

Abstract 1. (1) The objective of this investigation was to improve yields of prostaglandin Es using the prostaglandin synthetase system derived from sheep seminal vesicular glands so that a broad program of biological evaluation of prostaglandin E 2 and derivatives could be undertaken and supported. 2. (2) The synthetase associated with the microsomes can be precipitated by either acidification to pH 4.8–5.0, or alternatively by adding acetone to a concentration of 30%, and then recovered by centrifugation at 3000–4000 × g . An acetone-pentane powder prepared from this microsomal precipitate retains full prostaglandin synthetase activity. 3. (3) The conversion of arachidonic acid to prostaglandin E 2 by this acetonepentane powder preparation proceeds rapidly and approaches an asymptote within 10 min at 30°. At this point, the enzyme may be readily removed from the incubation mixture by centrifugation and added to fresh medium. The reaction begins again and continues to nearly the same extent as the initial incubation. This, and other evidence discussed, suggests that the prostaglandin synthetase system is inhibited by one or more products of the reaction. Cycling the enzyme 10 times with judicious additions of fresh enzyme to compensate for losses results in yields 3-fold higher than were hitherto possible using whole tissue homogenates. 4. (4) Some other important properties of this enzyme preparation are discussed.

Studies on the arachidonic acid cascade—I: Inhibition of phospholipase A2in vitro and in vivo by several novel series of inhibitor compounds

Abstract A series of compounds have been discovered which are potent inhibitors in vitro of hog pancreas, cobra venom, and bee venom phospholipase A 2 . Collagen-induced aggregation of human platelets was prevented by representatives of this series. The inhibition was reversed by aggregatory concentrations of arachidonate, indicating the hydrolysis of esterified arachidonate had been prevented. In the isolated perfused guinea pig lung sensitized to ovalbumin, the release of prostanoids, resulting from a challenge dose of the antigen, was prevented by exemplars of these compounds. Subsequent administration of arachidonate in the presence of the inhibitor resulted in full prostanoid synthesis and secretion, again indicating that the block was at the phospholipase level. Administration of some of these compounds to guinea pigs by subcutaneous or intraperitoneal routes delayed the onset, and decreased the severity, of erythema induced in depilitated skin by the controlled application of ultraviolet light. This result was, also, consistent with phospholipase A 2 inhibition. Kinetic experiments with hog pancreas phospholipase A 2 demonstrated that, with representatives of this series, the inhibition induced was noncompetitive, and appropriate dissociation constants have been calculated.

A novel preparation of human platelet lipoxygenase: Characteristics and inhibiton by a variety of phenyl hydrazones and comparisons with other lipoxygenases

Acetone-pentane powder preparations of human blood platelets were prepared and the characteristics of the 12L-lipoxygenase were studied using measurements of oxygen consumption. No sharp pH optimum with equal reaction velocities was observed over a range of pH 7.5-8.5 in a variety of buffers. The enzyme could be easily solubilized in 1% deoxycholate, and in this form was moderately stable to heat. Of 10 divalent cations tested at a concentration of 3.7 . 10(-3) M, only zinc and tin were inhibitory. None of he other ions was stimulatory. The products of the oxidation of arachidonate were characterized from both soluble and insoluble enzyme preparations. With the insoluble suspension, 55% of the added arachidonate was recovered as 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and 27% appeared as trihydroxyeicosatetraenoic acid isomers (THETEs). With the soluble preparation, 84% of the arachidonate appeared as 12-HETE and only 5% as THETEs. The Michaelis constants for dihomo-gamma-linolenic aicd, arachidonic acid, and eicosapentaenoic acid substrates are presented. A series of phenyl hydrazone inhibitors of various structural types were discovered to be potent inhibitors of the human platelet lipoxygenase. The sensitivities of this enzyme to these inhibitors were compared to soybean lipoxygenase and sheep seminal vesicular cyclodioxygenase. In general, the soybean enzyme was most sensitive; the sheep seminal vesicular cyclodioxygenase was the least sensitive and the human platelet lipoxygenase was intermediate between the two. The Ki values for two phenyl hydrazone inhibitors with both soybean and human platelet lipoxygenases is presented.

Purification and properties of a tween-hydrolyzing enzyme from rat adipose tissue.

Abstract A method is described for the preparation with good yields and approx. 75-fold purification of a hydrolase preparation from rat adipose tissue. Ultracentrifuge studies indicate that the best preparations are approx. 90% pure enzyme protein. The purified enzyme can be electrophoretically resolved into at least two components which appear to be very similar molecular forms of the same enzyme. Provisional substrate-specificity studies demonstrate that the enzyme will hydrolyze a number of different ester bonds including simple aliphatic esters, “Tween” esters, and certain triglyceride esters. With simple aliphatic esters, under the conditions employed, a marked preference is shown by the enzyme for esters in which the alcohol component is small, and the fatty acid moiety is 5–6 carbon atoms in length.

Biosynthesis of 6-keto PGF1α by microsomal acetone-pentane powder preparations from hog aorta, ram seminal vesicles, and bovine corpora lutea: Properties of same

Acetone-pentane powders of microsomal rich acetone precipitated fractions, have been prepared from hog aortas, ram seminal vesicles, and bovine corpora lutea. These preparations are all active in converting C14 labelled PGH2 to prostacyclin. The reaction was followed by quantitation of the spontaneous hydrolytic product, 6-keto PGF1alpha. The heat stability, pH optima, reactions with inhibitors, and other properties of these types are discussed. The comparative behavior of the respective enzyme preparations shows that while qualitatively they behave in a similar manner, quantitatively, there are significant differences between them, particularly with respect to heat treatment, and response to inhibitors.

Studies of the inhibition of lipolytic enzymes: I. the inhibition of a canine kidney and liver lipase in vitro and in vivo BY n-butyl carbamic acid methyl ester (U-14641)

Abstract n-Butyl carbamic acid methyl ester has been shown to be a potent inhibitor of canine liver and kidney lipases both in vitro and in vivo. The nature of this inhibition has been investigated and mechanisms consistent with the data are proposed. When this inhibitor is intravenously administered to dogs in suitable doses, a marked degree of inhibition of liver and kidney lipases has been demonstrated both by direct assay and by the greatly diminished rate of elimination of procaine from the plasma of dogs treated with this compound. A remarkable species specificity has been demonstrated, since butyl carbamic acid methyl ester does not inhibit liver and kidney lipases obtained from five other species.