Purification of multiple forms of the soluble 17alpha-hydroxy steroid dehydrogenase or rabbit liver.

Eight distinct forms of the soluble 17alpha-hydroxy steroid dehydrogenase of rabbit liver were resolved by DEAE-cellulose chromatography and isoelectric focusing. Five of these enzymes were homogeneous as judged by polyacrylamide-gel electrophoresis. Substrate-specificity studies carried out with oestradiol-17alpha and oestradiol-17alpha 3-glucuronide revealed a variation in activity toward these substrates among the different purified enzyme forms. Three forms of the 17alpha-hydroxy steroid dehydrogenase exhibited equal activity toward both oestrogen substrates, whereas three forms of the enzyme displayed a greater activity toward the glucuronide derivative of oestradiol-17alpha. One enzyme in particular is essentially specific for oestradiol-17alpha 3-glucuronide, its activity toward oestradiol-17alpha being only one-thirtieth that observed with the 3-glucuronide derivative.     

Properties of the multiple forms of the soluble 17alpha-hydroxy steroid dehydrogenase of rabbit liver.

The six forms of the 17alpha-hydroxy steroid dehydrogenase purified from rabbit liver cytosol have very similar physical properties. The molecular weights of all the enzymes were within 3% of the average mol.wt of 39 600. Only one of the six enzymes showed a significant difference in amino acid composition. All but one form of the 17alpha-hydroxy steroid dehydrogenases exhibited greater activities towards the androgen, epitestosterone, than towards oestrogen substrates. With oestrogen substrates one enzyme displayed a high specificity towards the substrate oestradiol-17alpha 3-glucuronide. This high activity was lost if the glucuronic acid moiety was removed or replaced by glucose or galacturonic acid. The other enzyme forms had approximately equal activity toward oestradiol-17alpha and its glucuronide or glucoside derivative. However, substitution of galacturonic acid at C-3 of oestradiol-17alpha substantially decreased the activity of all but one enzyme form.     

Studies of mammalian glucoside conjugation

The mammalian glucoside-conjugation pathway was studied by using p-nitrophenol as the model substrate and mouse liver microsomal preparations as the source of enzyme. The microsomal preparations supplemented with UDP-glucose glucosylated p-nitrophenol; p-nitrophenyl glucoside was identified by chromatography in six solvent systems. The unsolubilized glucosyltransferase of fresh microsomal preparations did not follow the usual Michaelis-Menten kinetics and was easily inhibited by many steroids. All the steroids tested inhibited glucosylation of p-nitrophenol to a greater degree than glucuronidation of p-nitrophenol when assayed in the same microsomal preparations. The steroids inhibited glucosylation with the following decreasing effectiveness: pregnan-3alpha-ol-20beta-one (3alpha-hydroxypregnan-20-beta-one)>oestradiol-17beta 3-methyl ether>oestradiol-17beta>oestriol>pregnane-3alpha,20beta-diol>oestrone. Pregnan-3alpha-ol-20beta-one, pregnane-3alpha,20beta-diol and oestrone had negligible effect on glucuronidation.     

The transfer of glucose to steroids by chimpanzee liver microsomes.

Microsomal preparations from chimpanzee liver can transfer glucose from UDPglucose to the 17alpha-hydroxyl group of 17alpha-estradiol and of 17alpha-estradiol-3-glucuronide. A phenolic glucoside of estrone, but not of either 17alpha- or 17beta-estradiol is also formed. No formation of glucosides of p-nitrophenol, or of diethylstilbestrol was demonstrated. The specificity of glucosyl transfer in the chimpanzee is not identical to that in either the human, the rabbit, or the sheep.     

Purification and properties of a β-glucuronidase from pig kidney

A β-glucuronidase has been isolated from pig kidney and purified 1600-fold using sodium desoxycholate precipitation, ammonium sulphate fractionation, heat treatment and chromatography on Sephadex G200, DEAE-cellulose (DE-52) and hydroxyapatite. The enzyme activity was assayed using oestrone 3-glucuronide as substrate; the final specific activity was 254 nmol oestrone/min/mg of protein. The purified enzyme showed apparent homogeneity in gel filtration and polyacrylamide gel electrophoresis. The pig kidney β-glucuronidase has a single pH optimum of 4.0–4.4 in acetate- and 5.4 in citrate-buffer; an activation energy of 16,800 cal/mol and a molecular weight of 275,000 were estimated. The K_M for oestrone 3-glucuronide was 22.6 μM. The enzyme was not inhibited by N-ethylmaleimide nor by dithioerythritol, however, it was strongly inhibited by Hg²⁺. Oestradiol-17β 3-glucuronide and oestriol 3-glucuronide acted as competitive inhibitors, whereas oestradiol-17β 17β-glucuronide, oestriol 16α-glucuronide, testosterone 17-glucuronide and cholesteryl 3-glucuronide were uncompetitive, pregnanediol 3-glucuronide was noncompetitive, and Cortisol 21-glucuronide gave a mixed type inhibition. The synthetic β-d-glucuronides of phenolphthalein, p-nitrophenol, naphthol, 6-bromo-naphthol and methylumbelliferone all inhibited the hydrolysis of oestrone 3-glucuronide; the inhibition was of a more complex type than simple competitive inhibition.     

Oestradiol-17 alpha dehydrogenase from chicken liver.

The NADP+-linked oestradiol-17 alpha dehydrogenase (EC 1.1.1.148) present in cell-free extracts of chicken liver was investigated with the aim of separating it from a closely related oestradiol-17 beta dehydrogenase (EC 1.1.1.62) found in the same subcellular fraction. However, its chromatographic behaviour on CM-cellulose and DEAE-cellulose was almost identical with that previously reported for the latter enzyme, including resolution into two peaks on the anion-exchanger. Both peaks contained oestradiol-17 alpha dehydrogenase and oestradiol-17 beta dehydrogenase activity. Further attempts to separate the putative enzymes by dye-ligand chromatography with the use of the dyes Procion Yellow, Reactive Red and Cibachron Blue linked to Sepharose were unsuccessful, and they behaved identically on affinity columns of adenosine 2',5'-bisphosphate-agarose and 17 beta-oestradiol 3-hemisuccinate bound to Sepharose. A previous report of partial separation on Sephadex G-200 was not confirmed. Slab gel electrophoresis of enzyme preparations after affinity chromatography on adenosine 2',5'-bisphosphate-agarose revealed multiple bands in systems containing sodium dodecyl sulphate, whereas analysis by rod gel electrophoresis gave two major and one minor bands that stained coincidently for oestradiol-17 alpha dehydrogenase, oestradiol-17 beta dehydrogenase, epitestosterone dehydrogenase and testosterone dehydrogenase activities. Isoelectric focusing gave four enzymically active peaks that each oxidized oestradiol-17 alpha and -17 beta. Apparent Km values for the two forms of oestradiol-17 alpha dehydrogenase obtained by DEAE-cellulose chromatography were 17 and 23 microM for oestradiol-17 alpha, and 8.7 and 11.0 microM for NADP+. Limited kinetic studies with oestradiol-17 alpha and -17 beta with the use of the mixed-substrate method showed that the total velocity was equal to the sum of the separate velocities. The active-site inhibitor-alkylating agent 17 beta-(1-oxoprop-2-ynyl)androst-4-en-3-one did not cause time- or temperature-dependent inhibition, in contrast with the reported case of the oestradiol-17 beta dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase activities of the human placental oestradiol dehydrogenase. NADP+ appeared to afford some protection against inhibition. Investigation of substrate specificity with a limited range of steroids suggests that the enzyme(s) from chicken liver differs substantially from the oestradiol-17 beta dehydrogenase from human placenta, and although the evidence is not conclusive it suggests the existence of one enzyme.     

Androgen glucuronyl transferase activity in rat liver, evidence for the importance of hepatic tissue in 5 alpha-reduced androgen metabolism

To elucidate the role of the liver in 5 alpha-reduced androgen metabolism, we used a rat liver glucuronyl transferase assay to determine the conversion of 17 beta-hydroxy-5 alpha-androstane-3-one (DHT), 5 alpha-androstane-3 alpha, 17 beta-diol (androstanediol), and androsterone to their glucuronide metabolites. Serum levels of the two isomers of androstanediol glucuronide (androstanediol 3- and 17-glucuronide) were also measured. Using 5 microM unconjugated steroid as substrate, the production rate (pmol/mg/min) for each product from its respective unconjugated steroid was 6.9 +/- 0.4 for DHT glucuronide, 101 +/- 3.3 for androstanediol 3-glucuronide, 71 +/- 2.0 for androstanediol 17-glucuronide, and 181 +/- 11 for androsterone glucuronide. Production rates for androstanediol glucuronide were 800 times greater for rat liver than for rat prostate, when examined under similar conditions. In the presence of either 0 or 5 microM unlabeled androstanediol, about 60% of the androstanediol glucuronide formed by rat liver was androstanediol 3-glucuronide. In normal male rat serum, 69 +/- 8% (mean +/- SEM) of total androstanediol glucuronide was androstanediol 3-glucuronide. We have previously shown that rat prostate forms androstanediol 17-glucuronide, but not androstanediol 3-glucuronide. The results from the present study indicate that rat liver forms both androstanediol glucuronide isomers, and does so in about the same ratio as is found in rat serum. The rate of glucuronidation is also much greater in rat liver than in rat prostate. While other sites of glucuronidation are possible, these results are consistent with the hypothesis that DHT and other unconjugated androgens formed in rat prostate are conjugated to glucuronic acid mainly in the liver.     

An improved assay for hepatic glycogen synthase in liver extracts with emphasis on synthase R

An assay for measurement of optimal amounts of glycogen synthase R, the physiologically active form of the enzyme, in liver tissue extracts is described. Tissue extracts enriched in synthase R had a pH profile different from those reported for synthase D and synthase I. In tissue extracts, synthase I had a broad pH optimum but maximal activity was present at pH 7.0-9.0. Synthase D had a sharp pH optimum at pH 8.5 and had little activity at pH 7.0, either in the presence or in the absence of glucose 6-phosphate (G6P). In extracts enriched in synthase R, the pH optimum was 7.0-8.0 without G6P, but 8.0 with G6P. The synthase R activity without G6P rapidly decreased at a higher pH. The proportion of synthase in the physiologically active form traditionally has been reported as an activity ratio based upon the activity in the presence and absence of G6P. The assay has been performed at a single pH. Because of the differences in pH profile, we recommend that the enzyme be measured at pH 7.0 in the absence of G6P and pH 8.5-8.8 in the presence of G6P. In previous assays the substrate UDP-Glc concentration used often has been less than saturating, and the G6P concentration generally has been excessive. A substrate concentration of 11 mM UDP-Glc was found to be necessary for maximal activity. A G6P concentration of 2 mM is adequate for measurement of the D form of the enzyme.     

Lipolytic enzymes of Trichophyton rubrum.

Trichophyton rubrum cells contain lipase, phospholipases A and B and acyl CoA lysolecithin acyl transferase activities. This dermatophyte excretes lipase and phospholipase A into the growth medium when cultivated in Sabouraud's broth. Extracellular lipase has optimum activity at pH 8.0 whereas the intracellular lipase is maximally active at pH 8.0 whereas the intracellular lipase is maximally active at pH 7.0. The optimum pH of phospholipase A and B activities which are localized in 15000 g sedimentable cell fragments are 7.0 and 6.0 respectively. Supernatant obtained after removal of 1,005,000 g sedimentable fragments from cell extract contains acyl CoA lysolecithin acyl transferase which requires ATP, CoA, Mg2+ and an unsaturated fatty acid for its activity.     

Transfer of xylose to steroids by rabbit liver microsomes.

Rabbit liver microsomal preparations can transfer xylose from UDP-xylose to estron, 17alpha-estradiol, and 17beta-estradiol, and, in poorer yield, to diethylstilbestrol and p-nitrophenol. No transfer of xylose to estriol, testosterone, epitestosterone or 17alpha-estradiol 3-glucuronide could be demonstrated. The xyloside of [6,7-3H]estrone which was formed by liver microsomes crystallized to constant specific activity with estrone beta-D-xylopyranoside, the chemical preparation of which is described.     

Effect of conjugation to glucose on the uptake of 17 beta-(6,7-3-H)estradiol by rabbit liver subcellular fractions.

Homogenates and nuclear fractions from rabbit liver were incubated with 17β-[6,7-³H] estradiol and with the 3-glucoside and 3-glucuronide of this steroid. Localization of 17β-estradiol in the cell nucleus was enhanced when the glucoside rather than the free steroid was incubated with homogenates. It is suggested that conjugation to glucose may facilitate the transport of the steroid into the liver cell, or may protect it from metabolism. The effect was not observed in uterine tissue fractions.     

EFFECT OF THE HYDROGEN ION CONCENTRATION ON THE PHAGOCYTOSIS AND ADHESIVENESS OF LEUCOCYTES

1. Immediately after coming into contact with glass, leucocytes are most adhesive at pH 8.0 or > 8.0. 2. Agglutination of leucocytes increases with increasing H ion concentration from pH 8.0 to 6.0. 3. In phagocytosis experiments where leucocytes creep about on the slide picking up articles the optimum pH is 7.0. Here ameboid movement is probably the limiting factor. 4. The optimum for phagocytosis of quartz from suspension is on the acid side of neutrality at or near pH 6.7. 5. Phagocytosis of quartz increases with the acidity, while adhesiveness of leucocytes to glass increases with the alkalinity.     

Changes in the plasma concentrations of free and conjugated oestrogens in heifers after treatment to induce superovulation and the relationship with number of ovulations

Oestradiol-17 beta and conjugated oestrone, oestradiol-17 beta and oestradiol-17 alpha were measured in peripheral plasma of heifers treated with PMSG/PGF-2 alpha to induce superovulation. Changes in the concentrations of each hormone were synchronous, the highest level being near oestrus. For a given number of ovulations the hormone with the highest concentration was total oestradiol-17 alpha, then came total oestrone, total oestradiol-17 beta and oestradiol-17 beta. For each oestrogen, the maximum preovulatory concentration measured was significantly correlated with the number of ovulations; the regression line for total oestradiol-17 alpha was twice as steep as that for oestradiol-17 beta. It is concluded that in animals treated to induce superovulation assay of total oestradiol-17 alpha gives a better induction of the number of follicles induced to ovulate than does the more conventional assay of oestradiol-17 beta.     

Distinct pH modulation for dual function of Galphah (transglutaminase II)

Galpha(h), also known as transglutaminase II, has GTPase as well as transglutaminase activities. To better understand the factors affecting these dual enzymatic activities, we examined the optimal pH (at 25 degrees C) and thermal stability (at 37 degrees C) of the activities using membranous Galpha(h) from mouse heart. The optimum pH for the GTPase activity of Galpha(h) is approximately 7.0. As well, the GTP binding activity of Galpha(h) is more thermostable at pH 7.0 than that at pH 9.0. Consistent with these observations on the GTPase function of Galpha(h), both the phospholipase C-delta1 activity and the yield of co-immunoprecipitation of Galpha(h)-coupled phospholipase C-delta1 in alpha(1)-adrenoceptor/Galpha(h)/phospholipase C-delta1 complex preparations were enhanced by incubation with an alpha(1)-agonist, phenylephrine, at pH 7.0. On the other hand, the transglutaminase activity of Galpha(h) is higher in the basic pH range with an optimum activity at pH approximately 9.0. Also, the transglutaminase activity of Galpha(h) is more thermostable at pH 9.0 than that at pH 7.0. These results indicate not only pH as a modulator for the dual functions of Galpha(h), but also provide direct evidence for the involvement of pH in the Galpha(h)-mediated alpha(1)-adrenoceptor signaling system in vitro.     

有机磷农药在水生态系中生物净化机理研究——1.对硫磷的酶解

从氧化塘系统中分离出能降解对硫磷的细菌Pseudomonas sp.代号CTP-01,能将对硫磷分解成对硝基酚和二乙基硫代磷酸酯,并进一步分解对硝基酚。在有Cu++存在的情况下,酶比活可以达到1×104毫微克分子/毫克蛋白/分钟,Cu++对酶有激活作用,并对温度和pH影响有保护作用。对硫磷水解酶反应最适温度为40—50℃,超过50℃活性急剧降低,80℃完全失活。 CTP-01的对硫磷水解酶大部分是同膜片结合状态存在,超声破碎的无细胞酶制剂中,只有37.2%的活力存在于可溶性蛋白部分。       

Studies on a testosterone glucuronyltransferase from the cytosol fraction of human liver

An enzyme that conjugates the 16alpha-hydroxyl group of oestriol with glucuronic acid was found in the cytosol fraction of human liver. The enzymic activity could not be sedimented when the cytosol fraction was centrifuged at 158000g(av.) for 120min. The oestriol 16alpha-glucuronyltransferase was purified 100-fold by 0-30% saturation of the cytosol fraction with ammonium sulphate followed by filtration of the precipitate through Sephadex G-200. The activity was eluted at the void volume. The product of the reaction, oestriol 16alpha-monoglucuronide, was identified by paper chromatography and by crystallization of radioactive product to constant specific radioactivity. The optimum temperature was 37 degrees C, and the activation energy was calculated to be 11.1kcal/mol. The apparent Michaelis-Menten constants for oestriol and UDP-glucuronic acid were 13.3 and 100mum respectively. Cu(2+), Zn(2+) and Hg(2+) inhibited, whereas Mg(2+), Mn(2+) and Fe(2+) stimulated the enzyme. Substrate-specificity studies indicated that the amount of oestradiol-17beta, oestradiol-17alpha and oestrone conjugated was not more than about 5% of that found for oestriol. Oestriol 16alpha-monoglucuronide, a product of the reaction, did not inhibit the 16alpha-oestriol glucuronyltransferase; in contrast, UDP, another product of the reaction, inhibited the enzyme competitively with respect to UDP-glucuronic acid as the substrate, and non-competitively with respect to oestriol as the substrate. ATP and UDP-N-acetylglucosamine did not affect the oestriol 16alpha-glucuronyltransferase. 17-Epioestriol acted as a competitive inhibitor and 16-epioestriol as a non-competitive inhibitor of the glucuronidation of oestriol. 5alpha-Pregnane-3alpha,20alpha-diol also inhibited the enzyme non-competitively. It is most likely that the oestriol 16alpha-glucuronyltransferase described here is bound to the membranes of the endoplasmic reticulum.     

Comparative studies on thymidylate synthetase from P. berghei and mouse reticulocytes

Partially purified thymidylate synthetase from Plasmodium berghei and mouse reticulocytes was characterized. The mol. wt of the enzyme from P. berghei was about twice that from mouse reticulocytes. The optimum pH of the enzyme from P. berghei was found to be 6.5-7.5 while that from the host was 7.0-8.0. The enzyme from P. berghei was more susceptible to pH denaturation than the enzyme from reticulocytes. The enzyme from both sources differed in their Km values for substrates. The enzyme from reticulocytes was less sensitive to inhibition by substrate analogs than that from P. berghei.     

Proteolytic enzymes of Saccharomyces carlsbergensis

1. Of four proteolytic enzymes isolated from autolysing Saccharomyces carlsbergensis, one is inactivated at about 45 degrees C, whereas the others are stable at 50 degrees C. pH optima for activity are from 3.0 to 8.0 but maximum stability is between pH6.0 and 6.5. All appear to be glycoproteins, the carbohydrate moiety containing glucose and mannose residues. 2. Lysed protoplasts of the same yeast release four proteolytic enzymes each of which have two pH optima at pH3.0 and 7.0 approximately. Compared with the enzymes from autolysed yeast, resistance to high temperature is much less, and they are not glycoprotein in nature. 3. The same yeast grown with N-acetyltyrosine ethyl ester as nitrogen source secretes into the medium four proteases believed to be glycoprotein in nature. Generally they resemble the enzymes from lysed protoplasts more than those from autolysing yeast.     

Characteristics and identity of obligately aerobic spoilage yeasts from fish silage

Yarrowia (Candida) lipolytica was the predominant organism isolated from the surface film of growth derived from ground hake gurry to which only phosphoric acid was added to give a pH of 4.0. The optimum pH for the crude extracellular protease activity of two distinguishable strains of Y. lipolytica, designated CL1 and CL2, with casein as substrate was 7.0. The optimum temperature of the crude extracellular protease activity from both strains was 50 degrees C. The addition of 2.0% glucose to broth cultures resulted in a significant increase in final cell mass and extracellular protease activity but resulted in a reduction in the units of protease activity per mg of dry cell mass at initial pH values of 5.6 and 7.0 but not an initial pH of 8.0.     

Synthesis of a precursor for the preparation of 9 alpha,11 alpha-tritiated 5 alpha-androstane-3 alpha,17 beta-diol 17-glucuronide

Starting from 11 beta-hydroxytestosterone, we achieved the synthesis of a strategic precursor, C-9 (11) unsaturated 5 alpha-androstane-3 alpha, 17 beta-diol 17-glucuronide (9a), for the preparation of 9 alpha,11 alpha-tritiated 5 alpha-androstane-3 alpha, 17 beta-diol 17-glucuronide. We optimized the reaction conditions for catalytic reduction employing hydrogen and subsequent base hydrolysis followed by purification on Amberlite XAD-2 resin to obtain the saturated 5 alpha-androstane-3 alpha, 17 beta-diol 17-glucuronide.