Merle Mason

EFFECTS OF STEROID AND NONSTEROID METABOLITES ON ENZYME CONFORMATION AND PYRIDOXAL PHOSPHATE BINDING

Studies on the effects of steroid and nonsteroid metabolites on enzy me conformation and pyridoxal phosphate binding were reviewed. The results indicate that conjugated steroids can cooperate with or oppose the actions of specific nonsteroid effectors in determining enzyme conformation activity stability and coenzyme binding. Evidence is presented that these actions of steroid and nonsteroid metabolites can cause a redistribution of pyridoxal phosphate between apoenzymes. It was considered that similar actions may account for the ability of estrogens to cause changes in tryptophan metabolism resembling those occurring during pyridoxine deficiency. Effects of estrogen-treatment and of dietary pyridoxine deficiency on the kynurenine transaminase levels in liver and kidneys of male rats were compared. Pyridoxine deficiency resulted in a moderate decline of the % saturation of the renal supernatant enzyme with pyridoxal phosphate but also caused an apparent decline in apoenzyme levels. Renal mitrochondrial apotransaminase levels decreased slightly during pyridoxine deficiency but were markedly decreased by estrogen treatment. Female rats displayed similar results to those of estrogen-treated males. These results indicate that the estrogens influence kynurenine transaminase in vivo in a way similar to that observed in vitro but also demonstrate that they do not cause the same pattern of change in apoenzyme levels and pyridoxal phosphate distribution as the dietary pyridoxine deficiency.

L-kynurenine aminotransferase and L-α-aminoadipate aminotransferase. I. Evidence for identity

Summary The following observations strongly suggest that L-kynurenine aminotransferase (KAT) and L-α-aminoadipate aminotransferase (AAT) activities are associated with the same protein: 1. a similar distribution in the supernatant and mitochondrial fractions of kidney and liver of male rats, 2. a similar distribution in these fractions of kidney and liver of female rats, 3. a similar sex difference in the supernatant and mitochondrial fractions of kidney with no sex difference in liver fractions, 4. a similar pattern of inhibition with a homologous series of dicarboxylic acids with adipate being most effective, 5. competitive inhibition of KAT with d , l -α-aminoadipate (αAA), 6. substrate inhibition of KAT with α-ketoadipate (αKA), and 7. AAT activity in a highly purified preparation of KAT. A scheme for the interaction of the two activities in lysine and tryptophan metabolism is presented.

The enzymic synthesis of the sulfate esters of estradiol-17β and diethylstilbesterol

Abstract The present study demonstrates the formation of three sulfate esters of estradiol and three of diethylstilbesterol by a microsome-free extract of rat liver. The sulfate esters were separated by paper chromatography using a potassium phosphate buffer solvent and detected by radioautography using 35S. Evidence is presented that the three sulfate esters of estradiol are estradiol-3-sulfate, estradiol-17-sulfate and estradiol-3,17-disulfate and that those of diethylstilbesterol are two isomeric monosulfate esters and the disulfate ester of diethylstilbesterol.

Testicular steroid sulfatase: Substrate specificity and inhibition

Abstract Kinetic studies of the cleavage of dehydroepiandrosterone-sulfate (1) and androstenediol-3-sulfate by a particulate enzyme preparation from a rat testicular microsomal fraction gave K m values of 2.04 × 10 −6 M for DHA-S and 0.935 × 10 −6 M for androstenediol-3-sulfate with identical V max values. Inhibition studies with equimolar concentrations of substrate and inhibitor demonstrated that 5α-androstane-3β,17β-diol was the most potent inhibitor among fifteen C-19 and C-18 unconjugated steroids investigated. Substitution of: 1) a Δ 4 or Δ 5 bond or phenolic A ring for a saturated A ring, 2) 17α-hydroxyl group for a 17β-hydroxyl group, or 3) a 3α-hydroxyl group for a 3β-hydroxyl group, markedly decreased the inhibitory effect of the steroid. K i values of 1.7 × 10 −6 M, 3.3 × 10 −6 M and 11.8 × 10 −6 M were found with 5α-androstane-3β, 17β-diol, 5α-androstane-3α, 17β-diol and testosterone, respectively. The kinetic data related to inhibition are consistent with partial competitive inhibition.

Conversion of dehydroepiandrosterone sulfate to androst-5-enediol-3-sulfate by soluble extracts of rat testis

Abstract 3H-Androst-5-ene-17-one-3β-y1 sulfate is converted to 3H-androst-5-ene-17β-o1-3β-y1 sulfate by soluble extracts of rat testis in the presence of added NADH2 or NADPH2.The conversion was approximately 4 times greater than the conversion of 3H-androst-5-ene-17-one-3β-o1 to 3H-androst-5-ene-3β, 17β-diol under similar incubation conditions. The same extracts converted 3H-androst-5-ene-17-one-3β-o1 to 3H-androst-5-ene-17-one-3β-y1 sulfate in the presence of added ATP and Mg++.

Sulfurylation of estradiol-17B by extracts of ovary, corpus luteum, testis and adrenal cortex1

Abstract Soluble enzyme preparations from bovine ovary, corpus luteum and adrenal cortex and from rat testis sulfurylated estradiol-17β in the presence of ATP and Mg++. Two major products were tentatively identified by chromatographic comparisons as estradiol-3-sulfate and estrone sulfate2. In addition, two unidentified, highly-polar derivatives were formed from estradiol-17β in relatively small amounts by ovary, corpus luteum and testis. Estradiol-17-sulfate, which was formed readily in similar extracts of rat liver, was not formed in detectable amounts by the extracts of ovary, corpus luteum, testis, and adrenal cortex under the conditions described.

Tyrosine-α-ketoglutarate transaminase effect of the administration of sodium benzoate and related compounds on the hepatic enzyme level

Summary Intragastric administration of sodium benzoate caused increased levels of tyrosine-α-ketoglutarate transaminase ( L -tyrosine : 2-oxoglutarate aminotransferase, EC 2.6.1.5) in the livers of intact and adrenalectomized rats. Maximal increases occurred 3 h after benzoate administration to adrenalectomized rats. The response increased as the benzoate dosage was varied from 5 to 20 mg per 150 g body weight, reaching a maximal level 4-fold greater than that of saline-injected controls. -Higher dosage levels gave little if any further increase and in many cases resulted in toxic manifestations. Thirty-one cyclic compounds were compared with benzoate as inducing agents. Hydrocortisone, hydrocortisone hemisnccinate and diethylstilbestrol disulfate were more effective than benzoate. Cyclohexanoate, p-aminobenzoate, and α-naphthoate were as effective. Benzene, phenol, α-naphthol, β-naphthoate, phenylacetate, α-naphthylacetate, nicotinate, α-picolinate, salicylate, p-nitrobenzoate, cyclohexane, pyridoxine, pyridoxoate, DL -phenyllactate, o-phthalate, terephthalate, o-diphenate, pyromellitate, isonicotinate, benzene sulfonate, pyridine 3-sulfonate, sulfanilate, anthranilate, p-hydroxybenzoate, and o-nitrobenzoate were either substantially less effective or were ineffective. Strong inhibition of the increase by injected puromycin and actinomycin D, compounds which inhibit protein and RNA synthesis respectively, suggests that the benzoate-mediated effect occurred by a mechanism involving increases in protein and RNA synthesis. In this respect, the effect of benzoate resembles that of the glucocorticoids.

Hydrogenation of testosterone sulfate by the Δ4-3-ketosteroid reductase of rat liver microsomes

Abstract TPNH2 is oxidized in the presence of testosterone or testosterone sulfate and a microsomal preparation from the liver of female rats. In the presence of iorganic phosphate, DPNH2 also acts as a hydrogen donor. Evidence is presented that testosterone sulfate is converted predominantly to the 4,5-dihydro derivative, 3-keto-5α-androstane - 17-yl sulfate.

Studies of the in vitro and in vivo effects of conjugated steroids and carboxylic acids on hepatic tyrosine transaminase in the rat

Abstract 1. 1. Tyrosine transaminase ( l -tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) activity was lost rapidly in fresh rat-liver homogenates (pH 6.9), that were incubated at 38°. The inactivation was paralled by the loss of the coenzyme but was not reversed by the subsequent addition of pyridoxal 5-phosphate. 2. 2. The coenzyme, the keto acid substrates, and their anionic analogs retarded the inactivation and dissociation. Various anionic steroids and diethylstilbestrol disulphate (5·10−4−5·10−5 M) also retarded the inactivation and dissociation; free steroids were ineffective at saturation levels. Aromatic carboxylic acids were effective at 10−2−10−3 M, 5-hydroxytryptophan at 10−3 M, and l -glutamate, bicarbonate, and Pi at 10−2 M. Several other amino acids and NaCl were ineffective at 10−2 M. Many of the in vitro stabilizing agents caused elevated levels of hepatic tyrosine transaminase when injected into adrenalectomized rats. In general, the most potent stabilizers were also the most effective agents in causing the elevated enzyme levels in vivo. 3. 3. Estradiol disulfate and diethylstilbestrol disulfate also retarded the inactivation and dissociation that occured when the homogenates were incubated at 25° in 1.1 or 2.2. M urea or when partially-purified tyrosine transaminase was incubated with trypsin (EC 3.4.4.4) or chymotrypsin (EC 3.4.4.5). The rate of inactivation in homogenates was not significantly changed by the presence of 0.001 M EDTA or mercaptoethanol nor by incubation with alkaline phosphatase (EC 3.1.3.1). 4. 4. A small but significantly greater degree of association of the tyrosine transaminase with its coenzyme was found in rat-liver homogenates prepared 1 h after cortisol administration than in the injected controls that were sacrificed immediately. There was also a significantly slower rate of coenzyme dissociation in the 1-h animals. Similar doses of cortisone were ineffective in the latter case.

Effects of calcium ions and quinolinic acid on rat kidney mitochondrial kynurenine aminotransferase

Abstract At pH 6.4, rat kidney mitochondrial kynurenine aminotransferase activity is enhanced several-fold by the addition of CaCl2, apparently because Ca++ facilitates the translocation of α-ketoglutarate, one of the substrates, across the mitochondrial inner membrane. Chloride salts or Mg++, Mn++, Na+, K+, and NH4+ did not have this effect. At pH 6.8, the enzyme activity was near maximal even without added Ca++ but was strongly depressed by either of two calcium chelating agents, quinolinic acid (Q.A.) and ethyleneglycol-bis(β-aminoethyl ether)N,N′-tetraacetic acid (EGTA). These observations support the view that Ca++ is involved in regulating kidney mitochondrial translocation of α-ketoglutarate and that the reported interference of polycarboxylate anion translocation by Q.A. in vivo depends on the ability of that agent to chelate Ca++.