Influence of castration and testosterone replacement on hypothalamic tyrosine hydroxylase activity in the rat

Summary Hypothalamic tyrosine hydroxylase (TH) activity of castrate rats is modulated by testosterone propionate (TP) in vivo. Kinetic studies revealed that bothV _max andK _m were virtually unaltered for substrate tyrosine in the presence of an excess of DMPH₄ cofactor. TP replacement to castrate rats increased theK _m for added DMPH₄ cofactor, whileV _max decreased. These results suggest that TP decreases TH activity of castrate rats by inhibiting the enzymereduced pteridine cofactor complex.     

Hypothalamic tyrosine hydroxylase activity, plasma gonadotropin and prolactin levels after aminooxyacetic acid in ovariectomized rats

Plasma concentrations of gonadotropin, prolactin and hypothalamic tyrosine hydroxylase (TH) activity were measured in ovariectomized rats treated with aminooxyacetic acid (AOAA), a drug which elevates brain GABA levels. Hypothalamic TH activity was significantly increased with a significant decrease in prolactin (Prl) release. Plasma levels of gonadotropins were not modified by AOAA. These results support an inhibitory action of GABA on Prl release possibly mediated through hypothalamic dopamine.     

Hypothalamic tyrosine hydroxylase activity,plasma gonadotropin and prolactin levels after aminooxyacetic acid in ovariectomized rats

Summary Plasma concentrations of gonadotropin, prolactin and hypothalamic tyrosine hydroxylase (TH) activity were measured in ovariectomized rats treated with aminooxyacetic acid (AOAA), a drug which elevates brain GABA levels. Hypothalamic TH activity was significantly increased with a significant decrease in prolactin (Prl) release. Plasma levels of gonadotropins were not modified by AOAA. These results support an inhibitory action of GABA on Prl release possibly mediated through hypothalamic dopamine.Supported by grants from Indian Council of Medical Research (ICMR), New Delhi. RIA kits for the estimation of LH, FSH and Prl were kindly supplied by Dr A.F. Parlow, NIAMDD-NIH, Bethesda, Maryland, USA. GNB is a UGC research fellow.     

Characterization of D-fructose transport by rat kidney brush-border membrane vesicles: changes in hypertensive rats

D-fructose transport was characterized in renal brush-border membrane vesicles (BBMVs) from both spontaneously hypertensive rats (SHR) and normotensive genetic control Wistar-Kyoto (WKY) rats. Kinetic studies indicated that the maximal rate (Vmax) of D-fructose transport was significantly lower in SHR compared with WKY rats. No differences were observed in the Michaelis constant (Km) or the diffusion constant (Kd) between the two groups of animals. D-fructose inhibited its own transport, whereas the presence of D-glucose, D-galactose, phlorizin, and cytochalasin B did not inhibit the transport of D-fructose in either animal group. To explain the reduction in D-fructose transport in SHR, the density of the D-fructose transporter, GLUT5, was analyzed by Western blot. GLUT5 levels were lower in SHR, a reduction similar to that of the Vmax. Thus, there appears to be a high-affinity, low-capacity, GLUT5-type fructose carrier in the apical membranes of rat kidney cortex, and the decrease in the Vmax of D-fructose transport in renal BBMVs from hypertensive rats correlates well with a reduction in the expression of GLUT5 protein.     

Tyrosine hydroxylase activity and tyrosine titers during cockroach metamorphosis

Summary Tyrosine hydroxylation (TH) to dopa in vivo in nymphs and pharate adults ofPeriplaneta americana was very low, but increased over 30-fold beginning at ecdysis. Free tyrosine increased in pharate, adults and peaked at ecdysis, whereas TH activity peaked 6 h later. Both TH activity and tyrosine titers declined the next 24 h as the cuticle tanned. TH appears to regulate tanning substrate biosynthesis in cockroaches.Contribution No. 81-294-j, Department of Entomology, Kansas Agricultural Experiment Station, Manhattan KS. Research supported in part by National Science Foundation grant No. PCM-8003859. I wish to thank Dr. M.A.J. Al-Izzi for assistance with the enzyme analyses.     

Oxidation of synephrine by type A and type B monoamine oxidase.

Synephrine (SP) was found to be a substrate for monoamine oxidase (MAO) in rat brain mitochondria, showing the Km and Vmax values of 250 microM and 32.6 nmoles/mg of protein/30 min respectively. The inhibition studies showed that the SP oxidation was carried out by both type A and type B MAO and a major part of the activity was due to type A MAO.     

A sensitive and inexpensive high-performance liquid chromatographic assay for tyrosine hydroxylase

We describe a highly sensitive assay method for tyrosine hydroxylase (TH) using high-performance liquid chromatography with amperometric determination. This assay method could be applicable to any tissues with low enzyme activity, such as rat cerebellum. We also describe the kinetic properties of TH in rat cerebral cortex.     

3'5'-Cyclic AMP phosphodiesterase activity in the internal and external layers of human myometrium at the end of pregnancy]

The hydrolysis of cAMP by phosphodiesterase was studied in whole homogenate from human myometrium at the end of the pregnancy before onset of labour. Tissue samples were taken from outer and inner layers of placental and anti-placental sites. Kinetic analysis shows in every case two apparent Km values for low and high cAMP concentrations in the order of 1 x 10(-5) M and 1 x 10(-4) M. On the other hand Vmax values are lower for the enzyme isolated from the placental site than for the one isolated from the anti-placental area. In the 4 zones studied, an appreciable proportion of the low Km enzyme is present.     

A sensitive and inexpensive high-performance liquid chromatographic assay for tyrosine hydroxylase

Summary We describe a highly sensitive assay method for tyrosine hydroxylase (TH) using high-performance liquid chromatography with amperometric determination. This assay method could be applicable to any tissues with low enzyme activity, such as rat cerebellum. We also describe the kinetic properties of TH in rat cerebral cortex.     

Occurrence of mitochondrial monoamine oxidase in human semen.

Mitochondrial monoamine oxidase (MAO) was found in human semen, showing its Km and Vmax values of 91.7 microM and 290 pmoles/mg of protein/60 min, respectively, with kynuramine as substrate. A major part of the activity was due to type A MAO.     

NADP-isocitric dehydrogenase of gerbil adrenal mitochondria: support of steroid hydroxylation

In gerbil adrenal cortex the activity of intramitochondrial NADP-linked isocitric dehydrogenase (IDH) is up to 10-fold greater than the NAD-linked IDH. The NADP-IDH, apparent Km 0.58 mM, Vmax 280 nmoles/min/mg mitochondrial protein, appears to be the major source of reducing equivalents to support adrenal mitochondrial steroid 11B- and 19-hydroxylation in this species.     

Correlation of immunogenicity with suppression of lymphocyte adenosine 3',5'-monophosphate-dependent protein kinase.

Cyclic-AMP-dependent protein kinase activity was depressed in whole spleen as well as in isolated splenic lymphocytes from 3-methylcholanthrene (MCA), R3230 AdCa mammary adenocarcinoma, N-hydroxy-2-acetylaminofluorene, and 4-dimethylaminoazobenzene (DMAAB) tumor-bearing Fischer rats as compared to control animals. The magnitude of depression increased with the immunogenicity of the tumor. The depressed enzyme activity was the result of a reduced Vmax for adenosine 3',5'-monophosphate (cAMP)-stimulated histone phosphorylation.     

S-nitrosoglutathione reductase activity of human and yeast glutathione-dependent formaldehyde dehydrogenase and its nuclear and cytoplasmic localisation

S-nitrosoglutathione (GSNO) formation represents a mechanism for storage and transport of nitric oxide. Analysis of human liver and Saccharomyces cerevisiae extracts has revealed the presence of only one enzyme able to significantly reduce GSNO, identified as glutathione-dependent formaldehyde dehydrogenase (FALDH). GSNO is the best substrate known for the human and yeast enzymes (kcat/Km = 444,400 and 350,000 mM(-1) min(-1), respectively). Although NADH is the preferred cofactor, some activity with NADPH (Km = 460 microM) can be predicted in vivo. The subcellular localization demonstrates a cytosolic and nuclear distribution of FALDH in living yeast cells. This agrees with previous results in rat, and suggests a role in the regulation of GSNO levels in the cytoplasmic and nuclear compartments of the eukaryotic cell.     

Partial purification and characterization of acid phosphatase from sporulated oocysts of Eimeria tenella

Acid phosphatase of Eimeria tenella oocysts (Peak II) was purified 77-fold with a recovery of 26% using protamine sulfate precipitation, DEAE-cellulose chromatography and Sephadex G-200 gel filtration. This enzyme occurs in multiple forms as indicated by two peaks which can be separated by DEAE-cellulose chromatography and polyacrylamide gel electrophoresis. The partially purified enzyme has optimal activity at pH 4.5. With p-nitrophenyl phosphate the Km and Vmax values for (Peak II) were 25 mM and 1.57 mumol/min/mg protein, respectively. The enzyme (Peak II) is strongly inhibited by Hg++, Cu++, iodoacetamide, fluoride and molybdate. Tartrate and other divalent metal ions have no effect on enzyme activity. The partially purified Peak II phosphatase is not a glycoprotein as it is not absorbed on concanavalin-A Sepharose and its treatment with bacterial neuraminidase does not alter its elution profile through DEAE cellulose.     

Effect of progesterone on human corticosteroid 21-hydroxylation.

Progesterone inhibits the 21-hydroxylation of 17alpha-hydroxyprogesterone by human adrenal cortex microsomes. The possible light this finding may shed on the genetic condition, the 'adrenogenital syndrome' is discussed. Km and Vmax data for the above hydroxylation reaction are given.     

Somatic cell hybrids producing inhibitors of melanotic melanoma tyrosine hydroxylase

Summary Splenic lymphocytes from BALB/c mice pre-immunized with purified tyrosine hydroxylase (TH) were fused with murine myeloma N/1 cells. Supernatants of only 2 from large number of cloned cell hybrids contained an inhibitor of TH.     

Normetanephrine and metanephrine oxidized by both types of monoamine oxidase

Both normetanephrine and metanephrine were found to be oxidized by both types of monoamine oxidase in mouse liver mitochondria. Both Km and Vmax values of type B MAO for both substrates were higher than those of type A MAO, which caused the shift of inhibition curves with clorgyline and deprenyl according to the increase in substrate concentration.     

Cleavage of des-Arg9-bradykinin by angiotensin I-converting enzyme from pig kidney cortex

Fast and very slow hydrolyses of des-Arg9-bradykinin and angiotensin II by angiotensin I-converting enzyme were detected by high performance liquid chromatography. The Michaelis constants of the enzyme, Km values, for des-Arg9-bradykinin and bradykinin were found to be 0.24 mM and 4.4 microM, and the maximum velocities, Vmax values (mumol . min-1 . mg protein-1) for these compounds to be 3.24 and 0.34, respectively. The enzyme also hydrolyzed Z-Gly-Pro-Gly-Gly-Pro-Ala to a tripeptide that was identified as dansyl-Gly-Pro-Ala by TLC on polyamide. These observations show that the enzyme hydrolyzes the peptides at the bond before the prolyl residue in the penultimate position.     

Dinemorphan N-demethylation by mouse liver microsomes

Dinemorphan, an antitussive drug, is N-demethylated in vitro by mouse liver microsomes with biphasic kinetics showing two apparent Km and Vmax. Moreover, dinemorphan N-demethylation is inhibited by CO, SKF-525A, metyrapone and it is specifically catalyzed by a phenobarbital-inducible form of cytochrome P-450.     

Nature of inhibition of rat testicular alkaline phosphatase by isatin

Isatin has been found to inhibit rat testicular alkaline phosphatase (EC 3.1.3.1). That the inhibition is non-competitive as well as non-allosteric is evident from a) the hyperbolic curve relating inhibition as a function of inhibitor concentration; b) the small change in enthalpy, free energy and entropy; c) the number of isatin molecules associating with 1 molecule of the enzyme (n=1.29); and, d) the decrease in the values of both Km and Vmax in the presence of isatin.