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1.
This study was conducted to define the pattern of metabolism of 3H-progesterone in incubates of rat testicular tissue at various time intervals after hypophysectomy and to determine the effect of gonadotropin treatment on the metabolism of 3H-progesterone in posthypophysectomy regressed testes. Formation of tritium labeled testosterone, androstenedione, 5α-androstanediol and androsterone was markedly diminished within two weeks and only traces of these substances were formed between the 23rd and 54th day after hypophysectomy. The major metabolite throughout this time period was 3H-20α-dihydroprogesterone. These data demonstrate that in posthypophysectomy-regressed testes 3H-progesterone metabolism does not revert to that observed in fetal testes or testes from immature animals. Treatment with HCG, commencing on the 33rd day after hypophysectomy resulted first in formation of 5α-reduced androgens and marked decrease in 20α-dihydroprogesterone. Additional treatment produced increased formation of radiolabeled testosterone and androstenedione and diminution of 5α-reduced androgens. This metabolic pattern is reminiscent of that observed in normally developing testes. Treatment with PMS commencing on the 33rd day after hypophysectomy resulted in formation of large amounts of androstenedione and testosterone and decrease of 20α-dihydroprogesterone to trace amounts within 10 days of initiation of treatment. After additional 10 days of treatment the formation of androstenedione diminished, testosterone remained unchanged. The possibility is suggested that FSH activity in PMS may be responsible for the different pattern of progesterone metabolism. The data of an three experiments suggest that the 20α-hydroxysteroid oxidoreductase activity may be influenced by gonadotropins. 相似文献
2.
Steven J. Scheinman Gerard N. Burrow Theoharis C. Theoharides Zoe N. Canellakis 《Life sciences》1977,21(8):1143-1147
High titer antiserum to hepatic ornithine decarboxylase was prepared by employing enzyme·monospecific antibody complex as the immunizing antigen. This new antiserum preparation was successfully labeled with 125I and was found to retain its specific immune properties. Iodinated antiserum was used to precipitate thyroid ornithine decarboxylase induced by a mixture of thyroid stimulating hormone and methyl xanthine in rat thyroids . 125I-labeled antibody incorporation into the enzyme antibody complex after induction showed an increase which paralleled the increase in enzymatic activity and thus suggested synthesis of thyroid enzyme protein. 相似文献
3.
Lucio Annunziato Gianfranco Di Renzo Salvatore Amoroso Aldo Quattrone 《Life sciences》1984,35(4):399-407
Release of endogenous dopamine (DA) from arcuate-periventricular nucleus-median eminence fragments has been analyzed in an static incubation system.Exposure of these hypothalamic fragments to increasing concentrations of K+ ions produced a dose-dependent release of endogenous DA. The highest rate of K+-stimulated DA efflux occurred in the first 10 minutes, thereafter it progressively decline reaching prestimulated levels at 30 minutes. If two consecutive depolarizing stimuli of 40 mM KCl were applied to the same hypothalamic fragment, after a 40 minutes rest period, an equivalent release of endogenous DA occurred. Removal of Ca++ ions from the incubation medium containing the Ca++ chelator EGTA caused a decrease of basal DA efflux and completely prevented the K+-induced release of DA.Furthermore when verapamil, a blocker of Ca++ entrance, was added to the incubation medium in a concentration of 50 μM, the K+-induced DA efflux was completely counteracted, whereas spontaneous release was unmodified.Finally nomifensine, a potent blocker of DA uptake, added in a final concentration of 10 μM, significantly reinforced K+-induced release of endogenous DA. Since nomifensine did not modify basal DA release, this study confirmed its prevalent uptake blocking property rather than its releasing action on DA. 相似文献
4.
A single subcutaneous injection of folate, homofolate or MTX resulted in the inhibition of the activity of dihydrofolate reductase in homogenates prepared from the kidneys of normal mice. Stimulation of 3H-thymidine uptake occurred in the kidneys of treated animals approximately 30 hr after administration of either folate or homofolate, and reached a peak 72 hr after administration. The effects of folate and MTX on dihydrofolate reductase activity were also determined. One hr after administration of 15 mg/kg methotrexate (MTX) or 300 mg/kg folate, enzyme activity was inhibited by 90%.3H-deoxyuridine uptake was neither stimulated nor depressed after treatment with MTX. After administration of folate, uptake of 3H-deoxyuridine was stimulated at approximately 30 hr after drug-treatment and reached a peak at 72 hr after folate administration. Treatment with xanthopterin had no effect on the activity of dihydrofolate reductase . Xanthopterin stimulated uptake of both deoxyuridine and thymidine in an identical manner.The increased DNA synthesis that occurs in animals after treatment with agents that cause renal damage is distinct from the effect these agents have upon dihydrofolate reductase. Nucleoside incorporation after treatment with folate, homofolate, MTX or xanthopterin cannot be predicted on the basis of enzyme inhibition. Treatment with MTX, folate or homofolate results in enzyme inhibition which is not correlated with the uptake of deoxyuridine into DNA. 相似文献
5.
Pretreatment of Chang liver cells with (0.5 or 1 mM) stimulated Na+-independent uptake of leucine at low concentrations (?1 mM). The stimulatory effect of on the uptake of leucine measured in Na+-replete medium was completely blocked by the addition of (5 mM), which shows that the L system participates in the stimulation. The Na+-dependent uptake of glycine was depressed by pretreatment. The stimulation of the Na+-independent component of leucine uptake continued for at least 30 min after treatment, while the inhibition of glycine uptake was progressive with time and the Na+-dependent uptake of leucine became depressed later, after the treatment. It has been demonstrated that treatment of cells with is capable of increasing the Na+-independent influx of leucine and at the same time slightly decreasing the efflux of it. These results suggest that attacks the Na+-independent system of amino acid transport at the reactive SH groups(s) of relevant protein(s) in favor of specific activation of that system in this cell. 相似文献
6.
A study was made of the phosphorylation of chromatographically purified histone H1 subfractions from the liver of premetamorphic tadpoles (). Two H1 subfractions were obtained which differed in terms of net incorporation of [32P]phosphate . Analysis of N-bromosuccinimide cleavage products further revealed that the two subfractions also differed in the relative distribution of [32P]phosphate in N- and C-terminal regions of the molecule. Incorporation of [32P]phosphate into both regions of the molecule occurred virtually exclusively in serine residues. 相似文献
7.
An enzymatic method for [32P]phosphoenolpyruvate synthesis 总被引:7,自引:0,他引:7
A convenient method for the enzymatic synthesis of [32P]phosphoenolpyruvate from [γ-32P]ATP using partially pufified phosphoenolpyruvate carboxykinase from Escherichia coli is described. The synthesis was shown to convert essentially all the [γ-32P]ATP to [32P]phosphoenolpyruvate, which was subsequently separated from residual [γ-32P]ATP and by chromatography on AG-1-X8-bicarbonate resin. 相似文献
8.
A proteinase (called Proteinase I) present in myxamoebae of the cellular slime mold, , was labeled with [32P] by growth of cells on media containing [32P] orthophosphate. The labeled proteinase was purified to apparent homogeneity and characterized by dissociation chromatography and quantitative immune-precipitin analysis. Based upon the results of these studies it was concluded that phosphoryl moieties were tightly associated (presumably covalently bonded) with the polypeptide subunits of Proteinase I. 相似文献
9.
Seung-ki Lee Richard A. Jungmann 《Biochemical and biophysical research communications》1981,102(1):538-544
The phosphorylation of RNA polymerase II after isoproterenol stimulation of confluent rat C6 glioma cell cultures has been investigated. Glioma cells were incubated in the presence of Na2H32PO4 and stimulated for 1 hour with the β-adrenergic agonist isoproterenol. The phosphorylation pattern was analyzed after purification of RNA polymerase II by immunoprecipitation, SDS-polyacrylamide gel electrophoresis and autoradiography. Isoproterenol markedly increased [32P]phosphate incorporation into the 214,000 dalton RNA polymerase subunit. Analysis of the phosphate acceptor amino acid revealed the presence of only [32P]phosphoserine. The data demonstrates an isoproterenol-induced structural modification of RNA polymerase II. 相似文献
10.
Yvette J. Fernandez Rose-Anne M. Boigegrain Claudie D. Cambon-Gros Salvador E. Mitjavila 《生物化学与生物物理学报:生物膜》1984,770(2):171-177
The aim of our work is to show the importance of the role of hydrophobic bonds in maintaining Mg2+-ATPase or sucrase activity and Na+-coupled d-glucose uptake normal for the brush border of rat enterocytes. The activity of the two enzymes and the d-glucose uptake were therefore measured under the action of and related to the fluidity determined by ESR. Three concentrations were used for the first eight alcohols, those of octanol being about 1500-times lower than those of methanol. For each alcohol the d-glucose uptake and the fluidity were linear functions of the logarithm of the concentration, the linear regressions being practically parallel and equidistant. The concentrations () of the eight alcohols inhibiting the d-glucose uptake by 80% were similar to those increasing the membrane fluidity by 3%. The linear relationship which existed in both cases between log 1 / and log being octanol / water partition coefficients of the alcohols, was evidence of great sensitivity to the hydrophobic effect of the alcohols. Only the first alcohols, however, produced any notable inhibition of Mg2+-ATPase and sucrase. Hydrophobic bonds are thus shown to have little influence in maintaining the activity of Mg2+-ATPase and sucrase, but they modulate the Na+-coupled d-glucose uptake. 相似文献
11.
G Tunnicliff 《Biochemical and biophysical research communications》1980,97(3):1024-1030
Crude lysates from a strain of enterotoxigenic have been shown to catalyse the incorporation of [32P] from [adenylate-32P] NAD+ into an 11,000 dalton protein in rat liver membranes. [32P] incorporation paralleled adenylate cyclase activation and the results suggest that the mechanism of action of the heat-labile enterotoxin may involve ADP-ribosylation of an intracellular acceptor protein. 相似文献
12.
Acetylcholine stimulates hydrolysis of 32 P-labeled phosphatidic acid in guinea pig synaptosomes 总被引:3,自引:0,他引:3
[3H]-inositol or [3H]-arachidonate was injected intracerebrally into guinea pigs. Labeled nerve endings were incubated with Ach1 or CCh, both of which stimulate labeling of PhA and PhI from 32Pi by > 100% and 70% respectively. Their addition did not affect the labeled phosphatidyl-[3H]-inositol or [3H]-arachidonyl-diglyceride and -PhI. Enhanced hydrolysis of [3H]-inositol-PhiP and -PhIP2 in the presence of ACh, CCh or choline was not reversed by atropine. In a two-step experiment, PhA was labeled with 32Pi, and DNP was added to block further formation. Addition of ACh stimulated an atropine-sensitive in [32P]-PhA. 相似文献
13.
Neurotransmitter storage vesicles were isolated from rat brain by differential centrifugation and the uptake of (?) 3H-norepinephrine was determined . Uptake showed a marked temperature dependence, an absolute requirement for ATP-Mg2+, and was inhibited by reserpine. Uptake was linear for 5 min at 30°, but not at 37°. The uptake was saturable and displayed a single Km value of 4 × 10?7 M. Other phenylamines and indoleamines displayed competitive inhibition of norepinephrine uptake; the affinities followed the rank order: reserpine>harmaline>serotonin>epinephrine> dopamine>norepinephrine>metaraminol. Uptake was reduced in vesicles isolated from rats treated intracisternally with 6-hydroxydopamine but not from rats treated with 5,6-dihydroxytryptamine, suggesting that most of the uptake occurs in catecholaminergic, and not serotonergic, vesicles. This method provides a ready characterization of pharmacologic effects on rat brain storage vesicle properties, as demonstrated by the prompt and complete inhibition of uptake after administration of reserpine . 相似文献
14.
The addition of glucagon (10?6 M) to an incubation mixture containing 32Pi and hepatocytes isolated from livers of rats fed results in both a 3-fold increased incorporation of 32P into L-type pyruvate kinase and a decreased catalytic activity. The 32P incorporated into pyruvate kinase was covalently bound to the enzyme as evidenced by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In addition, exogenous cyclic AMP (10?3 M) stimulated the phosphorylation and the suppression of catalytic activity to a similar extent. On the other hand, insulin (10?7 M) had essentially no effect on the incorporation of 32P into pyruvate kinase or on its catalytic activity under the conditions used in this study. These results suggest that phosphorylation of pyruvate kinase is stimulated by glucagon via cyclic AMP and cyclic AMP-dependent protein kinase and that the activity of the enzyme is, at least in part, regulated by a phosphorylation-dephosphorylation mechanism. 相似文献
15.
The sarcolemmal membranes isolated from rat skeletal muscle are capable of incorporating 32P from [γ?32P]ATP. The membrane protein phosphorylation requires Mg2+. Cyclic AMP, cyclic GMP and their dibutyrul derivatives showed no marked effect on sarcolemmal phosphorylation.The Mg2+-dependent 32P labeling was significantly enhanced by Na+. The rate of Na+ -stimulated 32P incorporation was quite rapid reaching steady state levels within 5 s at 0 °C. K+ reduced the Na+ -stimulated 32P-incorporation but enhanced the 32Pi release. This inhibitory effect of K+ on Na+ -stimulated 32P incorporation was prevented by the cardiac glycoside, ouabain.The Na+ -dependent 32P labeling showed substrate dependency and the Na+ site was saturable. The apparent for ATP was 2 · 10?5 M. The optimum pH for 32P labeling was between 7 and 8.Na+ -dependent membrane phosphorylation showed a direct relationship with the (Na+ + K+ATPase activity. The high turnover rate of 32P intermediate (12 000 min ?1) suggested its functional significance in the overall transport ATPase reaction sequence.The predominate portion (> 90%) of the phosphorylated membrane complex was sensitive to acidified hydroxylamine and to alkaline pH suggesting an acylphosphate nature of the phosphoprotein.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that 32P incorporation occurred predominately into a 108 000 dalton subunit which is a major protein component of sarcolemmal membranes. A very low level of 32P incorporation was also observed into a 25 000 dalton subunit and Ca2+ slightly enhanced the phosphorylation of this component.The size () and some properties of the sarcolemmal phosphoprotein are closely similar to other (Na+ + K+ATPase preparations reported so far. 相似文献
16.
Phosphorylation of high mobility group proteins 14 and 17 by nuclear protein kinase NII in rat O6 glioma cells 总被引:1,自引:0,他引:1
J J Harrison R A Jungmann 《Biochemical and biophysical research communications》1982,108(3):1204-1209
High mobility group (HMG) proteins 14 and 17 of rat C6 glioma cells are phosphorylated on both serine and threonine. In HMG 14 about 60% of the total [32P]phosphate was identified as phosphoserine and 40% as phosphothreonine. In HMG 17, there was 88% phosphoserine and 12% phosphothreonine. Glioma cell nuclear protein kinase NII phosphorylates HMG 14 and 17 on serine as well as threonine and the relative percentages of [32P]phosphoamino acid are similar to those seen . Nuclear protein kinase NI and the type I and II cAMP-dependent protein kinases exhibit only minor phosphorylating activity towards HMG 14 and 17. We conclude that nuclear protein kinase NII is responsible for the phosphorylation of HMG 14 and 17 . 相似文献
17.
The properties of carnitine transport were studied in rat kidney cortex slices. Tissue: medium concentration gradients of 7.9 for L-[methyl-14C]carnitine were attained after 60-min incubation at 37°C in 40 μM substrate. L- and D-carnitine uptake showed saturability. The concentration curves appeared to consist of (1) a high-affinity component, and (2) a lower affinity site. When corrected for the latter components, the estimated Km for L-carnitine was 90 μM and per ml intracellular fluid; for D-carnitine, and per ml intracellular fluid. The system was stereospecific for L-carnitine. The uptake of L-carnitine was inhibited by (1) D-carnitine, γ-butyrobetaine, and (2) acetyl-L-carnitine. γ-Butyrobetaine and acetyl-L-carnitine were competitive inhibitors of L-carnitine uptake. Carnitine transport was not significantly reduced by choline, betaine, lysine or γ-aminobutyric acid. Carnitine uptake was inhibited by 2,4-dinitrophenol, carbonyl cyanide , N2 atmosphere, KCN, , low temperature (4°C) and ouabain. Complete replacement of Na+ in the medium by Li+ reduced L- and D-carnitine uptake by 75 and 60%, respectively. Complete replacement of K+ or Ca2+ in the medium also significantly reduces carnitine uptake. Two roles for the carnitine transport system in kidney are proposed: (1) a renal tubule reabsorption system for the steady-state maintenance of plasma carnitine; and (2) maintenance of normal carnitine levels in kidney cells, which is required for fatty acid oxidation. 相似文献
18.
A chloroplast tRNAmMet species from is very poorly 5′-end [] labelled using [γ-32P]ATP and T4 polynucleotide kinase. In sequencing the tRNA using standard 5′-labelled methods a very minor contaminating tRNA is preferentially labelled. The partial tRNA sequence determined by this method has an anticodon (CUC) for tRNAGlu. 相似文献
19.
Procedure for measurement of amino acid transport from blood to brain in small animals 总被引:3,自引:0,他引:3
The exponential plasma specific activity curve 2.5 to 12.5 min after injection (sc) of [14C]tyrosine was integrated and divided by time to obtain the mathematical relationship between the average equivalent specific activity and the measured specific activity S in any individual animal. is the constant, average value of S that is equivalent to the curvllinearly varying quantity that the body tissues are actually exposed to. Dividing the total brain radioactivity by gave the tissue Tyr uptake U. The function is linear from 2.5 to 12.5 min and represents the rate of uptake of the amino acid. Incorporation into protein was similarly measured. Brain uptake of Tyr averaged 7.06, and the apparent protein incorporation was 1.99 nmol/g of brain per min. The γ-glutamyl cycle inhibitor l-methionine-RS-sulfoximine reduced total brain uptake of tyrosine by 42.8% and the apparent rate of protein incorporation by 39.0%. 相似文献
20.
The effect of norepinephrine and acetylcholine on the 32P incorporation into phospholipids of normal and sympathetically denervated rabbit iris muscle was investigated. (1) In the absence of exogenously added neurotransmitters sympathetic denervation exerted little effect on the incorporation of 32P into the phospholipids of the excised iris muscle. thr iris muscle incorporated 32P into phosphatidylinositol, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin in that order of activity while phosphatidylinositol was followed by phosphatidylcholine. (2) Tension responses of iris dilator muscle from denervated irises exhibited supersensitivity to norepinephrine. Furthermore, norepinephrine at concentrations of 3 μM and 30 μM produced 1.6 times and 3 times stimulation of the phosphatidic acid of the denervated muscle respectively. In contrast at 30 μM it stimulated this phospholipid by 1.6 times in the normal muscle. This stimulation was completely blocked by phentolamine. (3) While in the normal muscle acetylcholine stimulated the labelling of phosphatidic acid and phosphatidylinositol by more than 2 times, in the denervated muscle it only stimulated 1.4 to 1.7 times. (4) Similarly when 32Pi was administered intracamerally, the labelling found in the various phospholipids of the denervated iris was significantly lower than that of the normal. (5) It was concluded that denervation decreases the 32P labelling in the presence of acetylcholine. (6) The norepinephrine-stimulated 32P incorporation into phosphatidic acid appears to be post-synaptic. 相似文献