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1.
Thyroliberin (TRH), dibutyryl cyclic AMP (db-cAMP), and 3-isobutyl-l-methylxanthine (MIX) had a stimulatory effect on prolactin (PRL) and growth hormone (GH) release from GH 3 cells. Half-maximal and maximal effects were observed for TRH at 2.5 nM and 10 nM; for db-cAMP at 0.6 mM and 5 mM, respectively. MIX (0.1 mM–1 mM) induced a dose-dependent accumulation of cellular cyclic AMP, while the hormone release was already maximally stimulated at 0.1 mM MIX. The maximal effects on hormone release of TRH and db-cAMP, but not of TRH and MIX, were additive.The Ca2+ channel blockers Co2+ (5 mM) and verapamil (100 M) and the Ca2+ chelator EGTA (4 mM) abolished the stimulatory effect of TRH (1 M) on hormone release. Co2+ and verapamil, but not EGTA, inhibited the stimulatory effect of db-cAMP (5 mM) on hormone release. The inhibitory effects of Co2+ and verapamil on GH release were counteracted by the combination of TRH and db-cAMP. For PRL release Co2+, but not verapamil, was able to inhibit the combined action of TRH and db-cAMP. Co2+, verapamil, and EGTA eliminated the stimulatory effect of MIX (1 mM) on PRL release while only Co2+ and EGTA affected the GH release. Hormone release in the presence of MIX plus verapamil or EGTA, but not Co2+, was increased by TRH.The calmodulin antagonist trifluoperazine (TFP) at 30 M inhibited basal hormone release and hormone release stimulated by TRH (1 M), db-cAMP (5 mM), and MIX (1 mM). The Ca2+ ionophore A23187 (5 M) had a stimulatory effect on basal hormone release which was abolished by 30 M TFP. 相似文献
2.
The effect of salts (nitrates, chlorides, and sulfates) of microelements, Cd2+, Ni2+, and Co2+ and the effect of boric acid and ammonium molybdate on phosphate uptake by maize root cortex segments were tested.
Higher concentration (0.1 mM) of Cu2+ salts caused enhancement of phosphate efflux to the extent that efflux was higher than influx.
Inhibitory action on phosphate uptake by maize root cortex segments was exerted by following salts: 0.01 mM Cu2+ salts (20–30% inhibition), 0.5 mM ZnSO4 (9.7%), 0.5 and 0.05 mM ZnCl2 (34.3% and 20.8%), 0.1 mM salts of Cd2+, Ni2+, Co2+ (35–78%).
1 mM FeSO4 had significant stimulatory effect (92%) on phosphate uptake. Much weaker stimulatory effect was exerted by 1 mM FeCl3 (14%), 0.05 mM ZnSO4 (9.6%), 0.005 mM ZnCla and ZnSO4 (8.4 and 18.5%) and 0.001 mM CdCl2 and CdSO4 (20.8 and 12.4%).
All other tested salts-salts of Mn2+ (0.1 and 0.01 mM), 0.01 and 0.001 mM salts of Co2+ and Ni2+, 0.001 mM salts of Cu2+, 0.001–10 mM boric acid, and 0.001–0.1 mM ammonium molybdate left phosphate uptake unaffected. 相似文献
3.
The adenylate cyclase activity of human ejaculated spermatozoa in broken-cell preparations was investigated. In the presence of 5 mM metal cations and 0.1 mM ATP, the relative enzyme activity with Mn2+, Ca2+, Mg2+, Ba2+ was 1.00, 0.28, 0.22, and 0.03, respectively. Added Ca2+ appeared to activate the enzyme in the presence of Mn2+ or Mg2+. The human sperm adenylate cyclase was stimulated by ~ 2-fold by free Ca2+ (lmM) in the presence of Mg2+ (5 mM). If the GTP analogue, 5′-guanylyl imidophosphate (Gpp(NH)p) was added to the sperm homogenate in the presence of 200 μM ethylene-glycol-bis (β-aminoethylether) N,N′-tetraacetic acid (EGTA), the adenylate cyclase activity was increased by approximately 25%, but with the addition of 280 μM Ca2+ there was a decrease in enzyme activity. A similar response to low concentrations of Ca2+ was obtained after complementation of the sperm enzyme with the guanine nucleotide regulatory component from human erythrocytes, where the addition of 40 μM Gpp(NH)p, 200 μM EGTA, and Ca2+ (≤ 160 μM) stimulated the sperm enzyme ~ 3–4-fold, but the further addition of Ca2+ (280 μM, final) neutralized the stimulatory effect. The addition of adenosine, and the nucleotides 5′-AMP and 5′-ADP inhibited the enzyme, whereas guanine and 5′-GMP had no appreciable effect. Human follicular fluid and serum also had little direct effect on the sperm adenylate cyclase. These resuls suggest that Ca2+ might be an important physiological modulator of the human sperm adenylate cyclase. 相似文献
4.
Buffalo milk galactosyltransferase is a single poly-peptide of molecular weight 55,000 to 56,000. The enzyme is specific for
glucose as an acceptor substrate in the presence of 8-lactalbumin, L-Arabinose. L-xylose, D-ribose and D-fructose did not
serve as acceptor substrates even at concentration as high as 0.13 M, while N-acetylglucosamine and ovalbumin served as good
acceptors of galactosyl moiety in the absence of ∞ -lactalbumin. UDP-galacturonic acid did not serve as a donor substrate;
on the contrary, it inhibited the reaction. Lactose synthetase reaction was inhibited by D-ribose, L-arabinose and L-xylose,
whereas D-fructose did not show any inhibition. Buffalo milk ∞ -lactalbumin enhanced the synthesis of lactose but inhibited
the synthesis of N-acetyllactosamine. Cations like Ca2+, Mg2+, Cu2+, Ba2+ and Co2+ could not replace Mn2+ in the N-acetyllactosamine synthetase reaction. Except Co2+, these cations had no effect on this reaction. Co2+ was found to be a competitive inhibitor of Mn2+. The observed inhibition of the reaction by-EDTA also confirmed the absolute requirement of Mn2+ for the reaction. Lactose synthetase reaction had an optimum pH of 8.5, whereas N-acetyllactosamine synthetase reaction was
maximal at pH 8.0. 相似文献
5.
The divalent cation requirements of NOS activity in bovine retina homogenate supernatant were investigated. Supernatants were assayed under standard conditions (in mM: EDTA 0.45, Ca2+ 0.25, Mg2+ 4.0). In order to investigate the enzyme's dependence on divalent cations, the tissue homogenate was depleted of di- and trivalent cations by passing it over a cation-exchange column (Chelex 100). Surprisingly, NOS activity was 50-100% higher in this preparation. However, addition of either EDTA (33 M) or EGTA (1 mM) almost fully inhibited NOS activity, suggesting a requirement for residual divalent metal cation(s). Phenanthroline or iminodiacetic acid at low concentrations had little effect on activity, suggesting no requirement for Fe2+, Zn2+ or Cu2+. Ca2+ had a moderate stimulatory effect, with an optimum activity around 0.01 mM. Mg2+ or Mn2+ had little effect at concentrations < 0.25 mM. However, in the presence of EDTA, Mn2+ or Ca2+ markedly stimulated NOS activity with the optimum at 0.1 mM. At high concentrations (> 0.1-0.2 mM), all divalent cations tested (Ba2+, Zn2+, Co2+, Mn2+, Mg2+, Ca2+), as well as La3+, dose-dependently inhibited NOS activity. We propose that retinal NOS requires low concentrations of naturally occurring divalent metal ions, most probably Ca2+, for optimal activity and is inhibited by high di- and trivalent metal concentrations, probably by competition with Ca2+. 相似文献
6.
Properties of partially purified NADP-malic enzyme (EC 1.1.1.40) from glumes of developing wheat grains were examined. The pH optimum for enzyme activity was influenced by malate and shifted from 7.3 to 7.6 when the concentration of malate was increased from 2 to 10 mM. The Km values, at pH 7.3, for various substrates were: malate, 0.76 mM; NADP, 20 μM and Mn2+, 0.06 mM. The requirement of Mn2+ cation for enzyme activity could be partially replaced by Mg2+ or Co2+. Mn2+ dependent enzyme activity was inhibited by Pb2+, Ni2+, Hg2+, Zn2+, Cd2+, Al3+ and Fe3+. During the reaction, substrate molecules (malate and NADP) reacted with enzyme sequentially. Activity of malic enzyme was inhibited by products of the reaction viz pyruvate, HCO3? and NADPH2. At a limiting fixed concentration of NADP, these products induced a positive cooperative response to increasing concentrations of malate. 相似文献
7.
The aminoacylation of tRNA catalysed by valyl-tRNA synthetase (EC 6.1.1.9) and isoleucyl-tRNA synthetase (EC 6.1.1.5) fromMycobacterium smegmatis is dependent on the presence of divalent metal ions. Polyamines alone, in the absence of metal ions, do not bring about aminoacylation.
In the presence of suboptimal concentrations of Mg2+, polyamines significantly stimulate the reaction. Of the cations tested, only Mn2+, Co2+ and Ca2+ can partially substitute for Mg2+ in aminoacylation, and spermine stimulates aminoacylation in the presence of these cations also. At neutral pH, spermine
deacylates nonenzymatically aminoacyl tRNA. AMP and pyrophosphate-dependent enzymatic deacylation of aminoacyl-tRNA (reverse
reaction) is also stimulated by spermine. The inhibitory effect of high concentration of KC1 on aminoacylation is counteracted,
by spermine. The low level of activity between pH 8.5–9.0 at 1.2 mM Mg2+ is restored to normal level on the addition of spermine. The inhibitory effect of high pH on aminoacylation in the presence
of low concentration of Mg2+ is also prevntedvby spemine. 相似文献
8.
Gordon B. Ward Richard T. Mayer Mark F. Feldlaufer James A. Svoboda 《Archives of insect biochemistry and physiology》1991,18(2):105-117
Gut chitin synthase was characterized and the sterols and ecdysteroids in the sugarcane rootstalk borer weevil, Diaprepes abbreviatus, were identified. An in vitro cell-free chitin synthase assay was developed using larval gut tissues from D. abbreviatus. Subcellular fractionation experiments showed that the majority of chitin synthase activity was located in 10,000g pellets. The gut chitin synthase requires Mg2+ to be fully active: 7–8-fold increases in activity were obtained with 10 mM Mg2+ present in reaction mixture. Calcium also stimulated activity (4–5-fold with 10 mM Ca2+), while Cu+2 completely inhibited at 1 mM. Other monovalent and divalent cations had little or no effect on activity. The pH and temperature optima were 7 and 25°C, respectively. Gut chitin synthesis was activated ca. 50% by trypsin treatments. GlcNAc stimulated chitin synthase activity, but Glc, GlcN and glycerin did not. Polyoxin D, UDP, and ADP inhibited the chitin synthase reaction with I50's of 75 μM, 2.3 mM, and 3.6 mM, respectively. Nikkomycin Z was a potent inhibitor of chitin synthase (91% inhibition at 10 μM). Tunicamycin and diflubenzuron had no effect on the enzyme. The apparent Km and Vmax for the gut chitin synthase were, respectively, 122.5 ± 7.4 μM and 426 ± 19.7 pmol/h/mg protein utilizing UDP-GlcNAc as the substrate. Sterol analyses indicated that cholesterol was the major dietary and larval sterol. HPLC/RIA data indicated that 20-hydroxyecdysone was the major molting hormone. 相似文献
9.
Cd2+, Mn2+, and Al3+ inhibited synaptosomal amine uptake in a concentration-dependent and time-dependent manner. In the absence of Ca2+, the rank order of inhibition of noradrenaline uptake was: Cd2+ (IC50 = 250 μM) > Al3+ (IC50 = 430 μM) > Mn2+ (IC50 = 1.50 mM), the IC50 being the concentration of metal ions that gave rise to 50% inhibition of uptake. In the presence of 1 mM Ca2+, the rank order of inhibition of uptake was: Al3+ (IC50 = 330 μM) > Cd2+ (IC50 = 540 μM) > (IC50 = 1.5 mM). The rank order of inhibition of serotonin uptake without Ca2+ was: Al3+ (IC50 = 370 μM) > Cd2+ (IC50 = 610 μM) > Mn2+ (IC50 = 3.4 mM) and the rank order in the presence of 1 mM Ca2+ was: Al3+ (IC50 = 290 μM) > Cd2+ (IC50 = 1.5 mM) > Mn2+ (IC50 = 4.0 mM). Ca2+, at 1 mM, definitely antagonized the inhibitory actions of Cd2+ on noradrenaline and serotonin uptake. Al3+ stimulated noradrenaline uptake at concentrations around 20–250 μM but inhibited this uptake at concentrations exceeding 300 μM in a dose-related fashion. Ca2+, at 1 mM, enhanced both the stimulatory and inhibitory effects of Al3+. Ca2+ also enhanced the inhibitory actions of Al3+ on seotonin uptake. These results, in conjunction with those we have previously published, suggest that Cd2+, Mn2+, and Al3+ exert differential and selective effects on the structure and function of synaptosomal membranes. 相似文献
10.
Eugene E. Quist 《Biochemical and biophysical research communications》1980,92(2):631-637
In the presence of 1.0 mM ATP and MgCl2, the specific viscosity of suspensions of human erythrocyte ghosts decreases 35% in 20 minutes at 22°C. The changes in viscosity are a sensitive index of Mg-ATP dependent shape changes in these membranes. Low concentrations of Ca2+ (1 to 5 μM) inhibit Mg-ATP dependent viscosity changes. If ghosts were preincubated with 1 mM Mg-ATP and 20 μM A23187 to produce a maximal decrease in viscosity, addition of 10 μM Ca2+ to the preincubated ghosts increased the viscosity to levels observed in ghosts preincubated without ATP. Ca2+ (1 to 5 μM) also inhibited Mg2+ dependent phosphorylation 30% and stimulated dephosphorylation 25% in ghost membranes. These effects of Ca2+ on viscosity and phosphorylation may be due to a membrane bound Ca2+ phosphatase activity which dephosphorylates membranes phosphorylated by a Mg2+ dependent kinase activity. 相似文献
11.
Purified rat epididymal nuclei possess a cyclic AMP-independent protein kinase activity that phosphorylates of casein. The
enzymic activity was solubilized by treating intact nuclei with 1 M (NH4)2SO4. One major peak of kinase activity was obtained when the solubilized enzyme preparation was subjected to diethylaminoethyl-Sephadex
chromatography. The activity of the kinase was dependent on a bivalent metal ion such as Mg2+, Co2+, Ca2+ or Mn2+. NaCl (0.3 M) caused a further activation (approx. 200%) of the metal (Co2+)-dependent enzyme. The apparentK
m values of the enzyme for casein, ATP and Co2+ are approx. 0.6 mg/ml, 10 ΜM and 2.2 mM respectively. The enzyme was maximally active at pH 5.5. The enzyme showed high specificity
for phosphorylation of the acidic protein casein but did not phosphorylate basic proteins, such as histones and protamine.
The properties of the nuclear protein kinase were clearly different from those of the cytosolic enzymes previously characterized. 相似文献
12.
Xiaolin Pei Qiuyan Wang Chenglu Li Xiaopu Yin Rong Chen Tian Xie 《Biotechnology letters》2013,35(9):1419-1424
A nitrile hydratase (NHase) gene from Aurantimonas manganoxydans, cloned and expressed in Escherichia coli, gave an enzyme that efficiently hydrated 3-cyanopyridine to nicotinamide with high thermal stability. We have now found that adding Co2+ at 0.1 mM to LB medium was essential for production of an active enzyme. However, ≥0.3 mM Co2+ inhibited the growth of host cells in LB medium and decreased the production of the recombinant NHase. Furthermore, β-mercaptoethanol promoted regeneration of the Co2+-defective apoenzyme in vitro possibly by breaking a key disulfide bond thereby promoting the incorporation of Co2+ into the apoenzyme. 相似文献
13.
Karl H. Jakobs Klaus Aktories 《Biochimica et Biophysica Acta (BBA)/General Subjects》1981,676(1):51-58
In hamster adipocyte ghosts, ACTH stimulates adenylate cyclase by a GTP-dependent process, whereas prostaglandin E E1, α-adrenergic agonists and nicotinic acid inhibit the enzyme by a mechanism which is both GTP- and sodium-dependent. The influence of the divalent cations Mn2+ and Mg2+, was studied on these two different, apparently receptor-mediated effects on the adipocyte adenylate cyclase. At low Mn2+ concentrations, GTP (1 μM) decreased enzyme activity by about 80%. Under this condition, ACTH (0.1 μM) stimulated the cyclase by 6- to 8-fold, and NaCl (100 mM) caused a similar activation. In the presence of both GTP and NaCl, prostaglandin E1 (1 or 10 μM) and nicotinic acid (30 μM) inhibited the enzyme by about 70–80% and epinephrine (300 μM, added in combination with a β-adrenergic blocking agent) by 40–50%. With increasing concentrations of Mn2+, the GTP-induced decrease and the NaCl-induced increase in activity diminished, with a concomitant decrease in prostaglandin E1?, nicotinic acid- and epinephrine-induced inhibitions as well as in ACTH-induced stimulation. At 1 mM Mn2+, inhibition of the enzyme was almost abolished and stimulation by ACTH was largely reduced, whereas activation of the enzyme by KF (10 mM) was only partially impaired. The uncoupling action of Mn2+ on hormone-induced inhibition was half-maximal at 100–200 μM and appeared not to be due to increased formation of the enzyme substrate, Mn · ATP. It occurred without apparent lag phase and could not be overcome by increasing the concentration of GTP. Similar but not identical findings with regard to adenylate cyclase stimulation and inhibition by hormonal factors were obtained with Mg2+, although about 100-fold higher concentrations of Mg2+ than of Mn2+ were required. The data indicate that Mn2+at low concentrations functionally uncouples inhibitory and stimulatory hormone receptors from adenylate adenylate cyclase in membrane preparations of hamster adipocytes, and they suggest that the mechanism leading to uncoupling involves an action of Mn2+ on the functions of the guanine nucleotide site(s) in the system. 相似文献
14.
《Biochimica et Biophysica Acta (BBA) - Enzymology》1976,422(1):87-97
Phosphorylase kinase (EC 2.7.1.38) activity in crude cytosol preparations of chicken adipose tissue was assayed using as substrate either the endogenous phosphorylase b in the preparation or added purified rabbit skeletal muscle phosphorylase b. The results obtained with the two substrates were similar. The phosphorylase kinase reaction was markedly inhibited by ethyleneglycol-bis-(β-aminoethylether)-N,N′,-tetraacetic acid (EGTA), maximum inhibition (about 90%) occurring at approx. 0.2 mM. This inhibition was readily reversed by addition of Ca2+. Full reversal was achieved with 0.3 mM Ca2+ in the presence of 0.5 mM EGTA; the estimated free Ca2+ concentration required was 4 μM. The activation of phosphorylase b was blocked immediately and completely by EGTA added during the course of the assay; reversal was achieved without a time lag by the addition of Ca2+. The Ca2+ requirement was also demonstrated directly by preparing an enzyme fraction from which Ca2+ had been removed and by using Ca2+-free reagents. Under these conditions the Ca2+ concentration needed for half maximum activation was 10 μM and maximum activation was obtained at about 100 μM. The possibility that the effects of EGTA and Ca2+ might be related to changes in phosphorylase phosphatase activity rather than phosphorylase kinase was considered unlikely since the phosphorylase phosphatase activity was inhibited during the phosphorylase kinase assay step by the inclusion of fluoride and β-glycerophosphate. Phosphorylase kinase activity in rat adipocytes, using endogenous phosphorylase as substrate, was also inhibited EGTA but, whereas the activity in chicken adipose tissue was inhibited by 90%, the activity in rat adipose tissue was inhibited only 60%. These data indicate that adipose tissue phosphorylase kinase has a Ca2+ requirement for optimal activity and is thus qualitatively similar to the enzyme in contractile tissues. 相似文献
15.
Joseph D. Robinson 《生物化学与生物物理学报:生物膜》1983,727(1):63-69
Na+-ATPase activity of a dog kidney (Na+ + K+)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 μM ATP and 50 μM MgCl2, but stimulated by 100 mM NaCl in the presence of 30 μM ATP and 3 mM MgCl2. The for the effect of MgCl2 was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na+-ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 μM MgCl2. Similar thimerosal treatment reduced (Na+ + K+)-ATPase activity by half but did not appreciably affect the for activation by either Na+ or K+, although it reduced inhibition by high Na+ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na+: Na+-discharge sites at which Na+-binding can drive E2-P back to E1-P, thereby inhibiting Na+-ATPase activity, and sites activating E2-P hydrolysis and thereby stimulating Na+-ATPase activity, corresponding to the K+-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na+-discharge and K+-acceptance sites. Mg2+ may stimulate Na+-ATPase activity by favoring E2-P over E1-P, through occupying intracellular sites distinct from the phosphorylation site or Na+-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na+-ATPase reaction and lessen Na+-inhibition of the (Na+ + K+)-ATPase reaction by increasing the efficacy of Na+ in activating E2-P hydrolysis. 相似文献
16.
Two acid phosphomonoesterases, 5′(3′)-ribonucleotide phosphohydrolase and 3′-ribonucleotide phosphohydrolase, were isolated from Tradescantia albiflora leaf tissue and purified by ammonium sulphate precipitation, gel filtration on Sephadex G-200 and repeated chromatography on DEAE-cellulose. The enzymes differed in their sensitivity to dialysis against 1 mM EDTA; the activity of 5′(3′)-ribonucleotide phosphohydrolase was unaffected, while 3′-ribonucleotide phosphohydrolase showed an increase of 60–90%. Both enzymes were rapidly inactivated above 50°. Their ion sensitivity was identical: 1 m M Zn2+ and Fe2+ were inhibitors for both by 20–80%; while Mg2+, Ca2+, Co2+, K+, Na+ at 1–10 mM had no significant effect on the activity of either enzyme. Inorganic phosphate inhibited both enzymes almost completely. EDTA (1 mM) did not inhibit either enzyme; none of the divalent cations tested were enzyme activators. 3′-Ribonucleotide phosphohydrolase hydrolysed both 3′- and 5′-nucleoside monophosphates (3′-AMP, 3′-CMP, 3′-GMP, 3′-UMP, 5′-AMP, 5′-CMP, 5′-GMP, 5′-UMP). 5′(3′)-Ribonucleotide phosphohydrolase showed a preference for the 3′-nucleoside monophosphates. Adenosine 3′,5′-cyclic monophosphate, purine and pyrimidine 2′,3′-cyclic mononucleotides at 0.1–1.OmM did not inhibit the enzymes. 相似文献
17.
The effect of Ca2+-binding protein regucalcin on protein kinase activity in the nuclei of normal and regenerating rat livers was investigated. Protein kinase activity in the nuclei isolated from normal rat liver was significantly increased by addition of Ca2+ (500 μM) and calmodulin (10 μg/ml) in the enzyme reaction mixture. Nuclear protein kinase activity was significantly decreased in the presence of EGTA (1.0 mM), trifluoperazine (TFP; 20 μM), dibucaine (10−4 M), or staurosporine (10−7 M), indicating that Ca2+-dependent protein kinases are present in the nuclei. Protein kinase activity was significantly elevated in the liver nuclei obtained at 6 to 48 h after a partial hepatectomy. Hepatectomy-increased nuclear protein kinase activity was significantly decreased in the presence of EGTA (1.0 mM), TFP (20 μM), or staurosporine (10−7 M) in the enzyme reaction mixture. The presence of regucalcin (0.1–0.5 μM) caused a significant decrease in protein kinase activity in the nuclei obtained from normal and regenerating rat livers. Meanwhile, the nuclear protein kinase activity from normal and regenerating livers was significantly elevated in the presence of anti-regucalcin monoclonal antibody (50–200 ng/ml). The present study suggests that regucalcin plays a role in the regulation of protein kinase activity in the nuclei of proliferative liver cells. J. Cell. Biochem. 71:569–576, 1998. © 1998 Wiley-Liss, Inc. 相似文献
18.
Edward W. Troyer Isabelle A. Hall James A. Ferrendelli 《Journal of neurochemistry》1978,31(4):825-833
Guanylate cyclase activity is present in both soluble and particulate fractions of homogenates of mouse cerebellum and retina. Soluble guanylate cyclases in cerebellum and retina have an apparent Km for GTP of approx 40 and 70 μM, respectively; are stimulated by Ca2+ and Mg2+ in the presence of low Mn2+; and do not respond to NaN3, NH2OH or detergent. The particulate guanylate cyclase found in brain has an apparent Km GTP of 237 7mu;M, is not stimulated by Ca2+ or Mg2+ in the presence of low Mn2+, but is stimulated by NaN3, NH2OH, and detergent. In particulate fractions of normal retina, guanylate cyclase has two apparent Km GTP values (42 and 225 μM); has higher activity at low concentrations of Mn2+ (0.5 mM) than at high concentrations (5.0 mM); is inhibited by Ca2+; and does not respond to NaN3, NH2OH, or detergent. Retinas essentially devoid of photoreceptor cells (from mice with photoreceptor dystrophy) have soluble guanylate cyclase activity which is similar to that in normal retina, but have only 4% as much particulate guanylate cyclase activity. This residual particulate guanylate cyclase has an apparent Km GTP value of 392 μM and other properties similar to particulate guanylate cyclase from brain. These data indicate the presence of three distinguishable guanylate cyclases in CNS: (1) a soluble enzyme present in both brain and retina: (2) a particulate enzyme which is also present in brain and in the inner or neural retina: and (3) another particulate enzyme which is apparently unique and confined to retinal photoreceptor cells. 相似文献
19.
《Journal of steroid biochemistry》1983,18(3):333-340
The cholesterol side chain cleavage enzyme (EC 1.14.15x) in mitochondria of a human term placenta was partially characterized. Enzyme activity was determined by separation of [26-14C]-cholesterol and [5-14C]-isocaproic acid formed by side chain cleavage. Since the amounts of unlabeled cholesterol were too large, a KM of cholesterol could not be determined. The apparent KM value of NADPH is 6.25 × 10−4 M. A pH optimum was found at pH 9.5 (Tris-buffer) and a temperature optimum at 40°C. The metal ions Sr2+ and Ba2+ showed no inhibition at 1 and 10 mM and a moderate inhibition at 100 mM. In low concentrations (1 mM), Mg2+ and Ca2+ slightly stimulated the enzyme whereas in higher concentrations (100 mM) an inhibitory effect was observed. A strong inhibition was achieved with 1 mM Zn2+, Cd2+, Cu2+ and by 10 and 100 mM Fe2+, Mn2+, Co2+ and Ni2+. During preincubation of the enzyme without radioactive substrate, a rapid loss in enzyme activity in relation to enzyme concentration was observed (initial activity = 100%) (preincubation time in hours): 0.5 h (97%), 1 h (55%) and 1.5 h (34%). A dose-dependent inhibition of the enzyme by the following proteins was achieved: bovine serum protein, human serum protein, human immunoglobulin G and ovalbumin. Furthermore, a dose-dependent inhibition was found with the membrane lipids lecithin and sphingosine. 相似文献
20.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1986,888(3):270-277
The transport of Cd2+ and the effects of this ion on secretory activity and metabolism were investigated in β cell-rich pancreatic islets isolated from obese-hyperglycemic mice. The endogenous cadmium content was 2.5 μmol/kg dry wt. After 60 min of incubation in a Ca2+-deficient medium containing 2.5 μM Cd2+ the islet cadmium content increased to 0.18 mmol/kg dry wt. This uptake was reduced by approx. 50% in the presence of 1.28 mM Ca2+. The incorporation of Cd2+ was stimulated either by raising the concentration of glucose to 20 mM or K+ to 30.9 mM. Whereas D-600 suppressed the stimulatory effect of glucose by 75%, it completely abolished that obtained with high K+. Only about 40% of the incorporated cadmium was mobilized during 60 min of incubation in a Cd2+-free medium containing 0.5 mM EGTA. It was possible to demonstrate a glucose-induced suppression of Cd2+ efflux into a Ca2+-deficient medium. Concentrations of Cd2+ up to 2.5 μM did not affect glucose oxidation, whereas, there was a progressive inhibition when the Cd2+ concentration was above 10 μM. Basal insulin release was stimulated by 5 μM Cd2+. At a concentration of 160 μM, Cd2+ did not affect basal insulin release but significantly inhibited the secretory response to glucose. It is concluded that the β cell uptake of Cd2+ is facilitated by the activation of voltage-dependent Ca2+ channels. Apparently, the accumulation of Cd2+ mimics that of Ca2+ also involving a component of intracellular sequestration promoted by glucose. 相似文献