Chloromercuriphenyl Sulfonate Research Articles

Uptake of biotin by native Xenopus laevis oocytes

The present study examined biotin uptake by Xenopus laevis oocytes in vitro. Uptake of low (0.03 microM) and high (10 microM) concentrations of biotin was linear with time for up to 4 h of incubation and occurred with little initial binding to oocytes. Uptake of biotin was dependent on extracellular Na+ concentration [Na+]o and was severely inhibited when Na+ was replaced by other monovalent cations [choline, tetraethylammonia, Li+, and tris(hydroxymethyl)aminomethane]. The initial rate of biotin uptake was saturable as a function of concentration with an apparent Michaelis constant of 3.9 +/- 0.5 microM and maximum velocity of 1,559 +/- 70 fmol.oocyte-1.h-1. Addition to the incubation medium of biotin structural analogues desthiobiotin and thioctic acid caused significant and concentration-dependent inhibition in the uptake of [3H]biotin. This inhibition was found to be competitive in nature with inhibition constant values of 9 and 17.5 microM. In contrast, neither the structural analogue biocytin nor biotin methyl ester (compounds in which the carboxyl group of the valeric acid moiety is blocked) showed any effect on the uptake of [3H]biotin. Biotin uptake was significantly blocked by the metabolic inhibitors dinitrophenol, cyanide, and azide and by incubation at 4 degrees C. Also, the sulfhydryl group blocker p-(chloromercuri)phenylsulfonate caused significant inhibition in biotin uptake. These results demonstrate that Xenopus oocytes possess an uptake system for biotin in its cell membrane that is Na+, energy, and temperature dependent. These characteristics of biotin uptake are similar to those reported in mammalian cells. It is suggested that Xenopus oocytes might be a useful in vitro model system to study the details of the mechanisms and regulation of biotin movement across biological membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of a human plasma lipid transfer protein complex with lipid monolayers

The interaction of a purified human plasma lipid transfer complex with cholesteryl ester, triacylglycerol and phosphatidylcholine in binary and ternary lipid monolayers was investigated. The lipid transfer complex, designated LTC, catalyzes the removal of cholesteryl oleate and triacylglycerol from phosphatidylcholine monolayers. Preincubation of LTC with p- chloromercuriphenyl sulfonate inhibits LTC-catalyzed removal of triacylglycerol; cholesteryl ester removal is not affected. The rate of LTC-facilitated removal of cholesteryl oleate from a phosphatidylcholine monolayer depends on the amount of LTC added to the subphase up to 100 μg protein. In addition, the rate of the LTC-catalyzed transfer of cholesteryl oleate to the subphase increases linearly as the amount of cholesteryl oleate in the monolayer increases to 6 mol%. LTC also removes cholesterol from phosphatidylcholine-cholesterol monolayers, albeit at a rate which is 15% of that for removal of cholesteryl oleate. The ability of LTC to facilitate triacylglycerol and cholesteryl ester removal depends on the composition of the monolayer. Phosphatidylcholine supports cholesteryl ester transfer whereas sphingomyelin-cholesteryl ester monolayers are almost refractory to LTC. In contrast, LTC removes triacylglycerol from either a phosphatidylcholine or a sphingomyelin monolayer. The results suggest the existence of at least two lipid transfer proteins, one of which catalyzes the removal of cholesteryl ester and the other triacylglycerol. The role of these proteins as they relate to lipoprotein metabolism is discussed.

The In Vitro effect of mercury compounds on noradrenaline output from guinea pig vas deferens

An increase in the noradrenaline output induced by organic and inorganic mercury compounds was investigated using isolated vas deferens from guinea pigs. Methylmercuric chloride (MMC, 2.5 × 10 −5 −2 × 10 −4 m ), p -chloromercuri phenyl sulfonate (PCMBS, 2.5 × 10 −5 −10 −3 m ) and HgCl 2 (5 × 10 −5 −2 × 10 −4 m ) were found to be effective in increasing the noradrenaline output in a concentration dependent manner. MMC and HgCl 2 did not require extracellular calcium, but the response to PCMBS was reduced in the absence of extracellular calcium. Thiols, such as cysteine and penicillamine reversibly inhibited the effects of mercury compounds, when added simultaneously with them in equal or in twofold concentrations. It is suggested that the mercury compounds may increase the noradrenaline output from adrenergic nerve terminals by modifying SH groups.

Effects of sulfhydryl reagents on synthetic and hydrolytic activities of multifunctional glucose-6-phosphatase

1. 1.Comprehensive, systematic studies of inhibitions of both Glc-6-P phosphohydrolase and carbamyl-P: glucose phosphotransferase activities of rat liver microsomal glucose 6-phosphatase (EC 3.1.3.9) by three classes of sulfhydryl reagents—mercaptideforming reagents, thiol-oxidants, and thiol-alkylating agents—have been carried out. 2. 2.Essentially equal, parallel patterns of time-dependent inhibitions of both activities were noted with all enzyme preparations tested—microsomal suspensions, detergent-dispersed microsomal preparations, and partially purified preparations—indicating that inhibitions by relatively low concentrations of thiol reagents may be produced regardless of the physical state of this membrane-bound enzyme. 3. 3.Inhibitions were dependent on (a) the nature of the inhibitor ( p- chloromercuriphenyl sulfonate > Hg 2+ > N- ethylmaleimide > iodosobenzoate > iodoacetate), (b) duration of exposure of the enzyme to inhibitor, and (c) reaction pH. Various thiol compounds— l-cysteine, GSH, and β-mercaptoethanol—partially reversed inhibitions, while phosphate substrates (but not d-glucose) slowed the rate of reaction of the enzyme with inhibitor. 4. 4.Exposure of the enzyme simultaneously to inhibitor and substrate for short periods of time produced kinetics resembling those of inhibition competitive with respect to phosphate substrates. Unit change in the slope of Dixon plots near pH 8.7 also implicates enzyme sulfhydryl in Glc-6-P binding. 5. 5.These observations, in toto, support the presence of functional sulfhydryl group or groups in glucose 6-phosphatase-phosphotransferase. Further, they suggest that such sulfhydryl may be located at or near the active site, and possibly might be involved in enzyme-phosphate-substrate binding.

Activation of guanyl cyclase and adenyl cyclase by secretin

Properties of rat liver guanyl cyclase and adenyl cyclase and the effects of hormones on the activity of these enzymes have been investigated. 1. 1. Secretin ( 2·10 −7–60·10 −7 M ) stimulates guanyl cyclase activity of 18 000 × g supernantants of rat liver homogenates with no change in the Michaelis-Menten constant for GTP ( 3.8·10 −5 M ). 2. 2. Insulin, glucagon, and hydrocortisone in vitro were found to have no effect on guanyl cyclase activity in the presence or absence of secretin. 3. 3. p- Chloromercuriphenyl sulfonate inhibits guanyl cyclase activity, and p- chloromercuriphenyl sulfonate inhibition is reversed by dithiothreitol. p- Chloromercuriphenyl sulfonate-dithiothreitol treatments have no effect on the sensitivity of the enzyme to secretin stimulation. These studies indicate that sulfhydryl groups may be involved in the regulation of the activity of liver guanyl cyclase. 4. 4. Adenyl cyclase activity of rat liver homogenate is stimulated by secretin to approximately 50% of the stimulation caused by glucagon. Using hormone concentrations that maximally stimulate homogenate adenyl cyclase, experiments with combinations of glucagon, secretin, and isoproterenol indicate that only secretin and isoproterenol do not have additive effects. Propranolol blocks stimulation due to isoproterenol, but does not affect stimulation by secretin. 5. 5. Insulin has no effect on either glucagon- or secretin-stimulated liver homogenate adenyl cyclase activity.