The effectiveness of inhibitors of soluble prolyl hydroxylase against the enzyme in the cisternae of isolated bone microsomes

Inhibitors of purified, soluble prolyl hydroxylase (K. Majamaa et al. (1984) Eur. J. Biochem. 138, 239-245; K. Majamaa et al. (1986) J. Biol. Chem. 261, 7819-7823) were tested against isolated chick embryo bone microsomes containing intracisternal prolyl hydroxylase and its radiolabeled, unhydroxylated procollagen substrate. Two groups of inhibitors were used which consisted of pyridine-2-carboxylate and 1,2-dihydroxybenzene (catechol) derivatives. The 2,4- and 2,5-pyridine dicarboxylic acids, which are potent inhibitors of the soluble enzyme (Ki values 2 and 0.8 microM, respectively), were effective in the same concentration range against intracisternal prolyl hydroxylase, although their relative affinities were reversed. Inhibition by pyridine-2,4-dicarboxylate in the microsomal system was reversed by increasing the concentration of 2-oxoglutarate. Pyridine-2,4-dicarboxylic acid did not inhibit the uptake of 2-[14C]oxoglutarate into microsomes, so it appears likely that the inhibitor must traverse the microsomal membrane and act directly at the enzyme level. Pyridine-2-carboxylic acid was ineffective in the microsomal system at 1 mM whereas it is a relatively potent inhibitor of the soluble enzyme with a Ki of 25 microM. This finding suggests that the second carboxyl group of the pyridine carboxylate derivatives may be required for their transport into the microsomal lumen. In the soluble system, 3,4-dihydroxybenzoic acid and 1,2-dihydroxybenzene had been found to be competitive inhibitors with relatively low Ki values of 5 and 25 microM, respectively. In the microsomal system, half-maximal inhibition was obtained at approximately 50-100 microM and inhibition was not reversed by increasing the concentrations of either 2-oxoglutarate or ascorbate, alone or together. These results imply that in situ these compounds do not inhibit prolyl hydroxylase directly. Thus, the microsomal system can assess the accessibility of the intracisternal enzyme to potential inhibitors and offers an insight into the in cellulo potential of such compounds.     

Calcium Transport in Sealed Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue : I. Characterization of a Ca-Pumping ATPase Associated with the Endoplasmic Reticulum

Calcium transport was examined in microsomal membrane vesicles from red beet (Beta vulgaris L.) storage tissue using chlorotetracycline as a fluorescent probe. This probe demonstrates an increase in fluorescence corresponding to calcium accumulation within the vesicles which can be collapsed by the addition of the calcium ionophore A23187. Calcium uptake in the microsomal vesicles was ATP dependent and completely inhibited by orthovanadate. Centrifugation of the microsomal membrane fraction on a linear 15 to 45% (w/w) sucrose density gradient revealed the presence of a single peak of calcium uptake which comigrated with the marker for endoplasmic reticulum. The calcium transport system associated with endoplasmic reticulum vesicles was then further characterized in fractions produced by centrifugation on discontinous sucrose density gradients. Calcium transport was insensitive to carbonylcyanide m-chlorophenylhydrazone indicating the presence of a primary transport system directly linked to ATP utilization. The endoplasmic reticulum vesicles contained an ATPase activity that was calcium dependent and further stimulated by A23187 (Ca(2+), A23187 stimulated-ATPase). Both calcium uptake and Ca(2+), A23187 stimulated ATPase demonstrated similar properties with respect to pH optimum, inhibitor sensitivity, substrate specificity, and substrate kinetics. Treatment of the red beet endoplasmic reticulum vesicles with [gamma-(32)P]-ATP over short time intervals revealed the presence of a rapidly turning over 96 kilodalton radioactive peptide possibly representing a phosphorylated intermediate of this endoplasmic reticulum associated ATPase. It is proposed that this ATPase activity may represent the enzymic machinery responsible for mediating primary calcium transport in the endoplasmic reticulum linked to ATP utilization.     

Calcium uptake and release by isolated cortices and microsomes from the unfertilized egg of the sea urchin Strongylocentrotus droebachiensis

Isolated cortices from unfertilized sea urchin eggs sequester calcium in an ATP-dependent manner when incubated in a medium containing free calcium levels characteristic of the resting cell (approximately 0.1 microM). This ATP-dependent calcium uptake activity was measured in the presence of 5 mM Na azide to prevent mitochondrial accumulation, was increased by oxalate, and was blocked by 150 microM quercetin and 50 microM vanadate (known inhibitors of calcium uptake into the sarcoplasmic reticulum). Cortical regions preloaded with 45Ca in the presence of ATP were shown to dramatically increase their rate of calcium efflux upon the addition of (a) the calcium ionophore A23187 (10 microM), (b) trifluoperazine (200 microM), (c) concentrations of free calcium that activated cortical granule exocytosis, and (d) the calcium mobilizing agent inositol trisphosphate. This pool of calcium is most likely sequestered in the portion of the egg's endoplasmic reticulum that remains associated with the cortical region during its isolation. We have developed a method for obtaining a high yield of purified microsomal vesicles from whole eggs. This preparation also demonstrates ATP-dependent calcium sequestering activity which increases in the presence of oxalate and has similar sensitivities to calcium transport inhibitors; however, the isolated microsomal vesicles did not show any detectable release of calcium when exposed to inositol trisphosphate.     

A high-affinity, calmodulin-sensitive (Ca2+ + Mg2+)-ATPase and associated calcium-transport pump in the Ehrlich ascites tumor cell plasma membrane

A unique cytoplast preparation from Ehrlich ascites tumor cells (G. V. Henius, P. C. Laris, and J. D. Woodburn (1979) Exp. Cell. Res. 121, 337-345), highly enriched in plasma membranes, was employed to characterize the high-affinity plasma membrane calcium-extrusion pump and its associated adenosine triphosphatase (ATPase). An ATP-dependent calcium-transport system which had a high affinity for free calcium (K0.5 = 0.040 +/- 0.005 microM) was identified. Two different calcium-stimulated ATPase activities were detected. One had a low (K0.5 = 136 +/- 10 microM) and the other a high (K0.5 = 0.103 +/- 0.077 microM) affinity for free calcium. The high-affinity enzyme appeared to represent the ubiquitous high-affinity plasma membrane (Ca2+ + Mg2+)-ATPase (calcium-stimulated, magnesium-dependent ATPase) seen in normal cells. Both calcium transport and the (Ca2+ + Mg2+)-ATPase were significantly stimulated by the calcium-dependent regulatory protein calmodulin, especially when endogenous activator was removed by treatment with the calcium chelator ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid. Other similarities between calcium transport and the (Ca2+ + Mg2+)-ATPase included an insensitivity to ouabain (0.5 mM), lack of activation by potassium (20 mM), and a requirement for magnesium. These similar properties suggested that the (Ca2+ + Mg2+)-ATPase represents the enzymatic basis of the high-affinity calcium pump. The calcium pump/enzyme system was inhibited by orthovanadate at comparatively high concentrations (calcium transport: K0.5 congruent to 100 microM; (Ca2+ + Mg2+)-ATPase: K0.5 greater than 100 microM). Upon Hill analysis, the tumor cell (Ca2+ + Mg2+)-ATPase failed to exhibit cooperative activation by calcium which is characteristic of the analogous enzyme in the plasma membrane of normal cells.     

The effect of trifluoroperazine on the sarcoplasmic reticulum membrane

The inhibitory effect of trifluoroperazine (25-200 microM) on the sarcoplasmic reticulum calcium pump was studied in sarcoplasmic reticulum vesicles isolated from skeletal muscle. It was found that the lowest effective concentrations of trifluoroperazine (10 microM) displaces the Ca2+ dependence of sarcoplasmic reticulum ATPase to higher Ca2+ concentrations. Higher trifluoroperazine concentrations (100 microM) inhibit the enzyme even at saturating Ca2+. If trifluoroperazine is added to vesicles filled with calcium in the presence of ATP, inhibition of the catalytic cycle is accompanied by rapid release of accumulated calcium. ATPase inhibition and calcium release are produced by identical concentrations of trifluoroperazine and, most likely, by the same enzyme perturbation. These effects are related to partition of trifluoroperazine ino the sarcoplasmic reticulum membrane, and consequent alteration of the enzyme assembly within the membrane structure, and of the bilayer surface properties. The effect of trifluoroperazine was also studied on dissociated ('chemically skinned') cardiac cells undergoing phasic contractile activity which is totally dependent on calcium uptake and release by sarcoplasmic reticulum, and is not influenced by inhibitors of slow calcium channels. It was found that trifluoroperazine interferes with calcium transport by sarcoplasmic reticulum in situ, as well as with the role of sarcoplasmic reticulum in contractile activation.     

Sulfation in vitro of mucus glycoprotein by submandibular salivary gland: effects of prostaglandin and acetylsalicylic acid

Enzymatic sulfation of mucus glycoprotein by rat submandibular salivary gland and the effect of prostaglandin and acetylsalicylic acid on this process were investigated in vitro. The sulfotransferase enzyme which catalyzes the transfer of sulfate ester group from 3'-phosphoadenosine-5'-phosphosulfate to submandibular gland mucus glycoprotein has been located in the detergent extracts of Golgi-rich membrane fraction of the gland. Optimum enzyme activity was obtained at pH 6.8 with 0.5% Triton X-100, 25 mM NaF and 4 mM MgCl2, using the desulfated glycoprotein. The enzyme was also capable of sulfation of the intact mucus glycoprotein, but the acceptor capacity of such glycoprotein was 68% lower. The apparent Km of the submandibular gland sulfotransferase for salivary mucus glycoprotein was 11.1 microM. The 35S-labeled glycoprotein product of the enzyme reaction gave in CsCl density gradient a 35S-labeled peak which coincided with that of the glycoprotein. This glycoprotein upon reductive beta-elimination yielded several acidic 35S-labeled oligosaccharide alditols which accounted for 75% of the 35S-labeled glycoprotein label. Based on the analytical data, the two most abundant oligosaccharides were identified as sulfated tri- and pentasaccharides. The submandibular gland sulfotransferase activity was stimulated by 16,16-dimethyl prostaglandin E2 and inhibited by acetylsalicylic acid. The rate of enhancement of the glycoprotein sulfation was proportional to the concentration of prostaglandin up to 2.10(-5) M, at which point a 31% increase in sulfation was attained. The inhibition of the glycoprotein sulfation by acetylsalicylic acid was proportional to the drug concentration up to 2.5.10(-4) M at which concentration a 48% reduction in the sulfotransferase activity occurred. The apparent Ki value for sulfation of salivary mucus glycoprotein in presence of acetylsalicylic acid was 58.9 microM. The results suggest that prostaglandins may play a role in salivary mucin sulfation and that this process is sensitive to such nonsteroidal anti-inflammatory agents as acetylsalicylic acid.     

Lipoxygenase-induced lipid peroxidation of isolated cardiac microsomes modulates their calcium-transporting function

We demonstrated previously that products of linoleic and arachidonic acids, arising from enzymatic or non-enzymatic oxidation, inhibit ATP-dependent calcium accumulation into and promote release of calcium from vesicles derived from sarcoplasmic reticulum of guinea-pig heart. In the present study, direct enzymatic peroxidation of cardiac membrane lipids was performed and the effect on calcium transport was examined. Vesicles were preincubated at 37 degrees C with soybean lipoxygenase-1 (linoleate:oxygen oxidoreductase, EC 1.13.11.12) for up to 1 h prior to the initiation of calcium accumulation. The extent of membrane peroxidation was assessed by monitoring the production of malondialdehyde. Pretreatment of vesicles with lipoxygenase for 40 and 60 min markedly depressed calcium accumulation. The lipoxygenase-induced suppression of calcium transport was completely antagonized by nordihydroguaiaretic acid (1 microM), not at all by indomethacin (1 microM), and only partially by 5,8,11,14-eicosatetraynoic acid (0.3 microM). Low concentrations of calcium (10(-5)-5 X 10(-5) M) enhanced, and a high concentration (10(-3) M) inhibited lipoxygenase-induced peroxidation of membrane lipids. The calcium-accumulating ability of the vesicles was inversely related to the extent of membrane peroxidation. The vesicles which showed the highest degree of peroxidation in the presence of 5 X 10(-5) M calcium, accumulated the lowest amount of calcium. In contrast, calcium at 10(-3) M suppressed lipid peroxidation, resulting in higher calcium uptake than in vesicles peroxidized in the absence of calcium. Thus, calcium transport is depressed in microsomes undergoing lipoxygenase-induced peroxidation, a process which in turn is modulated by calcium.     

Parotid microsomal Ca2+ transport. Subcellular localization and characterization.

Rat parotid gland homogenates were fractionated into mitochondrial, heavy microsomal and light microsomal fractions by differential centrifugation. ATP-dependent 45Ca2+ uptake by the subcellular fractions paralleled the distribution of NADPH-cytochrome c reductase, an enzyme associated with the endoplasmic reticulum. The highest rate of Ca2+ uptake was found in the heavy microsomal fraction. Ca2+ uptake by this fraction was dependent on the presence of ATP and was sustained at a linear rate by 5 mM-oxalate. Inhibitors of mitochondrial Ca2+ transport had no effect on the rate of Ca2+ uptake. Na+ and K+ stimulated Ca2+ uptake. At optimal concentrations. Na+ stimulated Ca2+ uptake by 120% and K+ stimulated Ca2+ uptake by 260%. Decreasing the pH from 7.4 to 6.8 had little effect on Ca2+ uptake. The Km for Ca2+ uptake was 3.7 microM free Ca2+ and 0.19 mM-ATP. Vanadate inhibited Ca2+ uptake; 60 microM-vanadate inhibited the rate of Ca2+ accumulation by 50%. It is concluded that the ATP-dependent Ca2+ transport system is located on the endoplasmic reticulum and may play a role in maintaining intracellular levels of free Ca2+ within a narrow range of concentration.     

Plasma membrane vesicles prepared from unadhered monocytes: Characterization of calcium transport and the calcium ATPase

We have purified unadhered human monocytes in sufficient quantities to prepare monocyte plasma membrane vesicles and study vesicular calcium transport. Monocytes were isolated from plateletpheresis residues by counterflow centrifugal elutriation. By combining this source and procedure, 7 x 10(8) monocytes of over 90% purity were obtained. The membranes, isolated on a sucrose step gradient, had an 18-fold enrichment in Na,K-ATPase, a 29-fold diminution of succinate dehydrogenase activity and were vesicular on transmission electron micrographs. The membrane vesicles loaded with oxalate accumulated calcium only in the presence of Mg and ATP. Calcium uptake did not occur if ATP was replaced by any of five nucleotide phosphates or if Mg was omitted. Calcium transport had a maximal velocity of 4 pmoles calcium/micrograms vesicle protein/min and a Km for calcium of 0.53 microM. The ionophore A23187 completely inhibited calcium accumulation while 5 mM sodium cyanide and 10 microM ouabain had no effect. A calcium-activated ATPase was present in the same plasma membrane vesicles. The calcium ATPase had a maximal velocity of 18.0 pmoles calcium/micrograms vesicle protein/min and a Km for calcium of 0.60 microM. Calcium-activated ATPase activity was absent if Mg was omitted or if (gamma - 32P) GTP replaced (gamma - 32P) ATP. Monocyte plasma membranes that were stripped of endogenous calmodulin by EGTA treatment showed a reduced level of calcium uptake and calcium ATPase activity. The addition of exogenous calmodulin restored the transport activity to that of unstripped monocyte plasma membranes. Thus, monocyte plasma membrane vesicles contain a highly specific, ATP-dependent calcium transport system and a calcium-ATPase with similar high calcium affinities.     

Nucleotidase activities in membrane preparations of nervous ganglia and digestive gland of the snail Helix aspersa

Nucleotide-metabolizing enzymes play an important role in the regulation of nucleotide levels. In the present report, we demonstrated an enzyme activity with different kinetic properties in membrane preparations of the nervous ganglia and digestive gland from Helix aspersa. ATPase and ADPase activities were dependent on Ca2+ and Mg2+ with pH optima approximately 7.2 and between 6.0 and 8.0 in digestive gland and nervous ganglia, respectively. The enzyme activities present in membrane preparations of these tissues preferentially hydrolyzed triphosphate nucleotides. In nervous ganglia, the enzyme was insensitive to the classical ATPases inhibitors. In contrast, in digestive gland, N-ethylmaleimide (NEM) produced 45% inhibition of Ca(2+)-ATP hydrolysis. Sodium azide, at 100 microM and 20 mM, inhibited Mg(2+)-ATP hydrolysis by 36% and 55% in digestive gland, respectively. The presence of nucleotide-metabolizing enzymes in these tissues may be important for the modulation of nucleotide and nucleoside levels, controlling their actions on specific purinoceptors in these species.     

Ionic and substrate requirements of the high affinity calcium pumping ATPase in endoplasmic reticulum of pancreas

1. Calcium transport and ATPase activities were determined in microsomal vesicles from pancreatic tissue enriched in endoplasmic reticulum membranes. 2. Calcium transport and ATPase share the following properties: (i) magnesium was required with a K0.5 of 0.7 mM and maximal pumping ATPase activity at 5 mM Mg-ATP; (ii) at saturating magnesium concentrations, calcium increased ATP splitting activity up to three times with an apparent K0.5 close to 0.3 microM calcium; (iii) potassium stimulated the high calcium affinity Mg2+-dependent ATPase and calcium transport. 3.The properties of the calcium pumping system fulfil the cationic and substrate requirements from a physiological point of view.     

Characterization and solubilization of membrane bound diacylglycerol lipases from bovine brain

Bovine brain contains two diacylglycerol lipases. One is localized in purified microsomes and the other is found in the plasma membrane fraction. The microsomal enzyme is markedly stimulated by the non-ionic detergent, Triton X-100, and Ca2+, whereas the plasma membrane diacylglycerol lipase is strongly inhibited by Triton X-100 and Ca2+ has no effect on its enzymic activity. Both enzymes were solubilized using 0.25% Triton X-100. The solubilized enzymes followed Michaelis-Menten kinetics. The apparent Km values for microsomal and plasma membrane enzymes are 30.5 and 12.0 microM respectively. Both lipases are strongly inhibited by RHC 80267, with Ki values for microsomal and plasma membrane diacylglycerol lipases of 70 and 43 microM, respectively. The retention of microsomal diacylglycerol lipase on a concanavalin A-Sepharose column and its elution by methyl alpha-D-mannoside indicates the glycoprotein nature of this enzyme.     

Calcium transport and phosphorylated intermediate of (Ca2+ + Mg2+)-ATPase in plasma membranes of rat liver

We have identified and characterized calcium transport and the phosphorylated intermediate of the (Ca2+ + Mg2+)-ATPase in plasma membrane vesicles prepared from rat liver. The calcium transport did not absolutely require the presence of oxalate and was completely inhibited by 1 microM of ionophore A23187. Oxalate, which serves as a trapping agent in calcium uptake of skeletal muscle and liver microsomes, was not absolutely required to maintain the net accumulation of calcium. The Vmax and Km for calcium uptake were 35.2 +/- 10.1 pmol of calcium/mg of protein/min, and 17.6 +/- 2.5 nM of free calcium, respectively. Ten mM magnesium was required for the maximal accumulation of calcium. Substitution of 5 and 10 mM ADP, CTP, GTP, and UTP for ATP could not support calcium uptake. The calcium uptake was not affected by 0.5 mM ouabain, 20 mM azide, or 2 micrograms/ml of oligomycin but was inhibited in a dose-dependent fashion by vanadate, with a Ki of approximately 20 microM for vanadate. The substrate affinities and specificities of this calcium-transport activity suggest that it is closely associated with the (Ca2+ + Mg2+)-ATPase reported in the plasma membranes of liver (Lotersztajn, S., Hanoune, J., and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215). A calcium-stimulated and magnesium-dependent phosphoprotein was also demonstrated in the same membrane vesicles. The free calcium concentration at which its phosphorylation was half-maximal was 15.5 +/- 5.6 nM. Sodium fluoride, ouabain, sodium azide, oligomycin, adriamycin, and N,N'-dicyclohexylcarbodiimide did not affect its formation while vanadate at 100 microM inhibited the calcium-dependent phosphorylation by approximately 60%. The properties of this phosphoprotein suggest that it may be the phosphorylated intermediate of the (Ca2+ + Mg2+)-ATPase in the plasma membranes of rat liver.     

Properties of yeast Saccharomyces cerevisiae plasma membrane dicarboxylate transporter

Transport of succinate into Saccharomyces cerevisiae cells was determined using the endogenous coupled mitochondrial succinate oxidase system. The dependence of succinate oxidation rate on the substrate concentration was a curve with saturation. At neutral pH the K(m) value of the mitochondrial "succinate oxidase" was fivefold less than that of the cellular "succinate oxidase". O-Palmitoyl-L-malate, not penetrating across the plasma membrane, completely inhibited cell respiration in the presence of succinate but not glucose or pyruvate. The linear inhibition in Dixon plots indicates that the rate of succinate oxidation is limited by its transport across the plasmalemma. O-Palmitoyl-L-malate and L-malate were competitive inhibitors (the K(i) values were 6.6 +/- 1.3 microM and 17.5 +/- 1.1 mM, respectively). The rate of succinate transport was also competitively inhibited by the malonate derivative 2-undecyl malonate (K(i) = 7.8 +/- 1.2 microM) but not phosphate. Succinate transport across the plasma membrane of S. cerevisiae is not coupled with proton transport, but sodium ions are necessary. The plasma membrane of S. cerevisiae is established to have a carrier catalyzing the transport of dicarboxylates (succinate and possibly L-malate and malonate).     

Inositol trisphosphate and thapsigargin discriminate endoplasmic reticulum stores of calcium in rat brain

ATP dependent Ca2+ accumulation into oxalate-loaded rat brain microsomes is potently inhibited by thapsigargin with an IC50 of 2 nM and maximal inhibition at 10 nM. Approximately 15% of the total A23187-releasable microsomal calcium store is insensitive to thapsigargin concentrations up to 100 microM. Inositol-1,4,5-trisphosphate (IP3) maximally inhibits 40% of the net Ca2+ accumulation by whole brain microsomes. Its effects are non-additive with thapsigargin suggesting that the IP3-sensitive Ca2+ pool is a subset of the thapsigargin sensitive Ca2+ pool. Marked regional differences occur in Ca2+ transport rates and sensitivity to both thapsigargin and IP3.     

Comparative study of the effect of aliphatic alcohols on energy-dependent accumulation of Ca2+ in intracellular membranes of smooth muscle cells

The effects of ethanol and other aliphatic alcohols on energy-dependent Ca2+ transport in endoplasmic reticulum and mitochondria were studied in digitonin-treated myometrium cells. The Ca2+ uptake in mitochondria increased (on 15-20%) with increasing methanol, ethanol and propanol concentrations in medium, whereas further rise of concentration inhibited this process. Treatments of myometrial cells with short-chain alcohols caused an inhibition of calcium uptake in endoplasmic reticulum. Butanol inhibited both calcium uptake in mitochondria and endoplasmic reticulum. Ca2+ accumulation in intracellular pools is inhibited by aliphatic alcohols in the following order of potency: butanol > propanol > ethanol > methanol. It is concluded that modifying effect of aliphatic alcohols on energy dependent calcium accumulation in intracellular membrane structures is defined as on origin of Ca(2+)-transporting system and (or) properties of these membrane structures so on properties of alcohols.     

Activation of detergent-solubilized rat hepatic 5'-iodothyronine deiodinase by NADPH and nonglutathione cytosolic components

An investigation was made of the possible role of the hepatic microsomal membrane in the activation of 5'-iodothyronine deiodinase (5'-DI) by a cytosolic activating system consisting of fraction A (relative mass (Mr) greater than 60,000), fraction B (Mr, approximately 13,000), and NADPH. Activation of 5'-DI in washed microsomes was compared with that of a microsome extract prepared by solubilization with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate and further purification by fractional precipitation with polyethylene glycol and by DEAE-Sephacel chromatography. All 5'-DI preparations exhibited qualitatively similar dependence upon NADPH and cytosolic factors in fractions A and B for 5'-DI activation and were relatively unresponsive to NADH. Activation of solubilized preparations, unlike that of intact microsomes, was more readily inhibited by low concentrations of detergent and not inhibited by NADPH concentrations above 0.25 mM. Attempted purification of 5'-DI failed to produce a substantial increase in specific activity of the enzyme. It is concluded that, while glutathione-independent cytosolic factors and NADPH can activate 5'-DI in the absence of an intact microsomal membrane, some membrane constituents removed during solubilization and purification of the enzyme are required for maximal activation.     

Taurine and cell volume maintenance in the shark rectal gland: cellular fluxes and kinetics

Tissue slices of shark rectal gland are studied to examine the kinetics of the cellular fluxes of taurine, a major intracellular osmolyte in this organ. Maintenance of high steady-state cell taurine (50 mM) is achieved by a ouabain-sensitive active Na+-dependent uptake process and a relatively slow efflux. Uptake kinetics are described by two saturable taurine transport components (high-affinity, Km 60 microM; and low-affinity, Km 9 mM). [14C]Taurine uptake is enhanced by external Cl-, inhibited by beta-alanine and unaffected by inhibitors of the Na+/K+/2Cl- co-transport system. Two cellular efflux components of taurine are documented. Incubation of slices in p-chloromercuribenzene sulfonate (1 mM) reduces taurine uptake, increases efflux of taurine and induces cell swelling. Studies of efflux in isotonic media with various cation and anion substitutions demonstrate that high-K+ markedly enhances taurine efflux irrespective of cell volume changes (i.e. membrane stretching is not involved). Moreover, iso-osmotic cell swelling induced in media containing propionate is not associated with enhanced efflux of taurine from the cells. It is suggested that external K+ exerts a specific effect on the cytoplasmic membrane to increase its permeability to taurine.     

Calcium transport mechanisms in basolateral plasma membrane-enriched vesicles from rat parotid gland.

Ca2+ transport was studied by using basolateral plasma membrane vesicles from rat parotid gland prepared by a Percoll gradient centrifugation method. In these membrane vesicles, there were two Ca2+ transport systems; Na+/Ca2+ exchange and ATP-dependent Ca2+ transport. An outwardly directed Na+ gradient increased Ca2+ uptake. Ca2+ efflux from Ca2+-preloaded vesicles was stimulated by an inwardly directed Na+ gradient. However, Na+/Ca2+ exchange did not show any 'uphill' transport of Ca2+ against its own gradient. ATP-dependent Ca2+ transport exhibited 'uphill' transport. An inwardly directed Na+ gradient also decreased Ca2+ accumulation by ATP-dependent Ca2+ uptake. The inhibition of Ca2+ accumulation was proportional to the external Na+ level. Na+/Ca2+ exchange was inhibited by monensin, tetracaine and chlorpromazine, whereas ATP-dependent Ca2+ transport was inhibited by orthovanadate, tetracaine and chlorpromazine. Oligomycin had no effect on either system. These results suggest that in the parotid gland cellular free Ca2+ is extruded mainly by an ATP-dependent Ca2+ transport system, and Na+/Ca2+ exchange may modify the efficacy of that system.     

Topography of rat hepatic microsomal enzymatic components of the fatty acid chain elongation system

The orientation of the condensing enzyme, the beta-hydroxyacyl-CoA dehydrase, and the trans-2-enoyl CoA reductase within the rat liver microsomal membrane was investigated by the use of impermeant inhibitors of enzyme activity: trypsin, chymotrypsin, subtilisin, mercury-dextran, and anti-beta-hydroxyacyl-CoA dehydrase IgG. The activity of the condensing enzyme was inhibited more than 70% by various proteases and was completely inhibited by 80 microM mercury-dextran. Similar results were obtained for the trans-2-enoyl-CoA reductase activity. On the other hand, in the absence of detergent, proteases inhibited beta-hydroxyacyl-CoA dehydrase activity by 25-40%, while in the presence of detergent the inhibition increased to 65-90%. Furthermore, anti-beta-hydroxyacyl-CoA dehydrase IgG, which in the absence of detergent produced no inhibition, in the presence of detergent inhibited beta-hydroxyacyl-CoA dehydrase activity by more than 80%; under identical conditions, preimmune IgG caused a 13% inhibition. Microsomes used throughout this study displayed greater than 90% latency with respect to mannose-6-phosphatase activity, indicating that the microsomes were intact. Latency was not affected by the proteases, by mercury-dextran, or by the presence of the enzyme assay components. These results suggest that both the condensing enzyme and the reductase are present on the cytoplasmic surface of the membrane, whereas the beta-hydroxyacyl-CoA dehydrase is embedded in the microsomal membrane.