Effect of sodium glycocholate and polyoxyethylene-9-lauryl ether on the hydrolysis of varying concentrations of insulin in the nasal homogenates of the albino rabbit

Protease inhibition has been postulated to be one of the several mechanisms by which penetration enhancers promote the mucosal absorption of peptide and protein drugs. The objective of this study was to determine whether protease inhibition by Na glycocholate and polyoxyethylene-9-lauryl ether, two extensively studied enhancers, led to suppression of insulin proteolysis over a range of insulin concentrations. To this end, the rate of insulin proteolysis in nasal tissue supernatants of the albino rabbit was determined in the presence of 0.1-2% Na glycocholate and polyoxyethylene-9-lauryl ether and at insulin concentrations ranging from 5 to 100 microM. Partly due to self-association, insulin was self-stabilizing against nasal proteolysis as its concentration was raised from 5 to 100 microM. At insulin concentrations lower than 50 microM, both Na glycocholate and polyoxyethylene-9-lauryl ether reduced the rate of insulin proteolysis. By contrast, at 100 microM insulin concentration, both enhancers accelerated insulin proteolysis. Such an effect was attributed to the deaggregation of insulin by the enhancers, increasing the proportion of monomers available for nasal proteolysis. The incorporation of 0.1 mM PCMPS, a potent inhibitor of insulin proteolysis, partly overcame the accelerating effect of Na glycocholate on insulin proteolysis.     

Prevention of thermal inactivation and aggregation of lysozyme by polyamines.

Proteins tend to form inactive aggregates at high temperatures. We show that polyamines, which have a relatively simple structure as oligoamids, effectively prevent thermal inactivation and aggregation of hen egg lysozyme. In the presence of additives, including arginine and guanidine (100 microM), more than 30% of 0.2 mg x mL(-1) lysozyme in sodium phosphate buffer (pH 6.5) formed insoluble aggregates by heat treatment (98 degrees C for 30 min). However, in the presence of 50 mm spermine or spermidine, no aggregates were observed after the same heat treatment. The residual activity of lysozyme after this heat treatment was very low (< 5%), even in the presence of 100 microM arginine and guanidine, while it was maintained at approximately 50% in the presence of 100 microM spermine and spermidine. These results imply that polyamines are new candidates as molecular additives for preventing the thermal aggregation and inactivation of heat-labile proteins.     

Inhibition by phenylglyoxal of the sodium-coupled fluxes of glucose and phosphate in renal brush-border membranes

The coupling of phosphate and glucose transport to sodium in brush-border membrane vesicles from rat kidney cortex was studied after chemical modification of arginine residues by phenylglyoxal. Phosphate (10 mM) and sodium (20 mM) uptakes were linear for 6 s and stimulated in the presence of their cosubstrate. The sodium:phosphate stoichiometry measured by a direct method was 1.74. Sodium-independent phosphate and glucose influx were found to be unaffected by phenylglyoxylation. Phosphate- or glucose-independent sodium influx also remained unaltered by the treatment. However, phosphate influx measured with sodium was inhibited by 69% and sodium influx measured with phosphate was inhibited by 40%. When these values were corrected for uncoupled fluxes, the sodium influx coupled to phosphate and the phosphate influx coupled to sodium were inhibited by 93 and 95%, respectively. Glucose influx measured in the presence of sodium was inhibited by 36% and sodium influx in the presence of glucose was reduced by 39%. When the values were corrected for diffusion, these inhibitions were 95 and 100%, respectively. We conclude that the coupling of phosphate and glucose to sodium fluxes by the renal carriers requires the participation of arginine residue(s) in the translocation process. Modification of this arginine by phenylglyoxal leads to a marked inhibition of coupling. These results suggest the implication of arginine residues in the molecular coupling for both glucose and phosphate sodium symporters.     

Activation of 5-lipoxygenase by guanosine 5'-O-(3-thiotriphosphate) and other nucleoside phosphorothioates: redox properties of thionucleotide analogs

The stable nucleotide analog guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) was found to be a very potent activator of 5-lipoxygenase in cell-free preparations from rat polymorphonuclear (PMN) leukocytes, causing a 10-fold stimulation of arachidonic acid oxidation at concentrations as low as 0.5-1 microM. The enhancement of enzyme activity was not directly related to G protein activation since the effect of GTP gamma S could not be abolished by GDP nor replaced by GTP or guanylyl-imidodiphosphate (up to 100 microM). Furthermore, other phosphorothioate analogs, such as guanosine 5'-O-(2-thiodiphosphate), adenosine 5'-O-(3-thiotriphosphate), adenosine 5'-O-(2-thiodiphosphate), and adenosine 5'-O-thiomonophosphate all stimulated 5-lipoxygenase activity at concentrations of 10 microM or lower. This effect could not be detected with any of the corresponding nucleoside phosphate derivatives. The stimulation of 5-lipoxygenase activity by nucleoside phosphorothioates was observed under conditions where the reaction is highly dependent on exogenous hydroperoxides, such as in the presence of beta-mercaptoethanol or using enzyme preparations pretreated with sodium borohydride or glutathione peroxidase. GTP gamma S stimulated arachidonic acid oxidation by 5-lipoxygenase to the same extent as the activating hydroperoxides but had no effect on the reaction measured in the presence of optimal concentrations of 13-hydroperoxyoctadecadienoic acid (1-5 microM). Finally, sodium thiophosphate, but not sodium phosphate, markedly stimulated 5-lipoxygenase activity with properties similar to those of GTP gamma S. These results indicate that GTP gamma S and other phosphorothioate derivatives have redox properties that can contribute to increase 5-lipoxygenase activity by replacing the effect of hydroperoxides.     

Purification of a vanadate-sensitive ATPase from clathrin-coated vesicles of bovine brain

Clathrin-coated vesicle acidification is mediated by an N-ethylmaleimide-sensitive, vanadate-resistant proton-translocating ATPase. This enzyme is a 530-kDa hetero-oligomer which catalyzes ATP-dependent proton pumping when reconstituted (Xie, X. S., and Stone, D. K. (1986) J. Biol. Chem. 261, 2492-2495). We now report the purification of a second ATPase from bovine brain clathrin-coated vesicles which is inhibited by both N-ethylmaleimide (1 mM) and vanadate (10 microM). Localization of the ATPase to clathrin-coated vesicles was demonstrated by the precipitation of ouabain-resistant, vanadate-sensitive ATPase activity with anti-clathrin antibodies. The enzyme was solubilized with 0.1% polyoxyethylene 9-lauryl ether and has been purified 700-fold to a specific activity of 42 mumol of Pi.mg of protein-1.min-1. A molecular mass of 116 kDa was determined by centrifugation in sucrose gradients prepared in H2O and D2O, by high performance liquid chromatography using gel filtration, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed under reducing conditions. The ATPase is unlike any known mammalian E1E2-type ATPase in that it is not inhibited by ouabain or [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) and it is not activated by Na+, K+, or Ca2+.     

Bismuth citrate in the quantification of inorganic phosphate and its utility in the determination of membrane-bound phosphatases

This paper describes a rapid and sensitive method to determine inorganic phosphate, even in the presence of labile organic phosphate compounds and large quantities of proteins. The method eliminates the use of sodium arsenite, a highly toxic compound, substituting bismuth citrate for it to stabilize the phosphomolybdic acid complex formed during the interaction of inorganic phosphate and molybdate reduced by ascorbic acid. This method has also been adapted to microplates and has been used to determine the activities of Na/K ATPase and alkaline phosphatase of intestinal basolateral and luminal plasma membranes.     

Nutritional factors affecting nisin production by Lactococcus lactis subsp. actis NIZO 22186 in a synthetic medium

L. DE VUYST. 1995 A minimal synthetic medium (SM8) for nisin-producing Lactococcus lactis subsp. lactis strains has been designed; it consists of eight growth-stimulating amino acids (glutamic acid, methionine, valine, leucine, threonine, arginine, isoleucine and histidine), five vitamins (biotin, calcium pantothenate, nicotinic acid, pyridoxine and riboflavin) and the mineral salts dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, magnesium sulphate and trisodium citrate. Nisin biosynthesis is strongly dependent on the presence of a sulphur source, either an inorganic salt (magnesium sulphate or sodium thiosulphate) or the amino acids methionine, cysteine or cystathionine. The amino acids serine, threonine and cysteine highly stimulate nisin production without affecting the final cell yield, indicating their precursor role during nisin biosynthesis.     

Purification of the membrane-bound proton-translocating inorganic pyrophosphatase from Rhodospirillum rubrum

The proton translocating membrane-bound inorganic pyrophosphatase of Rhodospirillum rubrum S1, has been solubilized with good yield from chromatophores using Triton X-100 (9–10 oxyethylene groups) in the presence of high concentrations of MgCl₂ and ethyleneglycol. The enzyme has been purified 80-fold by hydroxylapatite column chromatography, to a state of near homogeneity, according to polyacrylamide-gelelectrophoresis. The enzyme appears to be a very hydrophobic integrally bound membrane protein. Phospholipids or Triton X-100 reconstitutes the enzyme activity after solubilization and purification. The purified enzyme preparation has a specific activity of 24 units. Both the purified and the chromatophore-bound enzyme are inhibited by N-ethylmaleimide, 4-chloro-7-nitrobenzo-2-oxo-1,3-diazol (NBF-Cl), sodium fluoride, imidodiphosphate, methylenediphosphonate and the antibiotic Dio-9 (energy-transfer inhibitor). In the solubilized state the purified enzyme is not stimulated by uncouplers or inhibited by dicyclohexylcarbodiimide in contrast to the chromatophore-bound pyrophosphatase. When reconstituted into liposomes the purified enzyme regains the stimulation by uncouplers.     

Phosphorylation of a chromaffin granule-binding protein by protein kinase C

Protein kinase C was detected in a group of Ca2+-dependent chromaffin granule membrane-binding proteins (chromobindins) on the basis of Ca2+-, phosphatidylserine-, 1,2-diolein-, and phorbol myristate acetate-stimulated histone kinase activity. When the chromobindins were incubated with [gamma-32P]ATP, Ca2+, and phosphatidylserine, 32P was incorporated predominantly into a protein of mass 37 +/- 1 kilodaltons (chromobindin 9, or CB9). Phosphorylation of this protein was also stimulated by diolein and phorbol myristate acetate, indicating that it is a substrate for the protein kinase C activity present in the chromobindins. Maximum phosphate incorporation into CB9 in the presence of 1 mM Ca2+, 75 micrograms/ml of phosphatidylserine, 2.5 micrograms/ml of diolein, and 12.5 micrograms/ml of dithiothreitol was 0.53 mol/mol of CB9 in 5 min. Eight 32P-labeled phosphopeptides were resolved in two-dimensional electrophoretic maps of trypsin digests of CB9. Phosphoamino acid analysis revealed that phosphorylation was exclusively on serine (94%) and threonine (6%) residues. Incubation of the chromobindins with chromaffin granule membranes in the presence of [gamma-32P]ATP resulted in the incorporation of 32P into eight additional proteins besides CB9 that could be separated from the membranes by centrifugation in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. We suggest that phosphorylation of CB9 or these additional eight proteins may regulate events underlying exocytosis in the chromaffin cell.     

Phosphorylation in vivo and in vitro of the arginine-ornithine periplasmic transport protein of Escherichia coli

The arginine-ornithine periplasmic binding protein, an essential component of the arginine-ornithine transport system of Escherichia coli, was isolated in a phosphorylated form and in a non-phosphorylated form from the periplasmic fluid, after incubation of intact cells with (32P)orthophosphate under conditions similar to those used for arginine transport studies. The binding protein could also be labeled with 32Pi by incubation in vitro of the periplasmic fluid with [gamma-32P]ATP, or by incubation in vitro of the purified binding protein with radioactive ATP, Mg2+ and a phosphokinase enzyme released by osmotic-shock treatment. The two forms of the protein were separated by DEAE-Sephacel chromatography. By several different criteria, which included binding studies, analyses of the amino acid composition of the two forms of the protein, analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and testing for other components of the periplasmic space with affinities for inorganic phosphate, it was concluded that the 32P-labeled protein corresponds to a phosphorylated form of the arginine-ornithine-binding protein. The phosphorylation reaction required Mg2+ and a phosphokinase from the periplasmic fluid. The dissociation constant of the phosphorylated protein for arginine was 5.0 microM (dissociation constant of the unmodified protein equals 0.1 microM), suggesting that the chemically modified protein is the active form of the molecule which releases the ligand for its translocation through the cytoplasmic membrane. The pH-stability profile of the phosphoprotein has a 'U'-shape characteristic of acyl phosphates. Reaction of the phosphorylated binding protein with hydroxylamine at pH 5.4, also released Pi from the phosphoprotein. These properties suggest that the phosphoryl group of the phosphoprotein is linked covalently to a carboxyl function of the protein. This information indicates that ATP is a direct energy donor for the active transport of arginine and ornithine in E. coli, and a step of phosphorylation of the arginine-ornithine-binding protein appears to be involved in the utilization of the phosphate bond energy by the arginine-ornithine transport system.     

Sodium-dependent phosphate and alanine transports but sodium-independent hexose transport in type II alveolar epithelial cells in primary culture

Inorganic phosphate, amino acids and sugars are of obvious importance in lung metabolism. We investigated sodium-coupled transports with these organic and inorganic substrates in type II alveolar epithelial cells from adult rat after one day in culture. Alveolar type II cells actively transported inorganic phosphate and alanine, a neutral amino acid, by sodium-dependent processes. Cellular uptakes of phosphate and alanine were decreased by about 80% by external sodium substitution, inhibited by ouabain (30 and 41%, respectively) and displayed saturable kinetics. Two sodium-phosphate cotransport systems were characterized: a high-affinity one (apparent Km = 18 microM) with a Vmax of 13.5 nmol/mg protein per 10 min and a low-affinity one (apparent Km = 126 microM) with a Vmax of 22.5 nmol/mg protein per 10 min. Alanine transport had an apparent Km of 87.9 microM and a Vmax of 43.5 nmol/mg protein per 10 min. By contrast, cultured alveolar type II cells did not express sodium-dependent hexose transport. Increasing time in culture decreased Vmax values of the two phosphate transport systems on day 4 while sodium-dependent alanine uptake was unchanged. This study demonstrated the existence of sodium-dependent phosphate and amino acid transports in alveolar type II cells similar to those documented in other epithelial cell types. These sodium-coupled transports provide a potent mechanism for phosphate and amino acid absorption and are likely to play a role in substrate availability for cellular metabolism and in regulating the composition of the alveolar subphase. The decrease in phosphate uptake with time in culture is parallel to decrease in surfactant synthesis reported in cultured alveolar type II cells, suggesting that phosphate availability for surfactant synthesis may be accomplished by a sodium-dependent phosphate uptake.     

Measurement of enzymes related to sucrose metabolism in permeabilized Chlorella vulgaris cells

Sucrose phosphate synthase (UDP-glucose: D-fructose-6-phosphate-2-glucosyl transferase, EC 2.4.1.14), sucrose synthase (UDP-glucose: D-fructose-2-glucosyl transferase, EC 2.4.1.13) and invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) were measured in toluene permeabilized cells of Chlorella vulgaris Beijerinck. All three activities were detected at all stages of the growth curve; sucrose synthase and sucrose phosphate synthase showed a zone of maximum activity, while invertase increased with time of growth. Sucrose phosphate synthase and sucrose synthase (sucrose synthesis direction) were stimulated by divalent cations and inhibited by UDP. This inhibition could be reversed by Mg²⁺ or Mn²⁺. Sucrose phosphate synthase activity was inhibited by inorganic phosphate and was enhanced by glucose-6-phosphate, but was insensitive to sucrose. Arbutine decreased sucrose synthase activity in both directions. Sucrose cleavage was inhibited by divalent cations and by pyrophosphate. The effects on the enzyme activities of the presence of 2,4-dichlorophenoxyacetic acid (2,4-D), gibberellic acid, abscisic acid and kinetin in the growth medium were investigated. Sucrose synthase activity was practically unaffected by all plant hormones tested, except for the presence of kinetin which stimulated the activity. Sucrose phosphate synthase activity was increased by both kinetin and abscisic acid. The effect of the latter was partially reversed by the presence of gibberellic acid. 2,4-D and kinetin were potent stimulators of invertase activity.     

Peroxisomal localization of glucose-6-phosphate dehydrogenase and pyrophosphate-stimulated dihydroxyacetone-phosphate acyltransferase in mouse kidney.

1. The subcellular localization of dihydroxyacetone-phosphate acyltransferase (DHAPAT) (assayed in the presence of pyrophosphate) and glucose-6-phosphate dehydrogenase (NADP+-dependent) activity in mouse kidney was investigated by density-gradient centrifugation. 2. DHAPAT has a predominantly peroxisomal distribution, and the activity in purified peroxisomes is stimulated by various organic and inorganic phosphate-containing compounds. The pH optimum is acid. 3. Approx. 10% of the cellular NADP+-dependent glucose-6-phosphate dehydrogenase activity is associated with peroxisomal fractions and may provide a source of NADPH for the peroxisomal reduction of acyl-dihydroxyacetone phosphate formed by DHAPAT activity.     

Polyamines and polyamino acids regulation of cytosolic tyrosine protein (Tyr-P) kinase from human erythrocytes.

In vitro regulation of cytosolic tyrosine protein (Tyr-P) kinase from human erythrocytes by polyamines, polyamino acids, negative charged compounds or by insulin using angiotensin II or poly (Glu-Tyr)4:1 as substrates was studied. All the three polyamines, putrescine (Put), spermidine (Spd) and spermine (Spm) stimulated the Tyr-P kinase activity in a dose dependent manner. Spm stimulated Tyr-P kinase activity higher than Put and Spd whether the substrate was angiotension II or poly (Glu-Tyr)4:1. Polyamino acids (polyornithine, polyarginine, polyglutamic acid and polyaspartic acid) did not affect significantly the Tyr-P kinase phosphorylation except polylysine which significantly stimulated the Tyr-P kinase activity. Negative charged compounds (chondroitin sulfate A, B and C) and heparin inhibited the Tyr-P kinase phosphorylation while insulin did not influence the enzyme activity in the presence of either substrates.     

Alkyl dihydroxyacetone phosphate synthase in human fibroblasts and its deficiency in Zellweger syndrome

The cerebro-hepato-renal (Zellweger) syndrome is an autosomal recessive disorder biochemically characterized by the absence of morphologically distinguishable peroxisomes. Key enzymes involved in the biosynthesis of ether phospholipids, i.e., dihydroxyacetone phosphate acyltransferase and alkyl dihydroxyacetone phosphate synthase, are located in mammalian (micro)peroxisomes. We have previously shown a strikingly reduced activity of dihydroxyacetone phosphate acyltransferase in liver, brain, and cultured skin fibroblasts from Zellweger patients (Schutgens et al. 1984. Biochim. Biophys. Res. Commun. 120: 179-184). We have now extended these investigations by studying alkyl dihydroxyacetone phosphate synthase in cultured human skin fibroblasts. Enzymatic activity was determined by measuring the formation of radioactive alkyl dihydroxyacetone phosphate from palmitoyl dihydroxyacetone phosphate and [1-14C]hexadecanol as substrates. The enzyme was optimally active at pH 8.5 and was stimulated (about 2-3-fold) by the presence of 0.05% (v/v) Triton X-100. The apparent KM values for the enzyme in control fibroblasts amounted to 35 microM for palmitoyl dihydroxyacetone phosphate and 90 microM for hexadecanol. The reaction became inhibited at higher concentrations of both Triton X-100 and palmitoyl dihydroxyacetone phosphate. Control skin fibroblasts showed alkyl dihydroxyacetone phosphate synthase activity of 69 +/- 28 pmol X min-1 X mg-1 (n = 7), while fibroblasts from patients had an activity of only 6.3 +/- 1.7 pmol X min-1 X mg-1 (n = 7). Alkyl dihydroxyacetone phosphate synthase was also found to be deficient in tissue homogenates of Zellweger patients. The specific activity of this enzyme in liver, kidney, and brain homogenates from Zellweger patients was less than 15% of that in the corresponding tissues from controls.     

Phosphorylation by inorganic phosphate of sodium plus potassium ion transport adenosine triphosphatase. Four reactive states.

Native solium and potassium adenosine triphosphatase from guinea pig kidney accepted a phosphate group from radioactive inorganic phosphate to form an acyl phosphate bond at the active site in the presence or absence of sodium ion. Magnesium ion was always required. In the presence of sodium ion and absence of adenosine triphosphate, there was no phosphorylation by inorganic phosphate. Addition of unlabeled adenosine triphosphate produced a potassium-sensitive phosphoenzyme which exchanged its phosphate-group with radioactive inorganic phosphate. The dephosphoenzyme was an intermediate in this exchange. The rate constant for dephosphorylation was about 0.05 per second. Addition of rubidium ion, a congener of potassium ion, to the potassium-sensitive phosphoenzyme produced a phosphoenzyme labeled from inorganic phosphate with a corresponding rate constant of 0.26 per s. This was a rubidium-complexed phosphoenzyme. Addition of magnesium ion to potassium-sensitive phosphoenzyme converted it into insensitive phosphoenzyme, the splitting of which was not accelerated by potassium ion or by adenosine diphosphate. Its rate constant was 0.07 per s. In the absence of sodium ion and adenosine triphosphate, inorganic phosphate was incorporated directly into a similar insensitive phosphoenzyme. In the presence of potassium ion or rubidium ion, inorganic phosphate was incorporated into a potassium-complexed or rubidium-complexed phosphoenzyme which exchanged 32-P with inorganic phosphate completely in less than 3 s. Incorporation of inorganic phosphate into a complex of the enzyme with the inhibitor, ouabain, is already described in the literature. Its rate constant was about 0.02 per s. Thus there appear to be at least four reactive states of the phosphoenzyme which equilibrate measurably with inorganic phosphate, namely, potassium-sensitive phosphoenzyme, potassium-complexed phosphoenzyme, insensitive phosphoenzyme, and ouabain phosphoenzyme. Two of these reactive states are functional intermediates in native sodium and potassium ion transport adenosine triphosphatase. The results are compatible with control of the reactivity of the active site by conformational changes in the surrounding active center and with regulation of the energy level of the phosphate group according to the kind of monovalent cation bound to the enzyme.     

Cell-free stimulation of the insulin-sensitive cAMP phosphodiesterase by the joint actions of ATP and the soluble fraction from insulin-treated rat liver

The insulin-sensitive cAMP phosphodiesterase (PDE) from rat adipocytes was stimulated 60-70% upon incubation with 2 mM ATP and the soluble fraction (Fraction S-1) from insulin-treated rat liver. The effect of ATP was partially mimicked by ATP-gamma-S or GTP, but not by AMP-PNP. The PDE-stimulating activity in Fraction S-1 was preserved in the presence of 50 mM sodium phenyl phosphate, 50 mM sodium fluoride, and 0.1 mM sodium vanadate. The PDE-stimulating activity was not inhibited with either 0.5 mM H-7 or 5 microM PKI-(5-24)-peptide, but was blocked with 1 mM Kemptide. The active component in Fraction S-1 may be a phosphorylated compound, which, in the presence of ATP, may mediate the hormonal action on PDE.     

The acid phosphatase with optimum pH of 2.5 of Escherichia coli. Physiological and Biochemical study

In Escherichia coli, the physiological conditions governing the expression of an acid phosphatase with an optimum pH of 2.5 were determined. By contrast with most enzymes, the synthesis of this phosphatase was turned off in exponentially growing bacteria and started as soon as cultures entered the stationary phase. A starvation for inorganic phosphate resulted in a premature full induction, while carbon, nitrogen, and sulfur limitations were inefficient. In the presence of nonlimiting amounts of inorganic phosphate, however, the transfer of the culture to anaerobic conditions led to an immediate accumulation of the acid phosphatase. Cyclic AMP exerted a strong negative control on the biosynthesis and of this enzyme for which the integrity of both the cya and the crp gene functions was necessary. The acid phosphatase was purified to apparent homogeneity and behaved as a monomeric protein with a molecular weight of about 45,000. It had predominantly a phosphoanhydride phosphatase activity and preferentially hydrolyzed the gamma-phosphoryl residue of GTP (Km = 0.35 mM) and the 5'-beta-phosphoryl residue of ppGpp (Km = 1.8 mM). The corresponding beta-phosphoryl residue of GDP was little hydrolyzed, while CTP, ATP, and UTP were not. The enzyme did not split most phosphomonoesters with the exception of the synthetic substrate p-nitrophenyl phosphate (Km = 2.7 mM), 2,3-bisphosphoglycerate (Km = 5 mM), and fructose 1,6-bisphosphate (Km = 5 mM). It was competitively inhibited by tartaric acid and by sodium fluoride (Ki = 60 microM). In addition, it was sensitive to the inhibitor of the translation elongation factor EF-G fusidic acid, and was also strongly inhibited by the triazine dye Cibacron Blue F3GA (Ki = 0.3 microM), suggesting the existence of a site able to recognize nucleotides.     

Mechanism of stimulation of cyclic-GMP level in a neuronal cell line mediated by serotonin (5-HT3) receptors. Involvement of nitric oxide, arachidonic-acid metabolism and cytosolic Ca2+

The mechanism by which serotonin (5-HT3) receptors mediate a rise in cyclic-GMP level was investigated in a neuronal cell line. Inhibitors of phospholipase A2 (mepacrine) and of lipoxygenase (eicosatetraynoic acid or nordihydroguaiaretic acid) suppressed the action of serotonin. On the other hand, inhibition by hemoglobin indicates a role for nitric oxide which could be in part responsible for the cyclic-GMP effect as an intercellular stimulant. The suppression of the serotonin effect by the arginine analogues N omega-methyl-L-arginine and canavanine is consistent with the notion that nitric oxide could be released from arginine. The serotonin-induced rise of cyclic-GMP level depends on the presence of extracellular Ca2+ with half-maximal stimulation at 0.3 mM Ca2+. The serotonin-stimulated rise of cyclic GMP was inhibited by (a) addition of inorganic blockers of Ca2(+)-permeable channels (La3+, half-maximal inhibitory concentration (IC50) 0.04 mM; Mn2+, IC50, 0.4 mM; Co2+, IC50, 0.9 mM; Ni2+, IC50, 1.2 mM) and (b) of organic blockers (diltiazem: IC50, 6 microM, methoxyverapamil: IC50, 3 microM and (c) intracellular application of the Ca2+ chelator bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (IC50, 2 microM). Thus, two pathways for the activation of soluble guanylate cyclase by serotonin are possible: (a) via lipoxygenase products of arachidonic acid and/or (b) via nitric oxide or a related nitroso compound. Serotonin mediates a rise of cytosolic Ca2+ activity due to entry of extracellular Ca2+. It still has to be investigated which step depends on a rise of cytosolic Ca2+ activity that appears to be a prerequisite for activation of guanylate cyclase.