Initial Rate Conditions Research Articles

Isotope trapping and positional isotope exchange with rat and chicken liver phosphoenolpyruvate carboxykinases

Isotope-trapping studies of the enzyme.MgGTP complex were carried out with rat liver cytosolic and chicken liver mitochondrial phosphoenolpyruvate carboxykinases. For the rat liver enzyme, MgGTP was partially trapped from both E.MgGTP and E.MgGTP.OAA complexes, consistent with a steady-state random mechanism. For the chicken liver enzyme, MgGTP was 100% trapped from the E.MgGTP.OAA complex, consistent with a steady-state ordered mechanism. The rate constants for the interaction of MgGTP with the free enzymes are approximately 10(7) M-1 S-1, somewhat lower than the diffusion limit for association. The dissociation rate for the enzyme.MgGTP complexes is 26-92 s-1, reflecting a tightly bound complex with high commitment to catalysis in the presence of oxaloacetate. Positional isotope-exchange studies were also carried out with phosphoenolpyruvate carboxykinases from rat and chicken. No exchange if the beta gamma-18O in [beta gamma-18O, gamma-18O3]GTP to form [beta-18O, gamma-18O3]GTP was detected in the absence of oxaloacetate. In the presence of oxaloacetate, no positional isotope exchange of [beta gamma-18O, gamma-18O3]GTP was detected during initial rate conditions. The results indicate that at least one of the products dissociates rapidly from the E.MgGDP.PEP.CO2 complex relative to the net rate of MgGTP formation from the E.MgGDP.PEP.CO2 complex. A rapid equilibrium between the central complexes in which the beta-phosphoryl of GDP is restricted with respect to torsional rotation cannot be excluded but is unlikely on the basis of the relative rates of catalysis and torsional rotation. The addition of Mn2+, an activator of phosphoenolpyruvate carboxykinase, did not influence the positional isotope-exchange results.(ABSTRACT TRUNCATED AT 250 WORDS)

Exchange concept in water-in-oil microemulsions: consequences for ‘slow’ chemical reactions

Water-in-oil (w/o) microemulsions are thermodynamically stable dispersions of water droplets in an oil-continuous medium. Water and solutes exchange between droplets in a random process whereby pairs of droplets fuse transiently and then separate again. An equation is derived for the rate of an irreversible chemical reaction between two water-soluble reactants A and B, initially isolated in separate droplets, by applying the steady-state approximation (i.e. by assuming that the concentration of intermediate droplet species containing both A and B, generated in the fusion process, is constant under initial rate conditions). The equation shows how the rate of the chemical reaction can be expected to depend on the rate of the fusion process and the size and concentration of the droplets. The theoretical basis for the utilization of very fast chemical reactions in the measurement of droplet fusion rates, exploited in earlier published studies, is discussed in the context of the rate equation. The analysis is extended to the case of chemical reactions involving a reversible step (i.e. of the type A + B ⇄ C → P). The potential for the additional perturbation of reaction rates arising from the influence of properties of the microemulsion aqueous environment not associated with dynamics of droplet interactions is emphasized. With the emphasis on ‘slow’ chemical reactions, a revision of nomenclature is recommended in order to distinguish the kinetics of the fusion process itself from those of exchange, which should be regarded as a multiple-step process incorporating both droplet fusion and solute-transfer events.

Molecular sizes of amino acid transporters in the luminal membrane from the kidney cortex, estimated by the radiation-inactivation method

Renal brush-border membrane vesicles from rat kidney cortex were irradiated in frozen state with a gamma-radiation source. Initial rates of influx into these vesicles were estimated for substrates such as L-glutamic acid, L-alanine, L-proline and L-leucine to establish the molecular sizes of their carriers. Transport was measured in initial-rate conditions to avoid artifacts arising from a decrease in the driving force caused by a modification of membrane permeability. Initial rates of Na(+)-independent uptakes for those four substrates appeared unaffected in the dose range used (0-6 Mrad), indicating that the passive permeability of the membrane towards these substrates was unaffected. However, at higher doses of irradiation the Na+ influx and the intravesicular volume evaluated by the uptake of glucose at equilibrium were altered by radiation. Thus Na(+)-dependent influx values were corrected for volume changes, and the corrected values were used to compute radiation-inactivation sizes of the transport systems. Their respective values for L-glutamic acid, L-proline, L-leucine and L-alanine carriers were 250, 224, 293 and 274 kDa. The presence of the free-radicals scavenger benzoic acid in the frozen samples during irradiation did not affect the uptake of glucose, phosphate and alkaline phosphatase activity. These results indicate that freezing samples in a cryoprotective medium was enough to prevent secondary inactivation of transporters by free radicals. Uptakes of beta-alanine and L-lysine were much less affected by radiation. The radiation-inactivation size of the Na(+)-dependent beta-alanine carrier was 127 kDa and that of the L-lysine carrier was 90 kDa.

Mitochondrial α-glycerol phosphate dehydrogenase activity in IIA fibres of the rat lateral gastrocnemius muscle; the effect of Ca2+ and ATP

Mitochondrial alpha-glycerol phosphate dehydrogenase is an important enzyme, but it is difficult to extract and purify. We have measured the activity of this enzyme in single type IIA skeletal muscle fibres under initial rate conditions by microdensitometry of the formazan reaction product. The Km (1.6 mM) for the substrate (L-alpha-glycerol phosphate) was lower than reported for the extracted enzyme. Further, at low substrate concentrations (3 mM), the enzyme was allosterically activated by free Ca2+ concentrations of 1 microM or greater, and half-maximal stimulation occurred at 0.3 microM free Ca2+. In the absence of Ca2+, there was negative cooperativity of substrate binding with a Hill constant of 0.57, but no cooperativity occurred in the presence of calcium. ATP (10 mM) inhibited enzyme activity in the presence of Ca2+ but not in its absence.

Effects of phospholipid or cholesterol enrichment of rat intestinal brush border membrane on membrane order and transport of calcium

Calcium uptake by brush border membrane vesicles from rat small intestine measured under initial rate conditions comprises both saturable and nonsaturable components. Because the brush border is a lipid bilayer and may be sensitive to changes in membrane lipid, vesicles were treated with liposomes to enrich phospholipid (PL) or cholesterol (C) content above that of the control (Reference) vesicle. The effects of the changes in lipid composition on membrane fluidity were determined from fluorescence anisotropy (r) of diphenylhexatriene. Compared with Reference vesicles, liposome-treated vesicles showed decreased V max for saturable and K D for nonsaturable uptakes. Liposome treatment changed vesicle phospholipid composition compared with Reference vesicles. Liposome-treated vesicles had similar phospholipid composition but differed in greater cholesterol content of C- compared with PL-vesicles. Mean V max and K D were lower in C-than PL-vesicles, but the difference did not reach statistical significance, although fluidity was significantly lower in C- than PL-vesicles. The mechanism of inhibition of saturable calcium uptake in PL- and C-vesicles was uncompetitive. Thus, lipid composition is crucial for determining calcium uptake: any change from native lipid composition decreased transport. Fluidity, measured by the conventional probe diphenylhexatriene, did not correlate with calcium uptake by Reference compared with liposome-treated vesicles.

Metabolism of triiodothyroacetic acid (TA3) in rat liver. I. Deiodination of TA3 and TA3 sulfate by microsomes.

The deiodination of the acetic acid side-chain analogs of T3 as well as 3,3'-diiodothyronine (3,3'-T2) was investigated by incubating 125I-labeled 3,3',5-triiodothyroacetic acid (TA3) and 3,3'-diiodothyroacetic acid (3,3'-TA2) with rat liver microsomes at 37 C and pH 7.2 in the presence of 5 mM dithiothreitol. TA3 sulfate (TA3S) and 3,3'-TA2S were also tested as substrate since sulfation is known to accelerate T3 and 3,3'-T2 conversion. Reaction products were analyzed on Sephadex LH-20 and HPLC. TA3 underwent only inner ring deiodination (IRD), but 3,3'-TA2 was equally converted by IRD and outer ring deiodination (ORD). TA3S was metabolized very rapidly by IRD to 3,3'-TA2S which was only observed transiently due to its rapid deiodination predominantly in the outer ring. Kinetic studies under initial reaction rate conditions yielded apparent Michaelis-Menten (Km) values (micromolar) of 1.8 for TA3, 0.8 for 3,3'-TA2, and 0.004 for TA3S, and 0.02 for 3,3'-TA2S and Vmax values (picomoles per min/mg protein) of 174 for TA3, 49 for 3,3'-TA2, 21 for TA3S, and 63 for 3,3'-TA2S. The Vmax/Km ratios for the IRD of TA3 and TA3S were 16 and 930 times higher, respectively, relative to T3. Deiodinations were sensitive to propylthiouracil inhibition, indicating the involvement of the type I iodothyronine deiodinase. Furthermore, the iodothyroacetic acid derivatives competitively inhibited the ORD of rT3 with apparent inhibition constant (Ki) values (0.45 microM for TA3, 4 nM for TA3S, and 0.04 microM for 3,3'-TA2S) in agreement with corresponding Km values. We conclude that 1) TA3 and 3,3'-TA2 are better substrates than T3 and 3,3'-T2 for the type I deiodinase of rat liver; 2) the IRD of TA3 and ORD of 3,3'-TA2 are markedly enhanced by sulfation similar to the parent iodothyronines; and 3) TA3S in the best known substrate for IRD due to its very high affinity for the type I deiodinase.

Electrogenicity of phosphate transport by renal brush-border membranes.

Phosphate uptake by rat renal brush-border membrane vesicles was studied under experimental conditions where transmembrane electrical potential (delta psi) could be manipulated. Experiments were performed under initial rate conditions to avoid complications associated with the dissipation of ion gradients. First, phosphate uptake was shown to be strongly affected by the nature of Na+ co-anions, the highest rates of uptake being observed with 100 mM-NaSCN (1.010 +/- 0.086 pmol/5 s per micrograms of protein) and the lowest with 50 mM-Na2SO4 (0.331 +/- 0.046 pmol/5 s per micrograms of protein). Anion substitution studies showed that potency of the effect of the co-anions was in the order thiocyanate greater than nitrate greater than chloride greater than isethionate greater than gluconate greater than sulphate, which correlates with the known permeability of the membrane to these anions and thus to the generation of transmembrane electrical potentials of decreasing magnitude (inside negative). The stimulation by ion-diffusion-induced potential was observed from pH 6.5 to 8.5, indicating that the transport of both monovalent and divalent phosphate was affected. In addition, inside-negative membrane potentials were generated by valinomycin-induced diffusion of K+ from K+-loaded vesicles and showed a 57% stimulation of phosphate uptake, at pH 7.5. Similar experiments with H+-loaded vesicles, in the presence of carbonyl cyanide m-chlorophenylhydrazone gave a 50% stimulation compared with controls. Inside-positive membrane potentials were also induced by reversal of the K+ gradient (outside greater than inside) in the presence of valinomycin and gave 58% inhibition of phosphate uptake. The membrane-potential dependency of phosphate uptake was finally analysed under thermodynamic equilibrium, and a stimulation by inside-negative potential was observed. The transport of phosphate was thus driven against a concentration gradient by a membrane potential, implicating the net transfer of a positive charge during the translocation process. These results indicate a major contribution of electrical potential to phosphate uptake in renal brush-border membranes.

Radiation-inactivation studies on brush-border-membrane vesicles. General considerations, and application to the glucose and phosphate carriers.

Radiation-inactivation studies were performed on brush-border-membrane vesicles purified from rat kidney cortex. No alteration of the structural integrity of the vesicles was apparent in electron micrographs of irradiated and unirradiated vesicles. The size distributions of the vesicles were also similar for both populations. The molecular sizes of two-brush-border-membrane enzymes, alkaline phosphatase and 5'-nucleotidase, estimated by the radiation-inactivation technique, were 104800 +/- 3500 and 89,400 +/- 1800 Da respectively. Polyacrylamide-gel-electrophoresis patterns of membrane proteins remained unaltered by the radiation treatment, except in the region of higher-molecular-mass proteins, where destruction of the proteins was visible. The molecular size of two of these proteins was estimated from their mobilities in polyacrylamide gels and was similar to the target size, estimated from densitometric scanning of the gel. Intravesicular volume, estimated by the uptake of D-glucose at equilibrium, was unaffected by irradiation. Uptake of Na+, D-glucose and phosphate were measured in initial-rate conditions to avoid artifacts arising from a decrease in the driving force caused by a modification of membrane permeability. Na+-independent D-glucose and phosphate uptakes were totally unaffected in the dose range used (0-9 Mrad). The Na+-dependent uptake of D-glucose was studied in irradiated vesicles, and the molecular size of the transporter was found to be 288,000 Da. The size of the Na+-dependent phosphate carrier was also estimated, and a value of 234,000 Da was obtained.

High sensitivity of the Na+, K+-pump of human red blood cells to genins of cardiac glycosides.

1. Four different cardiac glycosides (ouabain, digitoxin, digoxin and gitoxin) and their corresponding genins were tested on Na+, K+-pump fluxes measured under steady-state and initial rate conditions (non equilibrium conditions) in human and rat erythrocytes and in mouse macrophages. 2. In human red cells, Na+, K+-pump fluxes exhibited up to 8 fold higher sensitivity to genins than to glycosides. In addition genins, but not the corresponding glycosides, exhibited double reactivity with regard to the erythrocyte Na+, K+-pump (with the exception of gitoxigenin). A weak reactivity component was similar to the one of the corresponding glycosides (IC50 of about 10(-6) M) and a high reactivity component exhibited IC50 values varying from 0.1 to 0.5 X 10(-6) M for digitoxigenin and ouabagenin respectively. 3. In contrast with human red cells, the initial rate of Na+, K+-pump fluxes in rat erythrocytes and mouse macrophages was less sensitive to genins than to the corresponding cardiac glycosides. 4. Dihydroouabain was 3, 10 and 75 times less active than ouabain in inhibiting the initial rate of Na+, K+-pump fluxes in human and rat erythrocytes and in mouse macrophages respectively. 5. In conclusion, Na+, K+-pump fluxes measured under initial rate conditions in human erythrocytes exhibit an unusually high sensitivity to genins of cardiac glycosides. This property probably results from the fast binding rate constants of genins and the slow association rates of glycosides to human red cells.

Increased renal catabolism of 1,25-dihydroxyvitamin D3 in murine X-linked hypophosphatemic rickets.

The hypophosphatemic (Hyp) mouse, a murine homologue of human X-linked hypophosphatemic rickets, is characterized by renal defects in brush border membrane phosphate transport and vitamin D3 metabolism. The present study was undertaken to examine whether elevated renal 25-hydroxyvitamin D3-24-hydroxylase activity in Hyp mice is associated with increased degradation of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] by side chain oxidation. Metabolites of 1,25(OH)2D3 were separated by HPLC on Zorbax SIL and identified by comparison with standards authenticated by mass spectrometry. Production of 1,24,25-trihydroxyvitamin D3, 24-oxo-1,25-dihydroxyvitamin D3, and 24-oxo-1,23,25-trihydroxyvitamin D3 was twofold greater in mitochondria from mutant Hyp/Y mice than from normal +/Y littermates. Enzyme activities, estimated by the sum of the three products synthesized per milligram mitochondrial protein under initial rate conditions, were used to estimate kinetic parameters. The apparent Vmax was significantly greater for mitochondria from Hyp/Y mice than from +/Y mice (0.607 +/- 0.064 vs. 0.290 +/- 0.011 pmol/mg per protein per min, mean +/- SEM, P less than 0.001), whereas the apparent Michaelis-Menten constant (Km) was similar in both genotypes (23 +/- 2 vs. 17 +/- 5 nM). The Km for 1,25(OH)2D3 was approximately 10-fold lower than that for 25-hydroxyvitamin D3 [25(OH)D3], indicating that 1,25(OH)2D3 is perhaps the preferred substrate under physiological conditions. In both genotypes, apparent Vmax for 25(OH)D3 was fourfold greater than that for 1,25(OH)2D3, suggesting that side chain oxidation of 25(OH)D3 may operate at pharmacological concentrations of substrate. The present results demonstrate that Hyp mice exhibit increased renal catabolism of 1,25(OH)2D3 and suggest that elevated degradation of vitamin D3 hormone may contribute significantly to the clinical phenotype in this disorder.

Fine structure and permeability of the metacercarial cyst wall of Clinostomum marginatum (Digenea).

Encysted metacercariae of Clinostomum marginatum (Digenea) were obtained from tissues of yellow perch, Perca flavescens. The outermost wall (host response) as seen under electron microscopy consisted of a single, fibrous tissue layer, 10-25 micron thick. The tissue contained flattened fibrocytes, small fat deposits, and vacuoles embedded between layers of collagen fibers. The cyst cavity was filled with small vesicles, crystals, and debris. No layer corresponding to the primary (parasite-produced) cyst wall secreted by most species of metacercariae was noted. To determine the permeability of the cyst wall, encysted worms were incubated under initial rate conditions with [3H] glucose, with and without the glucose transport inhibitors phlorizin and phloretin. After incubation, the worms were mechanically excysted, washed, and processed to determine glucose uptake rates. Vmax and Kt were greater than those obtained for worms excysted prior to incubation with substrate. Moreover, the presence of phlorizin or phloretin in the incubation medium had no effect on glucose uptake by encysted worms. Thus, the selective permeability of the cyst wall permits free diffusion of glucose to the cutaneous transport systems of the worm, while restricting the movements of phlorizin and phloretin.

Regulation of carboxyl ester lipase adsorption to surfaces. 1. Chemical specificity

The chemical specificity of the adsorption of porcine pancreatic carboxyl ester lipase to pure lipid surfaces was examined. Adsorption of native and catalytically inactivated enzyme was measured at the argon-buffer interface by using lipid films near the point of collapse. Protein adsorbed readily to films of triolein, 1,3-diolein, methyl oleate, oleonitrile, oleyl alcohol, and 13,16-docosadienoic acid. However, recovery of enzyme activity was variable. These differences and the changes in surface pressure accompanying adsorption indicated the occurrence of enzyme denaturation at the interface. Denaturation was controlled largely by surface free energy but showed some chemical specificity at high surface pressures. Adsorption of protein to the lipids was comparable when measured under either equilibrium or initial rate conditions. Together with surface pressure changes that accompany adsorption, the data indicate a relative lack of specificity for the enzyme-surface interaction. Adsorption to 13,16-docosadienoic acid and 1,3-diolein obeyed the Langmuir adsorption isotherm. Dissociation constants ranged from 10 to 50 nM, depending on enzyme form, ionic strength, and pH. With both lipids, a monolayer of enzyme was adsorbed at saturation. In contrast to these results, adsorption of enzyme activity and protein to films of 1-palmitoyl-2-oleoyl-phosphatidylcholine was less than or equal to 5% of that observed with the other lipids under all conditions. Comparison of rate constants for adsorption to 13,16-docosadienoic and 1,3-diolein as a function of subphase pH indicated a marked dependence on the ionization state of the fatty acid. Overall, the data suggest that the presence of zwitterionic and anionic lipids may regulate the interaction of the enzyme with substrate-containing surfaces in vivo.

Side-chain oxidation of vitamin D3 in mouse kidney mitochondria: effect of the Hyp mutation and 1,25-dihydroxyvitamin D3 treatment.

Side-chain oxidation of vitamin D is an important degradative pathway. In the present study we compared the enzymes involved in side-chain oxidation in normal and Hyp mouse kidney. Homogenates of normal mouse kidney catalyze the conversion of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3, 24-oxo-25-hydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3. After subcellular fractionation, total side-chain oxidative activity, estimated by the sum of the three products synthesized per milligram protein under initial rate conditions, coincided with the mitochondrial enzyme marker succinate-cytochrome-c reductase. Treatment of normal mice with 1,25-dihydroxyvitamin D3 (1.5 ng/g) resulted in an eightfold increase in mitochondrial enzyme activity, with no change in apparent Km but a significant rise in Vmax. With 24,25-dihydroxyvitamin D3 as the substrate, normal renal mitochondria produced 24-oxo-25-hydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3, and the synthesis of these metabolites could be increased sixfold by pretreatment with 1,25-dihydroxyvitamin D3. In the Hyp mouse, the side-chain oxidation pathway showed similar subcellular distribution of enzyme activity. However, product formation from 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 was twofold greater in mutant than in normal mitochondria. Furthermore, 1,25-dihydroxyvitamin D3 pretreatment of Hyp mice resulted in a 3.4-fold increase over basal metabolism of both 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. These results demonstrate that (i) kidneys from normal and Hyp mice possess basal and 1,25-dihydroxyvitamin D3 inducible enzyme system(s) in the mitochondrial fraction, which catalyze the side-chain oxidation of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3, and (ii) the Hyp mutation appears to perturb the renal metabolism of both substrates only in the basal state.

Effect of pH on the kinetics of Na +-dependent phosphate transport in rat renal brush-border membranes

The kinetics of Na +-dependent phosphate uptake in rat renal brush-border membrane vesicles were studied under zero-trans conditions at 37°C and the effect of pH on the kinetic parameters was determined. When the pH was lowered it turned out to be increasingly difficult to estimate initial rates of phosphate uptake due to an increase in aspecific binding of phosphate to the brush border membrane. When EDTA or β-glycerophosphate was added to the uptake medium this aspecific binding was markedly reduced. At pH 6.8, initial rates of phosphate uptake were measured between 0.01 and 3.0 mM phosphate in the presence of 100 mM Na +. Kinetic analysis resulted in a non-linear Eadie-Hofstee plot, compatible with two modes of transport: one major low-affinity system ( K m ≈ 1.3 mM), high-capacity system ( V max ≈ 1.1 nmol/s per mg protein) and one minor high-affinity ( K m ≈ 0.03 mM), low-capacity system ( V max ≈ 0.04 nmol/s per mg protein). Na +-dependent phosphate uptake studied far from initial rate conditions i.e. at 15 s, frequently observed in the literature, led to a dramatic decrease in the V max of the low-affinity system. When both the extra- and intravesicular pH were increased from 6.2 to 8.5, the K m value of the low-affinity system increased, but when divalent phosphate is considered to be the sole substrate for the low-affinity system then the K m value is no longer pH dependent. In contrast, the K m value of the high-affinity system was not influenced by pH but the V max decreased dramatically when the pH is lowered from 8.5 to 6.2. These results suggest that the low-affinity, high-capacity system transports divalent phosphate only while the high-affinity, low-capacity system may transport univalent as well as divalent phosphate. Raising medium sodium concentration from 100 to 250 mM increased Na +-dependent phosphate uptake significantly but the pH dependence of the phosphate transport was not influenced. This observation makes it rather unlikely that pH changes only affect the Na + site of the Na +-dependent phosphate transport system.

Kinase A does not activate phosphatidylinositol phosphorylation in rat lymphocyte membranes.

The interaction of the dissociated catalytic subunit of adenosine 3',5'-cyclic monophosphate-dependent protein kinase (kinase A) and phosphatidylinositol metabolism has been studied in rat spleen lymphocyte membranes. As reported previously (Sarkadi et al., FEBS Lett. 152: 195-198, 1983) addition of kinase A increased by about twofold the phosphorylation of phosphatidylinositol in lymphocyte membranes at low ATP concentrations. However, we have found that this increase is an artifact of the assay conditions, and that the increase is a consequence of an inhibition of membrane adenosine triphosphatase activity by the protein kinase A. When lipid phosphorylation was measured under initial rate conditions, at high ATP concentrations, the increase was abolished. No effect of kinase A was observed on initial rates of the synthesis or hydrolysis of phosphatidylinositol phosphate. No phosphatidylinositol bisphosphate was produced in the membranes under any of the assay conditions used.

Assessment of binding of l-cystine and l-lysine by rat renal brush-border membranes

Cystine and lysine bind to isolated rat renal brush-border vesicles. Three methods to determine the extent of amino acid binding to the membranes have been compared, one relying on the osmotic reactivity of the vesicle, a second by trichloroacetic acid precipitation of membrane-bound material and a third by initial rate analysis. For cystine, all methods yield comparable results at early time points, indicating the trichloroacetic acid method is a simple and valuable tool for binding estimation under initial-rate or near initial-rate conditions. For lysine, initial rate analysis and osmotic perturbation are the methods of choice since lysine co-precipitates with trichloroacetic acid.

Inhibition of Escherichia coli glutamine synthetase by phosphinothricin

The inhibition of Escherichia coli glutamine synthetase by phosphinothricin [2-amino-4-(methylphosphinyl)butanoic acid] has been studied. This amino acid was observed to function as an active site directed inhibitor exhibiting time-dependent inhibition of glutamine synthetase in the presence of ATP or adenylylimidodiphosphate (AMPPNP) but not adenylyl(β,γ-methylene) diphosphonate (AMPPCP). The inactivation was observed to be pseudo-first order. Phosphinothricin was also found to inhibit the enzyme reversibly under initial rate conditions and was competitive with respect to glutamate with K 1S = 18 ± 3 μ m. The inactive enzyme inhibitor complex was found to contain approximately 11 molecules of ADP and of 32P per dodecamer using [γ- 32P]ATP. Reactivation of the inactive enzyme complex was achieved by incubating the enzyme complex in 50 m m acetate (pH 4.4), 1 m KCl, and 0.40 m (NH 4) 2SO 4. ADP, phosphinothricin, and P i were released upon reactivation.

Membrane potential dependency of glutamic acid transport in rabbit jejunal brush-border membrane vesicles: K + and H + effects

We have applied our recently developed approach for quantitative generation and estimation of membrane potential differences (Berteloot, A. (1986) Biochim. Biophys. Acta 857, 180–188) to the reevaluation of glutamic acid transport rheogenicity in rabbit jejunal brush-border membrane vesicles. Membrane diffusion-potentials were created by altering iodide concentrations in the intra- and extravesicular compartments while keeping isosmolarity, isotonicity and ionic strength constant by chloride replacement. The known value of ion permeabilities relative to sodium in this preparation also allows calculation of membrane potential differences using the Goldman-Hodgkin-Katz equation. This strategy appears superior to more classical methods involving ionophore-induced membrane diffusion-potentials of protons or potassium as both cations have been shown to participate in the transport mechanism. In this paper, we demonstrate that this approach is perfectly suitable for the investigation of membrane potential dependency of glutamic acid transport as our results showed that chloride replacement by iodide did not affect uptake in vesicles with membrane potential clamped to zero by gramicidin D (sodium conditions) or by gramicidin D plus valimonycin (sodium + potassium conditions). The method thus allows to dissociate membrane potential effects from possible effects that might be introduced by altering the anion species. In these conditions, our studies clearly demonstrate that glutamic acid uptake, whether analyzed over a 1 min time scale or under initial rate conditions, was sensitive to membrane potential differences. However, our results also show that the electrogenicity of the transport system varied depending upon the intravesicular presence or absence of potassium, its presence stimulating the membrane potential dependency of uptake. This effect is modulated by the internal pH and it is concluded that inside H + and K + are not equivalent as countertransported cations. The external pH also seems to modulate the response to potential by acting on the fully loaded form(s) of the transporter. The possibility that outside H + competes for (an) external Na + binding site(s) and/or precludes the attachment of (an) extra sodium ion(s) should be considered.

The application of continuous monitoring microdensitometry to an analysis of NAD+ binding and 3β-hydroxy-Δ5-steroid dehydrogenase activity in the regressing corpus luteum of the pro-oestrous rat ovary

A technique which allows for the continuous microdensitometric monitoring (at 3.3 s intervals) of an enzyme reaction has been applied to a study of 3 beta-hydroxy-delta 5-steroid dehydrogenase activity in regressing corpora lutea cells of unfixed tissue sections (5 micron thick) of the pro-oestrous rat ovary. Initial reaction rates for NAD+ reduction by the activity of the enzyme were maintained for less than 3 min at all the concentrations (0-500 mumol/l) of co-factor employed. The relationship between NAD+ concentration and enzyme activity under initial velocity rate conditions was hyperbolic and maximum enzyme activity was seen when the NAD+ concentration was greater than 250 mumol/l. The apparent Michaelis-Menten constant (Km) was 29 mumol/l and Vmax 0.63 mumol NAD+ reduced/min/cm3 corpora lutea.

A multi-route exposure assessment of chemically contaminated drinking water.

This report provides an example of how a single source of contamination could potentially contribute to all routes of exposure. A modeling approach was used to estimate multiple exposure routes in an attempt to assess the health significance of gasoline-contaminated drinking water supplies. This model consisted of a two-compartment, indoor air quality equation that calculates the contribution made by ambient and indoor air contaminated by a pollutant volatilized from drinking water to that pollutant's inhalation burden. In addition, the model uses the traditional equations for assessing a pollutant's oral and dermal burdens. Benzene, toluene and xylene were used as surrogates for gasoline contamination to determine the contribution of contaminated water to adult and child body burdens from indoor air, oral (drinking water and food) and dermal exposure routes. The contribution thus calculated for each chemical was compared to the EPA's Office of Drinking Water Health Advisories. In terms of acute exposure, the use of chemically contaminated water for showering purposes may generate vapor in the confined area of the bathroom at levels sufficient to cause or contribute to mucous tissue irritation, as commonly reported in affected homes. High temperatures and humidity may also contribute to these effects, especially in the bathroom. In terms of chronic exposure, the use of chemically contaminated water at EPA-recommended guideline amounts in an affected home may result in inhalation, oral and dermal exposures leading to cumulative doses exceeding adult and child total daily body burdens based on EPA's Health Advisories. Thus, this model indicates that the traditional standard/guidelines derivation processes should be reevaluated to consider the pollutant contribution from multiple routes of exposure. The New Jersey Departments of Health and Environmental Protection conducted a study in which concentrations of several pollutants including benzene in the breathing zone were measured during a 15-minute shower in homes with contaminated water. The findings suggest that the air quality model used in the present study may satisfactorily predict the airborne concentrations of pollutants in, at least, the bathroom after showering with contaminated water (Pearson rank correlation coefficient of 0.773 with p = 0.0012 for n = 14). The findings of the present study support the use of an adjustment factor for all exposure durations to account for exposures to other sources of the contaminant, i.e., urban, occupational, and food. A value of 20% seems appropriate based on the study's findings.(ABSTRACT TRUNCATED AT 400 WORDS)