Kinetic characterization of outer-ring deiodinase activity (ORD) in the liver, gill and retina of the killifish Fundulus heteroclitus

Conversion of T4 to T3 is the first step in TH action and deiodinases are the major determinants of TH tissue availability and disposal. We here report the kinetic characterization of the outer-ring deiodinating (ORD) enzymes in the liver, gill and retina of sea water-adapted killifish, by using both rT3 and T4 as substrates. In liver, by using rT3, we detected a high Km (84 nM) and a low Km (1.3 nM) component with kinetic characteristics similar to mammalian deiodinases DI and DII. In contrast, T4-ORD only generated a low Km (0.5 nM) component. As judged by its Vmax (920 fmol 125I/mg per h) this DII enzyme is very abundant, approximately five and 20 times higher than that found in trout liver and hypothyroid rat, respectively. Kinetic analysis in killifish gill showed only one enzymatic component, with a high rT3 Km (430 nM) and a relatively low Vmax (4.3 pmol 125I/mg per h). Our results in killifish retina show the expression of a T4-low Km (0.6 nM) deiodinase with high cofactor requirements akin to the mammalian DII. The Vmax value for this enzyme is 182 fmol 125I/mg per h, five times lower than the one found in killifish liver, but comparable to that in hypothyroid rat pituitary. The biochemical similarities between fish and mammalian deiodinases could reflect their high conservation during vertebrate evolution and thus their importance in the regulation of thyroid hormone action.     

One-proton catalysis by the alpha-L-arabinofuranosidase III of Monilinia fructigena.

1. Michaelis-Menten parameters for the hydrolysis of p-nitrophenyl alpha-L-arabinofuranoside were measured as a function of pL (pH or pD) in both 1H2O and 2H2O. 2. The variation of both Vmax. and Vmax./Km with pL is sigmoid, the pK governing Vmax. shifting from 6.34 +/- 0.05 in 1H2O to 6.84 +/- 0.07 in 2H2O, and that governing Vmax./Km from 5.89 +/- 0.03 in 1H2O to 6.38 +/- 0.05 in 2H2O. 3. In the plateau regions there is a small inverse solvent isotope effect on Vmax./Km (0.92), and one of 1.45 on Vmax. 4. The variation of Vmax. with isotopic composition is strictly linear, indicating that the isotope effect arises from the transfer of a single proton.     

Some properties of hog thyroidal membrane-bound adenosine tri-phosphatase: proteolytic activation of Mg-dependent activity.

The ATPase preparations from the hog thyroid was preincubated with various amounts of trypsin. The activity of Mg-ATPase was consistently elevated. On the contrary, the Na, K-ATPase activity decreased with increasing amounts of trypsin. The effects were similar to those which were observed in the enzyme preparations treated with basis polyamino acids as previously reported. This phenomenon seemed to be specific in the preparations from the thyroid. The Mg-dependent activity was increased after pretreatment with trypsin or poly-L-lysine (PLL) when CTP, ITP and UTP were used as substrate. Thus the substrate specificity of Mg-ATPase was low. The enzyme-kinetics using ATP as substrate showed that the increase in activity was due to an increase in Vmax and not to a change in Km. The activity of Mg-ATPase was increased even after 30 min of preincubation with trypsin, while the Na, K-ATPase activity was almost diminished. These results suggest that the activity of Mg-ATPase in the preparation from the thyroid is specifically changed by the modification of the molecular environment of the enzyme with trypsin or basic polyamino acids.     

Pattern of sulfate uptake during root elongation in maize: its correlation with productivity

The efficiency of sulfate uptake was evaluated in excised roots of 22 maize genotypes, 12 inbreds and 10 hybrids, in order to study the relationship between the kinetic characteristics of the uptake and the grain productivity. During root elongation, the uptake capacity showed a pulse which appeared when the root reached 1/3 to 1/2 of its final length. The size of the accumulated pool of sulfate was significantly correlated with the productivity. The kinetic parameters of the uptake, Vmax and Km, followed the same trend, showing pulses, whoxe maximum had the same position for Vmax and Km in each genotype. The variability with the genotype of the size and duration of the Vmax pulse was not strictly connected with that of Km. The main correlation between Vmax and Km patterns was the following; inbreds were generally characterized by low Vmax and low Km; hybrids by high Vmax and high Km. As a consequence, in most cases, the benefit of the heterotic stimulation of Vmax was contrasted by the loss of affinity of the transport system or the nutrients.     

Uncompetitive stimulation of rat brain Na-K ATPase activity by rapid eye movement sleep deprivation

Rapid eye movement sleep deprivation is associated with an increase in Na-K ATPase activity. In order to understand the possible biochemical mechanism of this increase, the kinetics of Na-K ATPase was studied. Although the enzyme activity increased after the deprivation, the catalytic efficiency of the enzyme remained unaltered. The rapid eye movement sleep deprivation increased both the Vmax and the Km suggesting an uncompetitive stimulation of the enzyme. While increase in norepinephrine resulted in an increased Vmax, that of calcium increased the Km. Since an increase in norepinephrine has been suggested after deprivation, the increased Vmax is attributed to increased norepinephrine level following deprivation. However, since rapid eye movement sleep deprivation is reported to be associated with a decrease in calcium levels, the increase in Km following deprivation may be attributed to changes in factor(s) other than calcium.     

Influence of vasoconstriction, temperature, and flow on the kinetics of serotonin uptake in rabbit lungs

In the process of estimating the kinetic parameters of the pulmonary endothelial serotonin (5-HT) uptake, it is critically important to distinguish the effects of hemodynamic changes from endothelial injury. Therefore, the effects of changes in flow rate (1.7-5.0 ml/s), hemodynamics (vasoconstriction by norepinephrine), and temperature (39 vs. 33 degrees C) were investigated in isolated rabbit lungs. Indicator-dilution data were expressed in terms of the Michaelis-Menten equation for the two 5-HT uptake pathways in the preparation. The maximum uptake velocity (Vmax1) and the 5-HT concentration at half-maximum velocity (Km1) of the first pathway as well as the first-order constant (Vmax2/Km2) of the linear part of the second pathway were determined. Neither vasoconstriction nor flow variations had any effect on Km1, whereas increasing the flow rate caused extensive recruitment, with a concomitant increase in Vmax1 and Vmax2/Km2. Furthermore, all the kinetic parameters were significantly decreased at the lower temperature. We conclude that Km1 is independent of organ hemodynamics (vasoconstriction and flow) but susceptible to changes in 5-HT uptake capacity caused by a change in temperature. Vmax1 and Vmax2/Km2 respond to alterations in 5-HT uptake capacity and perfused organ volume. These are prerequisites to apply kinetic modeling as a method for the investigation of pulmonary endothelial function and integrity.     

Studies on the subsite specificity of the rat brain puromycin-sensitive aminopeptidase

The specificity of the puromycin-sensitive aminopeptidase from rat brain was examined. Using L-alanyl-beta-naphthylamide as substrate Vmax of the reaction was shown to be pH independent over the range of 5.5-9.0, while Km exhibited a pKa of 7.7. This latter value corresponds to the pKa of the amino group of the substrate. Using X-Ala and X-Leu to examine the specificity of the P1 site it was found that Arg and Lys exhibit the highest affinity, followed by Met, Val, Leu, Trp, and Phe, which bind congruent to 5- to 20-fold less well. Although Km varied more than 20-fold within this series, Vmax showed considerably less variation. Significantly weaker binding was observed with a P1 Gly, Ala, Ser, or Pro with no binding detectable with a P1 Glu. The presence of a P'1 Leu compared to P'1 Ala results in an approximate 10-fold decrease in Km with little change in Vmax. The effect of varying P'1 residues was examined with the series Leu-X. In this case basic and hydrophobic amino acids, with the exception of Val, all exhibit nearly the same Km. The binding of Arg-Arg and Lys-Lys showed the same Km as obtained for Arg-Leu or Lys-Leu, respectively. When Leu-Ser-Phe was compared to Leu-Ser the P'2 residue led to a 100-fold decrease in Km and slightly less than a 5-fold increase in Vmax. In contrast the addition of a P'2 Met to Leu-Trp results in only a 3-fold decrease in Km and a 3-fold increase in Vmax. The results indicate a preference for a basic or hydrophobic residue in the P1 and P'1 sites and indicate subsite-subsite interactions which primarily affect binding.     

Distinction between the two basic mechanisms of cation transport in the cardiac Na(+)-Ca2+ exchange system

In order to distinguish between the Ping-Pong and sequential mechanisms of cation transport in the cardiac Na(+)-Ca2+ exchange system, the initial rates of the Nai-dependent 45Ca uptake (t = 1 s) were measured in reconstituted proteoliposomes, loaded with a Ca chelator. Under "zero-trans" conditions ([Na]o = [Ca]i = 0) at a fixed [Na]i = 10-160 mM with varying [45Ca]o = 2.5-122 microM for each [Na]i, the Km and Vmax values increased from 7.7 to 33.5 microM and from 2.3 to 9.0 nmol.mg-1.s-1, respectively. The Vmax/Km values show a +/- 2-10% deviation from the average value of 0.274 nmol.mg-1.s-1.microM-1 over the whole range of [Na]i. These deviations are within the standard error of Vmax (+/- 3-7%), Km (+/- 11-17%), and Vmax/Km (+/- 11-19%). This suggests that, under conditions in which Vmax and Km are [Na]i dependent and vary 4-5-fold, the Vmax/Km values are constant within the experimental error. In the presence of K(+)-valinomycin the Vmax/Km values are 0.85 +/- 0.17 and 1.08 +/- 0.18 nmol.mg-1.s-1.microM-1 at [Na]i = 20 and 160 mM, respectively, suggesting that under conditions of "short circuit" of the membrane potential the Vmax/Km values still exhibit the [Na]i independence. At a very low fixed [45Ca]o = 1.1 microM with varying [Na]i = 10-160 mM, the initial rates were found to be [Na]i independent. At a high fixed [45Ca]o = 92 microM the initial rates show a sigmoidal dependence on the [Na]i with Vmax = 13.8 nmol.mg-1.s-1, KmNa = 21 mM, and Hill coefficient nH = 1.5. The presented data support a Ping-Pong (consecutive) mechanism of cation transport in the Na(+)-Ca2+ exchanger.     

Deoxyglucose uptake by mouse astrocytes: Effects of temperature and retrovirus infection

Deoxyglucose uptake by FVB/N mouse astrocytes was studied before and after infecton by tsl retrovirus which causes a neurodegenerative disease in mice similar to HIV-1 encephalopathy in man. The Michaelis-Menten kinetic parameters, Km and Vmax, of 2-deoxy-D-glucose uptake by brain and cerebellar astrocytes were measured following culture at 34°C where tsl retrovirus replicates optimally, and at 37°C. Compared to astrocytes cultured at 37°C, astrocytes cultured at 34°C had increased Km and decreased deoxyglucose uptake despite increased or unchanged Vmax. Following ts1 retrovirus infection, brain astrocyte deoxyglucose uptake doubled [132%] associated with decreased Km but unchanged Vmax, whereas cerebellar astrocyte deoxyglucose uptake doubled [102%] associated with increased Vmax but unchanged Km. These observations of altered deoxyglucose uptake kinetic parameters following retrovirus infection indicate different neurochemical mechanisms for the regional variation in deoxyglucose uptake observed following retrovirus infection of the CNS in vivo.     

Effect of membrane lipid environment on the activity of bovine adrenal 3-oxo-delta 5-steroid isomerase

The 3-oxo-delta 5-steroid isomerase (EC 5.3.3.1) activity from bovine adrenal cortex microsomes can be extracted in soluble form by the use of appropriate detergents, although recovery of enzyme activity is low (ca. 2%). Activity is restored upon removal of detergent and reconstitution of the enzyme into phospholipid vesicles. Both Km and Vmax of 3-oxo-delta 5-steroid isomerase of intact microsomes increase as the pH is raised from 7.5 to 9.5, with a particularly sharp increase (6- to 8-fold) above pH 8.5. The kinetic parameters of a detergent-solubilized isomerase preparation show little increase from pH 7.5 to 9.0, but isomerase reconstituted into artificial phospholipid vesicles demonstrates a 6- to 10-fold increase in both Km and Vmax over this pH range. Addition of Ca++ (1 mM) enhances the pH dependence of both Km and Vmax of the membrane-bound isomerase, causing a slight rise in Vmax/Km.     

Importance of methionine residues in the enzymatic carboxylation of biotin-containing peptides representing the local biotinyl site of E. coli acetyl-CoA carboxylase

A biotin-containing hexapeptide Ac-Glu-Ala-Met-Bct-Met-Met (1) that represents the local biotin-containing site of Escherichia coli acetyl-CoA carboxylase has been prepared by the solid phase method. Peptide 1 is carboxylated by the biotin carboxylase subunit dimer of E. coli acetyl-CoA carboxylase with the following kinetic parameters; Km 12 mM, Vmax 2.8 microM X min-1. These compare with the parameters for biotin of Km 214 mM and Vmax 28 microM X min -1. Hence, the overall reactivity (Vmax/Km) of 1 is 1.8 times greater than that of free biotin. When all methionines in 1 are replaced by alanine, the resulting peptide (2) retains a similar binding ability but with a much decreased Vmax. It was also found that peptide 3, which carries an N epsilon-benzyloxycarbonyllysine in place of biocytin in 1, decreases the Km of biotin threefold.     

Modulation of basal glucose transporter Km in the adipocyte by insulin and other factors

We have previously described experimental conditions where basal methylglucose transport in adipocytes exhibited an apparent Km of approximately 35 mM. Under those conditions insulin stimulated transport predominantly by decreasing the transport Km (Whitesell, R. R., and Abumrad, N. A. (1985) J. Biol. Chem. 260, 2894-2899). Our findings were in contrast with earlier reports that the Km of basal glucose transport was low (3-5 mM) and similar to that of transport in insulin-treated cells. In this study we have investigated the effect of different experimental conditions on the kinetics of basal glucose transport in adipocytes. When transport was assayed at 37 degrees C, cell agitation for 10 min prior to the transport assay decreased the basal Km from 35 to 12 mM. Deprivation of metabolic substrate produced a further reduction down to 2 mM. Refeeding starved cells with 1 mM glucose returned the Km back up to 12 mM in agitated cells and to 40 mM in stabilized cells. The effects of agitation to lower and of glucose to raise the basal Km were prevented by preincubating cells with dinitrophenol. Cell agitation or substrate lack did not alter the Vmax of basal transport and were without effect on both Km and Vmax in insulin-treated cells. The temperature dependencies of the kinetics of basal and stimulated transport were studied. A decrease in the assay temperature from 37 to 23 degrees C caused both basal Km and Vmax to drop proportionately from 25 to 5 mM, and 13 to 3.6 nmol/(microliter X min), respectively. In insulin-stimulated cells, only the Vmax was decreased (Km went from 3.5 to 3 mM, Vmax from 45 to 17 nmol/(microliter X min]. The results support the concept that experimental conditions can produce large changes in the Km of basal glucose transporters. Furthermore they explain why, under certain assay conditions (with temperatures around 23 degrees C or with deprivation of metabolic substrate), the effect of insulin on transport Km is not observed. Our data also suggest that basal transport characteristics do not persist in insulin-treated cells. We would propose that one of the actions of insulin (in addition to raising Vmax) is to change the characteristics of basal transporters by overriding metabolic factors which keep the Km high. Alternatively, insulin could cause the disappearance of basal transporters as new and different ones are recruited from intracellular stores.     

Mutual Inhibition Kinetic Analysis of γ-Aminobutyric Acid, Taurine, and β-Alanine High-Affinity Transport into Neurons and Astrocytes: Evidence for Similarity Between the Taurine and β-Alanine Carriers in Both Cell Types

The transport kinetics of gamma-aminobutyric acid (GABA), taurine, and beta-alanine in addition to the mutual inhibition patterns of these compounds were investigated in cultures of neurons and astrocytes derived from mouse cerebral cortex. A high-affinity uptake system for each amino acid was demonstrated both in neurons (Km GABA = 24.9 +/- 1.7 microM; Km Tau = 20.0 +/- 3.3 microM; Km beta-Ala = 73.0 +/- 3.6 microM) and astrocytes (Km GABA = 31.4 +/- 2.9 microM, Km Tau = 24.7 +/- 1.3 microM; Km beta-Ala = 70.8 +/- 3.6 microM). The maximal uptake rates (Vmax) determined were such that, in neurons, Vmax GABA greater than Vmax beta-Ala = Vmax Tau, whereas in astrocytes, Vmax beta-Ala greater than Vmax Tau = Vmax GABA. Taurine was found to inhibit beta-alanine uptake into neurons and astrocytes in a competitive manner, with Ki values of 217 microM in neurons and 24 microM in astrocytes. beta-Alanine was shown to inhibit taurine uptake in neurons and astrocytes, also in a competitive manner, with Ki values of 72 microM in neurons and 71 microM in astrocytes. However, beta-alanine was found to be a weak noncompetitive inhibitor of neuronal and astrocytic GABA uptake, whereas in reverse experiments, GABA displayed weak noncompetitive inhibition of neuronal and astrocytic uptake of beta-alanine. Likewise, taurine was a weak noncompetitive inhibitor of GABA uptake in neurons and similarly, GABA was a weak noncompetitive inhibitor of taurine uptake into neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the phosphotyrosyl protein phosphatase activity of calmodulin-dependent protein phosphatase

Calmodulin-dependent protein phosphatase from bovine brain and heart was assayed for phosphotyrosine and phosphoserine phosphatase activity using several substrates: 1) smooth muscle myosin light chain (LC20) phosphorylated on tyrosine or serine residues, 2) angiotensin I phosphorylated on tyrosine, and 3) synthetic phosphotyrosine- or phosphoserine-containing peptides with amino acid sequences patterned after the autophosphorylation site in Type II regulatory subunit of the cAMP-dependent protein kinase. The phosphatase was activated by Ni2+ and Mn2+, and stimulated further by calmodulin. In the presence of Ni2+ and calmodulin, it exhibited similar kinetic constants for the dephosphorylation of phosphotyrosyl LC20 (Km = 0.9 microM, and Vmax = 350 nmol/min/mg) and phosphoseryl LC20 (Km = 2.6 microM, Vmax = 690 nmol/min/mg). Dephosphorylation of phosphotyrosyl LC20 was inhibited by phosphoseryl LC20 with an apparent Ki of 2 microM. Compared to the reactions with phosphotyrosyl LC20 as the substrate, reactions with phosphotyrosine-containing oligopeptides exhibited slightly higher Km and lower Vmax values. The reaction with the phosphoseryl peptide based on the Type II regulatory subunit sequence exhibited a slightly higher Km (23 microM), but a much higher Vmax (4400 nmol/min/mg) than that with its phosphotyrosine-containing counterpart. Micromolar concentrations of Zn2+ inhibited the phosphatase activity; vanadate was less potent, and 25 mM NaF was ineffective. The study provides quantitative data to serve as a basis for comparing the ability of the calmodulin-dependent protein phosphatase to act on phosphotyrosine- and phosphoserine-containing substrates.     

Reassessment of the translocation hypothesis by kinetic studies on hexose transport in isolated rat adipocytes

The effect of insulin and factors which have insulin-like activity on the kinetic parameters of 3-O-methyl-D-glucose (MeGlc) transport in rat adipocytes were assessed. Carrier-mediated uptake of MeGlc was estimated by the difference in the amounts of [14C]MeGlc and L-[3H]glucose taken up in cells under equilibrium exchange conditions at 37 degrees C. The Km and Vmax values in basal cells were 17.4 mM and 0.24 nmol/10(6) cells/s, respectively. Removal of endogenous adenosine by adenosine deaminase resulted in a 26% decrease in the basal rate due to a slight increase in the Km (19.6 mM) and a decrease in the Vmax value (0.20 nmol/10(6) cells/s). The maximum concentration (10 nM) of insulin decreased the Km to approximately one-half of the basal (7.1 mM) concomitant with an 8.5-fold increase in the Vmax value (2.04 nmol/10(6) cells/s). Submaximal concentrations (50 and 150 pM) of insulin, N6-phenylisopropyladenosine (1 microM), mechanical agitation of cells by centrifugal force (160 x g), low temperature (15 degrees C), 12-O-tetradecanoylphorbol-13-acetate (1 microM), and hydrogen peroxide (10 mM) all decreased the basal Km value to a range of 13.5-7.3 mM, concomitant with a 1.7-7.4-fold increase in the Vmax. A possible explanation for the alterations in the kinetic parameters may be that insulin and other factors cause the translocation of the mobile low-Km glucose transporters from an intracellular site to the cell surface, where the stationary high-Km transporters are located. Thus, when the Km and Vmax values of the hypothetical high-Km transporters were assumed to be 20 mM and 0.20 nmol/10(6) cells/s, respectively, and the Km of the low-Km transporters was assumed to be 7 mM, the theoretical Km decreased from 20 to 7.5 mM as the Vmax of the low-Km transporters increased from near 0 to 2.0 nmol/10(6) cells/s. The relation between empirical Km and Vmax values as affected by several agents and conditions followed closely the relation predicted by the above two-transporter model.     

Metabolic adaptation of the renal carbohydrate metabolism. III

Summary The adaptive response of renal metabolism of glucose was studied in isolated rat proximal and distal renal tubules after a high protein-low carbohydrate diet administration. This nutritional situation significantly stimulated the gluconeogenic activity in the renal proximal tubules (about 1.5 fold at 48 hours) due, in part, to a marked increase in the fructose 1,6-bisphosphatase (FBPase) and phosphoenolpyruvate carboxykinase (PEPCK) activities. In this tubular fragment, FBPase activity increased only at subsaturating fructose 1,6-bisphosphate concentration (30% at 48 hours) which involved a significant decrease in the Km (31%) for its substrate without changes in the Vmax. This enzymatic behaviour is probably related to modifications in the activity of the enzyme already present in the renal cells. Proximal PEPCK activity progressively increased at all substrate concentrations (almost 2 fold at 48h of high protein diet) which brought about changes in Vmax without changes in Km. These changes are in agreement with variations in the cellular concentration of the enzyme. Neither gluconeogenesis nor the gluconeogenic enzymes changed in the distal fractions of the renal tubules. On the other hand, a high protein diet did not apparently modify the glycolytic ability in any fragment of the nephron, although a significant increase in the phosphofructokinase (PFK) and pyruvate kinase (PK) activities was found in the distal renal tubules. This short term regulation involved a significant decrease from 24 hours in the Km value of distal PFK (almost 40%) without changes in Vmax. The kinetic behaviour of distal PK was mixed. In the first 24h after high protein diet a significant decrease in the Km for phosphoenolpyruvate was found (30%) without variation in the Vmax, however during the second 24 hours the activity of this glycolytic enzyme increased significantly (almost 1.3 fold) without modifications in its Km value. On the contrary, this nutritional state did not modify the kinetic behaviour of any glycolytic enzyme in the proximal regions of the renal tubules.     

Gamma glutamyltransferase ammonia production from glutamine: effect of physiological glutathione concentration

The formation of ammonia from physiological glutamine concentration catalyzed by gamma glutamyltransferase was studied in the presence of physiological glutathione concentration. The apparent Km for ammonia formation from glutamine was 1.6 mM some 2 fold greater than the actual plasma concentration. In the presence of 30 microM glutathione neither the apparent Km or Vmax were changed. At supraphysiological glutathione concentration, 1mM, the apparent Km was increased while the Vmax decreased to one third. Hippurate the physiological modulator of the enzyme's glutaminase activity reduced the Km to 0.9 mM, the physiological range, and elevated the Vmax 2.7 fold.     

Irreversible Binding and Recovery of the Norepinephrine Uptake System Using an Alkylating Derivative of Norepinephrine

The effects of bromoacetylaminomenthylnorepinephrine (BAAN) on the sodium-dependent, high-affinity norepinephrine (NE) uptake system in rat brain synaptosomes and CNS neuronal cultures were investigated. BAAN inhibited [3H]NE uptake into synaptosomes in a dose- and time-dependent manner (IC50, 6.5 microM). Pretreatment of cortical synaptosomes or neuronal cells with BAAN alone, followed by washing to remove free drug, reduced the Vmax but did not alter the Km value for [3H]NE uptake. The BAAN-induced reduction in Vmax was attenuated by concurrent pretreatment with desipramine and blocked by the reaction of BAAN with dithiothreitol or cysteine. In contrast, BAAN was 19-fold less potent at inhibiting [3H]dopamine uptake in striatal synaptosomes, and no change in the Vmax or Km value for [3H]dopamine uptake was observed after a pretreatment with BAAN followed by washing. Furthermore, the irreversible beta-antagonist, bromoacetylalprenololmentane, was equipotent to BAAN for inhibiting [3H]NE uptake into cortical synaptosomes, but did not alter the Vmax or Km for [3H]NE after pretreatment. In neuronal cultures, BAAN inhibited sodium-dependent uptake of [3H]NE (IC50, 5.6 microM) with no effect on sodium-independent uptake. After pretreatment of cultures with 30 microM BAAN followed by washing, there was a 74% decrease in the Vmax for [3H]NE uptake. Following a 24-h lag period, uptake recovered to the control level within 48 h; however, recovery was completely blocked by cycloheximide. The data indicate that BAAN irreversibly binds to the [3H]NE uptake system in both CNS synaptosomes and neuronal cultures and may be a useful probe for studying the turnover of the [3H]NE uptake system.     

The effects of enzyme concentration on the kinetic behavior of adrenal 21-hydroxylase and 11beta-hydroxylase in the pregnant rat.

Experiments were designed to study the kinetic behavior of 21-hydroxylase and 11beta-hydroxylase as a function of enzyme concentration (Et) during proestrus, dasy 5 (D5), 12 (D12), and 22 (D22) of pregnancy, and within 24 h post-partum. The enzymes were prepared from rat adrenal microsomes and mitochondria, respectively. The experiments consisted of measuring the initial velocity of each reaction for a series of substrate concentrations at three fixed Et. Double reciprocal plots were constructed and the slope (Km/Vmax) of each line estimated. Variation in the value of the slope as a function of enzyme dilution would predict the presence of an endogenous effector. The kinetic behavior of 21-hydroxylase was not altered throughout the range of Et (10-100 microgram protein) at any of the reproductive stages. In contrast, kinetic behavior of 11beta-hydroxylase was clearly dependent upon Et. Dilution of the enzyme preparation (25-200 microgram of protein) increased the slope of the double reciprocal plot at all reproductive stages, thus suggesting that an activator substance may be present within the mitochondrial preparation. A secondary plot of the slope (Km/Vmax) versus Et described a power function (Km/Vmax = a [Et]b) with the greatest rate of change in Km/Vmax occurring at low values of Et. The rate of change in Km/Vmax per mg rise in mitochondrial protein at all dilutions of enzyme was greatest for proestrus and post-partum, followed by D22 greater than D12 greater than D5. In addition, repeated washing of the enzyme preparation at 4 degrees C increased Km/Vmax to a greater extent at all Et than did the control preparation. These findings suggest the presence of a diffusible endogenous activator of 11beta-hydroxylase whose influence decreases markedly at D5 and D12. On the other hand, there is no evidence to suggest the presence of a diffusible endogenous effector for 21-hydroxylase.     

Towards the mechanism of altered nutrients uptake in renal brush border membrane vesicles from pyelonephritic rats

Affinity constant (Km) of D-glucose, L-alanine, L-aspartate, L-lysine, L-proline and nutrients coupled Na+ were determined in renal brush border membrane vesicles prepared from control and pyelonephritic rats. The Km of D-glucose, amino acids and nutrients coupled Na+ was noted to be significantly increased (p less than 0.001) in experimental animals. The Vmax of D-glucose and amino acids was determined at different concentrations of nutrients keeping extravesicular Na+ constant or at different concentrations of extravesicular Na+ keeping nutrient concentration constant. In the experimental rats the Vmax decreased significantly (p less than 0.01) when compared to control. The increased Km and decreased Vmax may be one of the underlying mechanism leading to decrease in the uptake of D-glucose and amino acids.