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The hypoglycemic sulfonylureas glyburide and tolbutamide were found to be excellent inhibitors of the rat liver, heart, and skeletal muscle carnitine palmitoyltransferases, but glyburide was by far the most potent inhibitor. Carboxytolbutamide, a sulfonylurea that has no hypoglycemic effect, produced little or no inhibition of the enzyme from the three tissues examined. Fasting decreased the degree of inhibition of carnitine palmitoyltransferase by the sulfonylureas, and in genetically diabetic BB Wistar rats, a decrease in sensitivity was also clearly demonstrated. Initial rate kinetics of the inhibition of carnitine palmitoyltransferase indicated that glyburide inhibits noncompetitively with respect to palmitoyl-CoA while inhibition by malonyl-CoA was cooperatively competitive. Inhibition by malonyl-CoA was noncompetitive with respect to carnitine, but inhibition by glyburide was uncompetitive. These studies indicate that the hypoglycemic sulfonylureas inhibit carnitine palmitoyltransferase by a mechanism that is much different from inhibition by malonyl-CoA, but are, nevertheless, potent inhibitors of the enzyme. These results have important implications for energy metabolism in the liver and heart in relation to the use of sulfonylureas and for understanding the mechanism by which the sulfonylureas act to lower blood glucose, but there are also important implications of these results on the study of the metabolic regulation of fatty acid oxidation.

Chronic metabolic acidosis increased the Na+-H+ exchange activity in isolated renal brush-border membrane vesicles. Treatment altered the initial rate of Na+ uptake by increasing Vm (acidotic, 15.3 +/- 0.7 nmol of Na+ X mg-1 X 2 s-1; normal, 11.3 +/- 0.9 nmol of Na+ X mg-1 X 2 s-1), and not the apparent affinity KNa+ (acidotic, 10.2 +/- 0.5 mM; normal 10.2 +/- 0.6 mM). Metabolic acidosis resulted in the proportional increase in 1 mM Na+ uptake at every intravesicular pH measured. A positive cooperative effect on Na+ uptake was found with increased intravesicular acidity in vesicles from both normal and acidotic rats. When the data were analyzed by the Hill equation, it was found that metabolic acidosis did not change the n (acidotic, 1.33 +/- 0.13; normal, 1.43 +/- 0.07) or the K'H+ (acidotic, 0.27 +/- 0.05 microM; normal, 0.28 +/- 0.06 microM), but increased the apparent Vm (acidotic, 1.10 +/- 0.08 nmol of Na+ X mg-1 X 2 s-1; normal, 0.81 +/- 0.07 nmol of Na+ X mg-1 X 2 s-1). The uptake of Na+ in exchange for H+ in membrane vesicles from normal and acidotic animals was not influenced by membrane potential. We conclude that metabolic acidosis leads to either an increase in the number of functioning exchangers or an increase in the turnover rate of the limiting step in the exchange.

Apoferritin catalyzes the oxidation of Fe(II) to Fe(III). Ferroxidase activity is assayed and characterized by coupling the oxidation with the binding of Fe(III) to transferrin. The initial rate of Fe(II) oxidation is dependent on apoferritin and initial Fe(II) concentration but independent of transferrin concentration. The ferroxidase activity is inhibited by Zn(II). Ferritins with varying loads of iron have the same ferroxidase activity level. It is suggested that the described oxidation process represents the initial step of iron deposition in apoferritin. Since transferrin can intercept Fe(III) before it is deposited in apoferritin, active sites for Fe(II) oxidation must be on or near the surface of apoferritin. This finding is contrary to the current view of apoferritin-catalyzed oxidation of Fe(II) which places active sites in the channels to the core or inside the central core.

Short microtubules can be formed by shearing a sample at polymerization steady state of microtubules formed by glycerol-induced assembly of pure tubulin dimer. Such short microtubules show a rapid increase in mean length. The rate of this increase is too fast to be accounted for by statistical redistribution of subunits between microtubules. We propose that the fast length changes are a result of the end-to-end annealing of microtubules demonstrated by Rothwell et al. (Rothwell, S. W., Grasser, W. A., and Murphy, D. B. (1986) J. Cell Biol. 102, 619-627). This proposal has been tested by measuring the rate of annealing of free microtubules to Tetrahymena axonemes under conditions identical to those used for the lengthening of sheared microtubules. That free microtubules anneal to axonemal microtubules is indicated by the following observations. Axonemes elongate at both ends in the presence of steady state microtubules, as predicted for a symmetrical annealing process; under conditions where the microtubule number concentration is greater than that for axonemes, the initial rate of axoneme elongation is more rapid with a low concentration of long microtubules at steady state than with a high number concentration of short microtubules at steady state. These observations are inconsistent with the predictions of a model based on microtubule dynamic instability (Mitchison, T., and Kirschner, M. (1984) Nature 312, 237-242). The annealing rate observed with axonemes can account for the rate of elongation of sheared steady state microtubules.

The pH dependence of binding of [TyrA14-125I]monoiodoinsulin to isolated rat adipocytes was investigated at 37 degrees C. Although the amount of tracer bound at steady state at pH 7.8 was 6 times that at pH 6.8, the fractional degradation of receptor-bound iodoinsulin remained the same. The decreased binding at acidic pH could be accounted for by a decrease in the affinity. The pH-induced change in affinity was similar for iodoinsulin and insulin. The half-time of dissociation was not changed in the pH range 7.0 to 7.8. The presence of 25 nM insulin in the wash out medium caused a small increase in the dissociation of tracer independent of pH. Kinetic experiments revealed that the decreased affinity at acidic pH was mainly due to a decrease in the association rate constant. The decreased binding at acidic pH was reflected in a shift to the right of the insulin dose-response curve for initial rate of uptake of 3-O-[methyl-14C]glucose and the rate of conversion of glucose to lipids. It is concluded that the degradation of bound iodoinsulin is independent of pH (6.8 to 8.8), that the decreased binding at acidic pH is due to a decreased association rate constant, and that cells in acidic milieu are relatively insulin-resistant.

The addition of 0.167 to 4.0 mM cAMP to gel-filtered rabbit reticulocyte lysates stimulates the initial rate and the extent of polypeptide synthesis. The stimulation is at the initiation step of polypeptide synthesis as measured by the (i) increased dipeptide, methionyl-valine, accumulation in the presence of the specific initiation inhibitor, pactamycin, and (ii) increased formation of the 40 S and 80 S initiation complex when gel-filtered lysates are incubated with [35S]Met-tRNAFMet. Furthermore, a synergistic stimulation of protein synthesis is observed when cAMP and hexose phosphates (which alone elicit a 1.8-fold stimulation of protein synthesis) are added simultaneously to gel-filtered rabbit reticulocyte lysates. These results indicate that cAMP and hexose phosphates are both essential to maintain the high rate of initiation.

Proton transport catalyzed by the sodium pump was demonstrated using proteoliposomes reconstituted with purified pig kidney Na+,K+-ATPase. Intravesicular pH was monitored with fluorescence from fluorescein isothiocyanate dextran introduced into the vesicles. An ATP-induced ouabain-sensitive acidification of the intravesicular medium was observed, when the vesicles were incubated with ATP and without Na+. The ATP-induced acidification was blocked by either extravesicular Na+ or pretreatment of the enzyme with ouabain before reconstitution. Protonophores, X-537A or carbonyl cyanide m-chlorophenylhydrazone, abolished the intravesicular acidification. The acidification was not inhibited by 3 mM tetra-n-butylammonium. The initial rate of the H+ uptake was increased with a decrease in pH of the extravesicular medium, and the maximum rate was obtained at pH 5.5-5.6. It is concluded that H+ can be transported in place of Na+ by the sodium pump.

Transport of 3-O-methylglucose by rat thymocytes occurs by facilitated diffusion and follows a biphasic time course. The half-times of the two phases of uptake are 0.8 min and 20 to 30 min; the rapid phase contributes 10 to 20% of the total 3-O-methylglucose taken up at equilibrium. Cells incubated under anaerobic conditions for 1 hour undergo a 3- to 4-fold increase in the initial rate of 3-O-methylglucose uptake. The relative contribution of the rapid phase of uptake increases nearly 4-fold in anaerobically incubated cells, although the half-time of the rapid phase remains the same. Anaerobiosis also reduces the half-time of the slow phase of uptake by a factor of three. In the absence of exogenous glucose, anaerobiosis reduces cellular ATP by 97% after 1 hour at 37 degrees. However, full stimulation of transport activity does not occur in cells with such low levels of ATP. When anaerobically incubated cells are re-exposed to oxygen, ATP synthesis proceeds and transport activity increases by 100% within 5 to 10 min. Adding 1 mM 2,4-dinitrophenol at the time the anaerobic cells are reexposed to oxygen completely blocks the subsequent ATP synthesis and the associated increase in transport activity. Cells incubated aerobically in the presence of 1 mM 2,4-dinitrophenol show a 90% reduction in ATP levels and a 2-fold increase in the rate of 3-O-methylglucose uptake. An additional 70% increase in transport activity is observed when the cells are washed free of uncoupler and incubated an additional 10 min. The results suggest that transport activity is stimulated when cellular ATP levels decline but that the stimulation process requires some minimal level of ATP for full expression.

Five diastereoisomeric cyclopentane-1,2,3-triol monophosphate analogues of sn-glycerol 3-phosphate have been studied as substrates and inhibitors of sn-glycerol-3-phosphate dehydrogenase (EC 1.1.18) of rabbit skeletal muscle. Of the five analogues, only two were substrates, DL-1,2,3/0-1P (analogue I) and DL-1,2/3-1P (analogue II). The rest were weak competitive inhibitors of the enzyme. Initial rate kinetic studies of the substrate-active analogues analyzed by a rapid equilibrium random-order mechanism showed that Ks (I) = 4.6 +/- 2.6 mM; Ks (II) = 2.2 +/- 0.6 mM compared with Ks (DL-glycerol-3-P) = 2.4 +/- 0.9 mM. Correlation of the structures of the five analogues with their activities indicates that the enzyme requires a syn orientation of the carbon-oxygen bonds about C-2 and C-3 for activity, which is optimized by an anti relationship between the hydroxyl groups at C-1 and C-2 of the cyclopentanetriol monophosphates. These results are used to deduce the conformation of sn-glycerol-3-P as it is bound in the active site of the enzyme.

The addition of from 80 to 120 mM NaCl to a recA protein-promoted DNA strand exchange results in a significant increase in both the initial rate and extent of reaction. This increase results from the formation of a relatively stable recA protein-single-stranded DNA complex that is similar to the complex formed in the presence of the single-stranded DNA-binding protein. In addition to increasing the rate and extent of strand exchange, the amount of recA protein and ATP hydrolysis needed to promote these reactions efficiently is reduced. However, single-stranded DNA-binding protein is significantly more effective than addition of 100 mM NaCl in promoting stable complex formation and stimulating strand exchange. These findings support the hypothesis that a stable recA protein-single-stranded DNA complex is required for efficient DNA strand exchange.

We used a stopped-flow method for determining the kinetic properties (between 10 ms and 10 s) of the Ca2+ release induced by inositol 1,4,5-trisphosphate (InsP3) in saponin-treated rat hepatocytes. Preliminary experiments ensured that the indicator was able to monitor rapid changes in free Ca2+ reliably. At 20 degrees C, a maximally efficient concentration of 10 microM InsP3 released Ca2+ with a half-time of 150-300 ms, the initial rate being about 1-2 nmol of Ca2+/mg of cell protein/s. The delay between the addition of 10 microM InsP3 and the onset of Ca2+ release was shorter than 20 ms, suggesting that the opening process of Ca2+ channels after binding of InsP3 to receptors is completed within a few milliseconds. Half-maximal initial rates for Ca2+ release occurred at about 1 microM InsP3 (Hill index was 1.6). The resulting Ca2+ efflux had a moderate temperature dependence. It could not be fitted to a single exponential. After low speed centrifugation of saponin-treated cells (1000 x g for 1 min), part of the InsP3-sensitive Ca2+ pool was recovered in the cell-free supernatant fraction, suggesting that the response to InsP3 arises from a vesicular fraction which may diffuse from the saponin-treated cells into the medium.

Plots of reciprocal initial rate of formaldehyde formation as a function of reciprocal ethylmorphine concentration provide parallel lines for oxygen concentrations ranging from 5.41 to 211 microM, and the apparent Km values for ethylmorphine at these extremes are 167 and 417 microM, respectively. Similarly, the apparent Km for O2 decreases from 7.69 microM at 2 mM ethylmorphine to 3.64 microM at 0.2 mM ethylmorphine. Reciprocal plots of 1/Km app and 1/Vm for ethylmorphine against 1/[O2] give Km values for O2 of 9.35 microM and 9.09 microM, respectively. Similar plots give Km values for ethylmorphine of 0.28 mM and 0.36 mM. Similarly, NADH causes an uncompetitive stimulation. These data suggest that hepatic microsomal ethylmorphine N-demethylase follows parallel plot sequential kinetics. These results, along with studies of other workers, suggest that cytochrome P-450 forms an active complex with O2 which is stable for at least many milliseconds.

The specificity of 2-keto-3-deoxy-6-phosphogluconic (KDP-gluconate) aldolase for azomethine formation was studied by incubating α-carbonyl compounds and borohydride with the aldolase and determining the amount of inactivation sustained. Both pyruvate and α-ketobutyrate produced complete inactivation, whereas monohydroxyacetone, α-ketoisovalerate, α-ketoglutarate, 5-keto-4-deoxyglucarate, 2-keto-4-hydroxyglutarate, and 2-keto-3-deoxygluconate were partially inhibitory. Hydroxypyruvate and 2-ketogluconate were not inhibitory; thus, it was concluded that the only major restriction upon azomethine formation was against the presence of a hydroxyl group at carbon 3. Formation of an azomethine with α-ketobutyrate prevented subsequent azomethine formation with pyruvate, and α-ketobutyrate under some conditions competed with pyruvate for azomethine formation. Thus, it was concluded that the same lysine residue is involved in azomethine formation for both keto acids. The curves of the effect of concentration of pyruvate and α-ketobutyrate show about a 40-fold lower affinity for α-ketobutyrate. The aldolase catalyzed the exchange of only 1 proton from water into α-ketobutyrate. The initial rate of exchange was at least 37-fold slower with α-ketobutyrate than with pyruvate. 2-Keto-4-hydroxyglutarate was cleaved at 0.1% of the rate with KDP-gluconate, whereas 2-keto-3-deoxygluconate was cleaved at a definite, but much slower, rate. Oxalacetate was decarboxylated at about 0.5% of the rate with KDP-gluconate. These data are interpreted as demonstrating a dissociation of azomethine formation, cleavage, and perhaps proton exchange phases of the over-all reaction catalyzed by KDP-gluconate aldolase.

Steady state product inhibition kinetic studies of mitochondrial malate dehydrogenase at pH 8.0 in 0.05 m acetate-Tris buffer at 25° indicate that substrate activation occurs at malate concentrations between 3 x 10-2 and 2 x 10-1 m. The mechanism of activation apparently involves malate binding at other than the catalytic site, with the induction of a 10-fold decrease in the Michaelis constant for DPN. This accounts for activation through increased prevailing velocity without altering the basic compulsory order mechanism or the maximum velocity.

The mechanism of ppGpp inhibition of adenylosuccinate synthetase (EC 6.3.4.4) was examined. Initial rate kinetic studies demonstrate the ppGpp inhibition is competitive with respect to GTP and noncompetitive with respect to L-aspartate and IMP. This is in contrast to an earlier report (Gallant, J., Irr, J., and Cashel, M. (1971) J. Biol. Chem. 246, 5812-5816), which suggested that ppGpp did not bind at the GTP site. Possible reasons for the discrepancy are discussed. The potency of the ppGpp inhibition is confirmed.

Zinc greatly stimulates the initial rate of in vitro synthesis of 5',5'-diadenosine tetraphosphate by sheep liver lysyl- and phenylalanyl-tRNMA synthetases. In the case of each enzyme, maximum stimulation of 50- to 100-fold is reached upon addition of 50 microM ZnC2 to a reaction mixture (37 degrees c, pH 7.8) containing 50 microM dithioerythritol, 150 mM KCl, 5 mM ATP, 10 mM MgCl2, 0.1 mM cognate L-aminoacid, catalytic amounts of the aminoacyl-tRNA synthetase, and unlimiting pyrophosphatase activity. This observation made with aminoacyl-tRNA synthetases of mammalian origin supports the proposal that changes in cellular free zinc ion concentration could contribute to 5',5'-diadenosine tetraphosphate variations in animal cells as a function of growth activity.