Purification and characterization of an oxygen-stable carbon monoxide dehydrogenase of Methanothrix soehngenii

Carbon monoxide dehydrogenase was purified to apparent homogeneity from Methanothrix soehngenii. In contrast with the carbon monoxide dehydrogenases from most other anaerobic bacteria, the purified enzyme of Methanothrix soehngenii was remarkably stable towards oxygen and it was only slightly inhibited by cyanide. The native molecular mass of the carbon monoxide dehydrogenase of Methanothrix soehngenii determined by gel filtration was 190 kDa. The enzyme is composed of subunits with molecular mass of 79.4 kDa and 19.4 kDa in an alpha 2 beta 2 oligomeric structure. The enzyme contains 1.9 +/- 0.2 (n = 3) mol Ni/mol and 19 +/- 3 (n = 3) mol Fe/mol and it constitutes 4% of the soluble cell protein. Analysis of enzyme kinetic properties revealed a Km of 0.7 mM for CO and of 65 microM for methyl viologen. At the optimum pH of 9.0 the Vmax was 140 mumol of CO oxidized min-1 mg protein-1. The enzyme showed a high degree of thermostability.     

Characterization and purification of carbon monoxide dehydrogenase from Methanosarcina barkeri.

Carbon monoxide-dependent production of H2, CO2, and CH4 was detected in crude cell extracts of acetate-grown Methanosarcina barkeri. This metabolic transformation was associated with an active methyl viologen-linked CO dehydrogenase activity (5 to 10 U/mg of protein). Carbon monoxide dehydrogenase activity was inhibited 85% by 10 microM KCN and was rapidly inactivated by O2. The enzyme was nearly homogeneous after 20-fold purification, indicating that a significant proportion of soluble cell protein was CO dehydrogenase (ca. 5%). The native purified enzyme displayed a molecular weight of 232,000 and a two-subunit composition of 92,000 and 18,000 daltons. The enzyme was shown to contain nickel by isolation of radioactive CO dehydrogenase from cells grown in 63Ni. Analysis of enzyme kinetic properties revealed an apparent Km of 5 mM for CO and a Vmax of 1,300 U/mg of protein. The spectral properties of the enzyme were similar to those published for CO dehydrogenase from acetogenic anaerobes. The physiological functions of the enzyme are discussed.     

Kinetics for formate dehydrogenase of Escherichia coli formate-hydrogenlyase

Kinetic parameters of the selenium-containing, formate dehydrogenase component of the Escherichia coli formate-hydrogenlyase complex have been determined with purified enzyme. A ping-pong Bi Bi kinetic mechanism was observed. The Km for formate is 26 mM, and the Km for the electron-accepting dye, benzyl viologen, is in the range 1-5 mM. The maximal turnover rate for the formate-dependent catalysis of benzyl viologen reduction was calculated to be 1.7 x 10(5) min-1. Isotope exchange analysis showed that the enzyme catalyzes carbon exchange between carbon dioxide and formate in the absence of other electron acceptors, confirming the ping-pong reaction mechanism. Dissociation constants for formate (12.2 mM) and CO2 (8.3 mM) were derived from analysis of the isotope exchange data. The enzyme catalyzes oxidation of the alternative substrate deuterioformate with little change in the Vmax, but the Km for deuterioformate is approximately three times that of protioformate. This implies formate oxidation is not rate-limiting in the overall coupled reaction of formate oxidation and benzyl viologen reduction. The deuterium isotope effect on Vmax/Km was observed to be approximately 4.2-4.5. Sodium nitrate was found to inhibit enzyme activity in a competitive manner with respect to formate, with a Ki of 7.1 mM. Sodium azide is a noncompetitive inhibitor with a Ki of about 80 microM.     

Properties of purified carbon monoxide dehydrogenase from Clostridium thermoaceticum, a nickel, iron-sulfur protein

Carbon monoxide dehydrogenase from Clostridium thermoaceticum has been purified to homogeneity using a strict anaerobic procedure. The enzyme has a molecular weight of about 440,000 and it consists of three each of two different subunits giving the composition alpha 3 beta 3. The molecular weight of the alpha-subunit is 78,000 and that of the beta-subunit is 71,000. Pore limit gel electrophoresis gave a molecular weight of 161,000 indicating that the enzyme dissociates to form a dimer with an alpha beta structure. The dimer apparently contains per mol 2 nickel, 1 zinc, 11 iron, and 14 acid-labile sulfur. The anaerobic enzyme has an iron-sulfur type spectrum, which is changed in the presence of the substrate, CO. In the presence of oxygen, which destroys the activity or CO2, the spectrum is that of a typical iron-sulfur protein. Under acidic conditions a low molecular weight nickel factor separates from the enzyme. Viologens, methylene blue, ferredoxin, flavodoxin, and rubredoxin serve as electron acceptors. Of these rubredoxin is by far the most efficient. The enzyme has a pH optimum around 8.4. At this pH and 50 degrees C under 100% CO atmosphere, the apparent Km for methyl viologen is 3.03 mM and Vmax is 750 mumols of CO oxidized min-1 mg-1. Cyanide and methyl iodide inhibit the enzyme. CO reverses the cyanide inhibition but promotes the reaction with methyl iodide. The pure enzyme has no hydrogenase or formate dehydrogenase activity.     

Purification and properties of an iron-sulfur-containing and pyridoxal-phosphate-independent L-serine dehydratase from Peptostreptococcus asaccharolyticus

L-Serine dehydratase with a specific activity of 15 nkat/mg protein was present in the anaerobic eubacterium Peptostreptococcus asaccharolyticus grown either on L-glutamate or L-serine. The enzyme was highly specific for L-serine with the lowest Km = 0.8 mM ever reported for an L-serine dehydratase. L-Threonine (Km = 22 mM) was the only other substrate. V/Km for L-serine was 500 times higher than that for L-threonine. L-Cysteine was the best inhibitor (Ki = 0.3 mM, competitive towards L-serine). The enzyme was purified 400-fold to homogeneity under anaerobic conditions (specific activity 6 mukat/mg). PAGE in the presence of SDS revealed two subunits with similar intensities (alpha, 30 kDa; beta, 25 kDa). The molecular mass of the native enzyme was estimated as 200 +/- 20 kDa (gel filtration) and 180 kDa (gradient PAGE). In the absence of oxygen the enzyme was moderately stable even in the presence of sodium borohydride or phenylhydrazine (5 mM each). However, by exposure to air the activity was lost, especially when the latter agent was added. The enzyme was reactivated by ferrous ion under anaerobic conditions. The inability of several nucleophilic agents to inactivate the enzyme indicated the absence of pyridoxal phosphate. This was confirmed by a microbiological determination of pyridoxal phosphate. However, the enzyme contained 3.8 +/- 0.2 mol Fe and 5.6 +/- 0.3 mol inorganic sulfur/mol heterodimer (55 kDa) indicating the presence of an [Fe-S] center. The enzyme was successfully applied to measure L-serine concentrations in bacterial media and in human sera.     

Characterization of the flavins and the iron-sulfur centers of glutamate synthase from Azospirillum brasilense by absorption, circular dichroism, and electron paramagnetic resonance spectroscopies.

Azospirillum brasilense glutamate synthase has been studied by absorption, electron paramagnetic resonance, and circular dichroism spectroscopies in order to determine the type and number of iron-sulfur centers present in the enzyme alpha beta protomer and to gain information on the role of the flavin and iron-sulfur centers in the catalytic mechanism. The FMN and FAD prosthetic groups are demonstrated to be non-equivalent with respect to their reactivities with sulfite. Sulfite reacts with only one of the two flavins forming an N(5)-sulfite adduct with a Kd of approximately 1 mM. The enzyme-sulfite complex is reduced by NADPH, and the complexed sulfite is competitively displaced by 2-oxoglutarate, which suggests the reactive flavin to be at the imine-reducing site. These data are in agreement with the two-site model of the enzyme active center proposed on the basis of kinetic studies [Vanoni, M.A., Nuzzi, L., Rescigno, M., Zanetti, G., & Curti, B. (1991) Eur. J. Biochem. 202, 181-189]. Each enzyme protomer was found, by chemical analysis, to contain 12.1 +/- 0.5 mol of non-heme iron. Electron paramagnetic resonance spectroscopic studies on the oxidized and reduced forms of glutamate synthase demonstrated the presence of three distinct iron-sulfur centers per enzyme protomer. The oxidized enzyme exhibits an axial spectrum with g values at 2.03 and 1.97, which is highly temperature-dependent and integrates to 1.1 +/- 0.2 spin/protomer. This signal is assigned to a [3Fe-4S]1+ cluster (Fe-S)I. Reduction of the enzyme with an NADPH-regenerating system results in reduction of the [3Fe-4S]1+ center to a species with a g approximately 12 signal characteristic of the S = 2 spin state of a [3Fe-4S]0 cluster. The NADPH-reduced enzyme also exhibits an [Fe-S] signal at g values of 1.98, 1.95, and 1.88, which integrates to 0.9 spin/protomer and is due to a second cluster (Fe-S)II. Reduction of the enzyme with the light/deazaflavin method results in a signal characteristic of [Fe-S] clusters with g values of 2.03, 1.92, and 1.86 and an integrated intensity of 1.9 spin/protomer. This signal arises from reduction of the (Fe-S)II center and from that of the third, lower potential iron-sulfur center (Fe-S)III. Circular dichroism spectral data on the oxidized and reduced forms of the enzyme are more consistent with the assignment of (Fe-S)II and (Fe-S)III as [4Fe-4S] clusters rather than [2Fe-2S] centers.     

Preparation of a one-subunit cytochrome oxidase from Paracoccus denitrificans: spectral analysis and enzymatic activity

Cytochrome c oxidase was isolated from Paracoccus denitrificans as a two-subunit enzyme. Chymotrypsin-catalyzed proteolysis reduced the molecular weight of each subunit by about 8000. The spectral properties of this preparation, as well as its Km for cytochrome c(1.7 muM), remained unchanged with respect to the native enzyme. Vmax was reduced by about 55% when assayed in Triton X-100 or in Triton X-100 supplemented with asolectin. Following further proteolysis by Staphylococcus aureus V8 protease, subunit I remained unchanged as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas subunit II was split into small peptides. These were removed by ion-exchange high-performance liquid chromatography. The one-subunit enzyme had an apparent molecular weight of 43,000. The reduction of molecular weight was also confirmed by the diminution of the ultraviolet/Soret absorption ratio. This value was 1.8-2.1 for the native enzyme and 1.3-1.5 for the one-subunit enzyme. The spectral properties (including the spectrum CO reduced minus reduced) were not modified by the proteolytic treatment, indicating that cytochromes a and a3 were present in equal amounts. The lack of spectral alteration and the known close association of the copper B atom with cytochrome a3 suggest that copper B is also contained within the one-subunit enzyme. The Km of the one-subunit oxidase was similar to that of the two-subunit enzyme; Vmax was decreased by about 50%. The activity of the one-subunit oxidase had a salt-dependent maximum at 30 mM KCl, almost identical with that of the undigested enzyme, and was inhibited by micromolar concentrations of KCN.     

Nickel is required for the transfer of electrons from carbon monoxide to the iron-sulfur center(s) of carbon monoxide dehydrogenase from Rhodospirillum rubrum

The role of nickel in CO oxidation and electron flow was investigated in carbon monoxide dehydrogenase from Rhodospirillum rubrum. The Fe-S centers of oxidized, nickel-containing (holo) CO dehydrogenase were completely reduced within 1 min of exposure to CO. The Fe-S centers of oxidized, nickel-deficient (apo) CO dehydrogenase were not reduced during a 35-min incubation in the presence of CO. Apo-CO dehydrogenase Fe-S centers were reduced by dithionite. The Fe-S centers of cyanide-inhibited, holo-CO dehydrogenase were not reduced in the presence of CO but were reduced by dithionite. Treatment of apo-CO dehydrogenase with cobalt(II), zinc(II), and iron(II) resulted in association of these metal ions (0.70, 1.2, and 0.86 mol of M2+/mol, respectively) with the protein but no increase in specific activity. Purified holo-CO dehydrogenase contained 1.1 mol of nickel/mol of protein and could not be further activated upon addition of NiCl2, suggesting the presence of one catalytic nickel site on the enzyme. The M2+-treated enzymes could not be further activated by addition of NiCl2 as opposed to the untreated apoenzyme, whose activity was stimulated 50-100-fold to the level of holoenzyme upon addition of NiCl2. When placed under CO, the Fe-S centers of the cobalt-treated enzyme became reduced over a 35-min time course, as opposed to the zinc- and iron-treated enzymes, which remained oxidized. We conclude that nickel, or an appropriate nickel analogue in the nickel site, mediates electron flow from CO to the Fe-S centers of CO dehydrogenase.     

Isotope effect studies of the role of metal ions in isocitrate dehydrogenase.

Pig heart NADP+-dependent isocitrate dehydrogenase requires a metal ion for activity. Under optimum conditions (pH 7.5, Mg2+ present), the carbon isotope effect is k12/k13 = 0.9989 +/- 0.0004 for the carboxyl carbon undergoing decarboxylation and hydrogen isotope effects are VmaxH/VmaxD = 1.09 +/- 0.04 and (Vmax/Km)H/(Vmax/Km)D = 0.76 +/- 0.12 with threo-D,L-[2-2H]isocitric acid. Deuterium isotope effects measured by the equilibrium perturbation technique under the same conditions are VH/VD = 1.20 for the forward reaction and 1.02 for the reverse reaction. Under these conditions the rate-determining step in the enzymatic reaction must be product release. Dissociation of isocitrate from the enzyme-isocitrate complex and the enzyme-NADP+ complex must be two or more orders of magnitude slower than the chemical steps. The catalytic activity of the enzyme is about tenfold lower in the presence of Ni2+ than in the presence of Mg2+. The carbon isotope effect in the presence of Ni2+ at pH 7.5 is k12/k13 = 1.0051 +/- 0.0012 and the hydrogen isotope effects are VmaxH/VmaxD = 0.98 +/- 0.07 and (Vmax/Km)H/(Vmax/Km)D = 1.11 +/- 0.14. Thus, the rate decrease caused by substitution of Ni2+ for Mg2+ must result from the effects of metal on substrate and product binding and dissociation, rather than effects of metal on catalysis. However, a more detailed analysis of the carbon isotope effects reveals that there is also a large metal effect on the rate of the decarboxylation step, consistent with the view that the carbonyl oxygen of the oxalosuccinate intermediate is coordinated to the metal during decarboxylation.     

Kinetics of 3-O-methyl-D-glucose transport in isolated rat hepatocytes.

3-O-Methyl-D-glucose transport across the plasma membrane of isolated rat hepatocytes was followed for net entry of the sugar into sugar-free cells (zero trans entry), net exit of sugar into sugar-free medium (zero trans exit) and for unidirectional entry and exit fluxes when cells had been equilibrated with sugar in the extracellular medium (equilibrium exchange entry and exit). These measurements were performed at 20 degrees C and pH 7.4 by the use of simple manual methods. Initial rates of transport showed a Michaelis--Menten dependency on the sugar concentration at the cis side of the membrane over the range of concentrations tested (100 microM to 100 mM). Transport was found to be symmetrical with no evidence of substrate stimulation of transport from the trans side of the membrane. Parameters (mean values +/- S.E.M.) of transport were estimated as Vmax. 86.2 +/- 9.7 mmol/litre of cell water per min and Km 18.1 +/- 5.9 mM for exchange entry, Vmax. 78.8 +/- 5.3 mmol/litre of cell water per min and Km 17.6 +/- 3.5 mM for exchange exit, Vmax. 84.1 +/- 8.4 mmol/litre of cell water per min and Km 16.8 +/- 4.6 mM for zero trans exit.     

Characterization of three aminopeptidases purified from maternal serum

The biochemical characteristics of aminopeptidase A (EC 3.4.11.7), oxytocinase (EC 3.4.11.3) and alanyl aminopeptidase (EC 3.4.11.2) purified from serum of pregnant women were compared. Aminopeptidase A hydrolysed only acidic amino acid derivatives, whereas oxytocinase and alanyl aminopeptidase had partially overlapping broad substrate specificities. Oxytocinase showed the highest Vmax value with LeuNA but the lowest Km value with ArgNA (Km 0.059 +/- 0.08 mmol/l). Alanyl aminopeptidase hydrolysed AlaNA most rapidly, but showed the highest affinity for LysNA (Km 0.054 +/- 0.006 mmol/l). The enzymes were sensitive to EDTA. Co2+, Ni2+ and Zn2+ were able to reactivate all suppressed enzymes, but Mn2+ reactivated only aminopeptidase A after EDTA inhibition. The alkaline earth metals were activators of aminopeptidase A, while Co2+ activated only alanyl aminopeptidase. This enzyme was the most sensitive to L-amino acids. Acidic amino acids inhibited aminopeptidase A but had no effect on the two other enzymes. Oxytocinase was most sensitive to thermal treatment. Amastatin did not inhibit oxytocinase, whereas aminopeptidase A was more resistant than alanyl aminopeptidase to this effector.     

Intestinal brush border calcium uptake in spontaneously hypertensive rats and their genetically matched WKY rats

The current studies were designed to characterize calcium transport by intestinal brush border membrane in the spontaneously hypertensive rat (SHR) and normotensive control, the Wistar-Kyoto (WKY) rat. The biochemical and functional purity of the intestinal brush border membranes in SHR and WKY rats was validated by marker enzymes and the ability to transiently transport D-glucose in the presence of Na+ gradient. Calcium transport into duodenal and jejunal vesicles represented a minor binding component and transmembrane movement as evident by initial rate studies, A23187 studies, and lanthanum displacement experiments. Initial rate and time course of calcium uptake was lower in SHR compared with WKY rats. Kinetic analysis of calcium uptake by the jejunum (total uptake minus binding component) showed a Vmax of 6.98 +/- 0.2 and 1.8 +/- 0.2 nmol/mg protein/7 sec in WKY rats and SHR, respectively (P less than 0.001), whereas Km values were 0.76 +/- 0.04 and 0.87 +/- 0.1 mM for WKY rats and SHR, respectively. Similar kinetic analysis of calcium uptake by the duodenal segments showed a Vmax of 10.3 +/- 0.8 and 2.8 +/- 0.2 nmol/mg protein/7 sec in WKY rats and SHR, respectively (P less than 0.01). Km values were 0.7 +/- 0.2 and 0.3 +/- 0.06 mM (P greater than 0.05). Vmax of calcium uptake in the 2-week-old rats (prehypertensive period) was 6.0 +/- 0.3 and 3.53 +/- 0.3 nmol/mg protein/7 sec in WKY rats and SHR, respectively (P less than 0.001), whereas Km values were 0.60 +/- 0.07 and 0.5 +/- 0.01 mM, respectively. These results suggest that calcium binding and uptake by duodenal and jejunal intestinal brush border membranes of SHR is significantly decreased compared with WKY rats. The decrease in transmembrane calcium uptake is secondary to decrease in Vmax and is present before the appearance of hypertension, implying a genetically determined defect in calcium uptake in intestinal brush border membranes of the SHR.     

Isolation and characterization of a cyanide dihydratase from Bacillus pumilus C1.

A cyanide-degrading enzyme from Bacillus pumilus C1 has been purified and characterized. This enzyme consisted of three polypeptides of 45.6, 44.6, and 41.2 kDa; the molecular mass by gel filtration was 417 kDa. Electron microscopy revealed a multimeric, rod-shaped protein approximately 9 by 50 nm. Cyanide was rapidly degraded to formate and ammonia. Enzyme activity was optimal at 37 degrees C and pH 7.8 to 8.0. Activity was enhanced by Sc3+, Cr3+, Fe3+, and Tb3+; enhancement was independent of metal ion concentration at concentrations above 5 microM. Reversible enhancement of enzymatic activity by azide was maximal at 4.5 mM azide and increased with time. No activity was recorded with the cyanide substrate analogs CNO-, SCN-, CH3CN, and N3- and the possible degradation intermediate HCONH2. Kinetic studies indicated a Km of 2.56 +/- 0.48 mM for cyanide and a Vmax of 88.03 +/- 4.67 mmol of cyanide per min/mg/liter. The Km increased approximately twofold in the presence of 10 microM Cr3+ to 5.28 +/- 0.38 mM for cyanide, and the Vmax increased to 197.11 +/- 8.51 mmol of cyanide per min/mg/liter. We propose naming this enzyme cyanide dihydratase.     

DNA polymerase insertion fidelity. Gel assay for site-specific kinetics

A quantitative assay based on gel electrophoresis is described to measure nucleotide insertion kinetics at an arbitrary DNA template site. The assay is used to investigate kinetic mechanisms governing the fidelity of DNA synthesis using highly purified Drosophila DNA polymerase alpha holoenzyme complex and M13 primer-template DNA. Km and Vmax values are reported for correct insertion of A and misinsertion of G, C, and T opposite a single template T site. The misinsertion frequencies are 2 X 10(-4) for G-T and 5 X 10(-5) for both C-T and T-T relative to normal A-T base pairs. The dissociation constant of the polymerase-DNA-dNTP complex, as measured by Km, plays a dominant role in determining the rates of forming right and wrong base pairs. Compared with Km for insertion of A opposite T (3.7 +/- 0.7 microM), the Km value is 1100-fold greater for misinsertion of G opposite T (4.2 +/- 0.4 mM), and 2600-fold greater for misinsertion of either C or T opposite T (9.8 +/- 4.2 mM). These Km differences indicate that in the enzyme binding site the stability of A-T base pairs is 4.3 kcal/mol greater than G-T pairs and 4.9 kcal/mol greater than C-T or T-T pairs. In contrast to the large differences in Km, differences in Vmax are relatively small. There is only a 4-fold reduction in Vmax for insertion of G opposite T and an 8-fold reduction for C or T opposite T, compared with the correct insertion of A. For the specific template T site investigated, the nucleotide insertion fidelity for Drosophila polymerase alpha seems to be governed primarily by a Km discrimination mechanism.     

The effects of bivalent cations on ribulose bisphosphate carboxylase/oxygenase.

The half-saturation constants for binding of the bivalent cations (Mg2+, Ni2+, Co2+, Fe2+ and Mn2+) to ribulose bisphosphate carboxylase/oxygenase from Glycine max and Rhodospirillum rubrum were measured. The values obtained were dependent on the enzyme and the cation present, but were the same for both oxygenase and carboxylase activities. Ribulose bisphosphate rather than its cation complex was the true substrate. The kinetic parameters Vmax.(CO2), Vmax.(O2), Km(CO2), Km(O2), and K1(O2) were determined for both enzymes and each cation activator. The evolutionary and mechanistic implications of these data are discussed.     

Formate dehydrogenase of Clostridium pasteurianum

Formate dehydrogenase was purified to electrophoretic homogeneity from N2-fixing cells of Clostridium pasteurianum W5. The purified enzyme has a minimal Mr of 117,000 with two nonidentical subunits with molecular weights of 76,000 and 34,000, respectively. It contains 2 mol of molybdenum, 24 mol of nonheme iron, and 28 mol of acid-labile sulfide per mol of enzyme; no other metal ions were detected. Analysis of its iron-sulfur centers by ligand exchange techniques showed that 20 iron atoms of formate dehydrogenase can be extruded as Fe4S4 centers. Fluorescence analysis of its isolated molybdenum centers suggests it is a molybdopterin. The clostridial formate dehydrogenase has a pH optimum between 8.3 and 8.5 and a temperature optimum of 52 degrees C. The Km for formate is 1.72 mM with a Vmax of 551 mumol of methyl viologen reduced per min per mg of protein. Sodium azide competes competitively with formate (K1 = 3.57 microM), whereas the inactivation by cyanide follows pseudo-first-order kinetics with K = 5 X 10(2) M-1 s-1.     

Activity of membranous dopamine beta-monooxygenase within chromaffin granule ghosts. Interaction with ascorbate

The role of intra- and extravesicular ascorbate has been investigated in dopamine beta-monooxygenase (D beta M) turnover using adrenal medulla chromaffin granule ghosts. Resealing of vesicle ghosts with high levels of intravesicular ascorbate leads to viable vesicles, as evidenced from the high rates of the ATP-dependent accumulation of tyramine, Vmax = 14 +/- 1 nmol/min.mg and Km = 20 +/- 6 microM. However, the D beta M-catalyzed conversion of tyramine to octopamine occurs slowly, Vmax = 0.50 +/- 0.13 nmol/min.mg and Km = 29 +/- 18 mM. When ascorbate is present instead in the external buffer, the D beta M rate increases 3.6-fold for a final Vmax = 1.8 +/- 0.2 and Km = 1.2 +/- 0.3 mM. This relatively high rate of enzyme turnover is retained in ghosts resealed with a large excess of ascorbate oxidase, ruling out contamination by intravesicular ascorbate as the source of enzyme activity. The synergistic effect of intravesicular ascorbate was examined under conditions of 2 mM external ascorbate, showing that the enzymatic rate increases 2.7-fold, from 1.2 (0 internal ascorbate) to 3.2 +/- 0.4 nmol/min.mg (saturating internal ascorbate). This result confirms that high levels of internal ascorbate are not damaging to intravesicular D beta M. These studies demonstrate very clearly that external ascorbate is the preferred reductant for the membranous form of D beta M in chromaffin granule ghosts.     

Kinetic characterization of the Ca2+-pumping ATPase of cardia sarcolemma in four states of activation

The Ca2+ dependence of the Ca2+-pumping ATPase of bovine cardiac sarcolemma was studied for four states of activation: (a) unactivated, (b) cAMP-dependent protein kinase (cAMP protein kinase C-subunit)-activated, (c) calmodulin (CAM)-activated, and (d) CAM plus cAMP protein kinase C-subunit-activated. Analysis of the Ca2+ dependence of active transport gave the following Vmax (nanomoles Ca2+/(mg x min], Km (nM) for Ca2+, and Hill coefficient values for the four states at pH 7.4, 37 degrees C: (a) 1.7 +/- 0.3, 1800 +/- 100, 1.6 +/- 0.1; (b) 3.1 +/- 0.5, 1100 +/- 100, 1.7 +/- 0.1; (c) 15.0 +/- 2.5, 64 +/- 1.4, 3.7 +/- 0.2; and (d) 36.0 +/- 6.5, 63 +/- 1.7, 3.7 +/- 0.1. CAM has the most dramatic effect, increasing the apparent Ca2+ affinity by a factor of 28, increasing the Hill coefficient 2.0 units to a value approaching 4 and increasing the Vmax by a factor of 9 or 12. The effective Ca2+ concentration (EC50) for the Ca2+-induced activation of the enzyme in the presence of 5 microM calmodulin is close to the Km for Ca2+ for the CAM-activated state (64 nM). Activation by cAMP protein kinase C-subunit had only minor effects on the Km and Hill coefficient, but increased the Vmax of both the unactivated and the CAM-activated forms of the pump by factor of 1.8 and 2.4, respectively. Analysis suggests that CAM activation is the result of direct binding of Ca2-CAM or high complexes, conferring higher Ca2+ affinity to the enzyme. Analysis suggests that regulatory phosphorylation (cAMP protein kinase C-subunit) increases the rates of processes subsequent to or distinct from Ca2+ binding. The CAM-activated form of the pump was further characterized. Unexpectedly, this form of the enzyme is stimulated a factor of 1.9 by ADP, with half-maximal stimulation between 0.4 and 0.7 mM. Analysis of the progress curves for uptake show that the CAM-activated enzyme is highly resistant to inhibition by transported Ca2+, with an IC50 of 32 mM. The implications of these findings for the pump mechanism and for its role in the regulation of cardiac contractility are discussed.     

Purification and properties of urease from bovine rumen.

Urease (urea amidohydrolase, EC 3.5.1.5) was extracted from the mixed rumen bacterial fraction of bovine rumen contents and purified 60-fold by (NH4)2SO4 precipitation, calcium phosphate-gel adsorption and chromatography on hydroxyapatite. The purified enzyme had maximum activity at pH 8.0. The molecular weight was estimated to be 120000-130000. The Km for urea was 8.3 X 10(-4) M+/-1.7 X 10(-4) M. The maximum velocity was 3.2+/-0.25 mmol of urea hydrolysed/h per mg of protein. The enzyme was stabilized by 50 mM-dithiothreitol. The enzyme was not inhibited by high concentrations of EDTA or phosphate but was inhibited by Mn2+, Mg2+, Ba2+, Hg2+, Cu2+, Zn2+, Cd2+, Ni2+ and Co2+. p-Chloromercuribenzenesulfphonate and N-ethylmaleimide inhibited the enzyme almost completely at 0.1 mM. Hydroxyurea and acetohydroxamate reversibly inhibited the enzyme. Polyacrylamide-gel electrophoresis showed that the mixed rumen bacteria produce ureases which have identical molecular weights and electrophoretic mobility. No multiple forms of urease were detected.     

Aromatic-L-amino-acid decarboxylase activity in mouse pancreatic islets

Aromatic-L-amino-acid decarboxylase activity has been measured in intact or homogenised pancreatic islets of ob/ob mice (Ume? ob/ob). The method used involves the trapping and measuring of the 14CO2 released from L-[1-14C]dihydroxyphenylalanine (L-dopa). Islets showed a decarboxylase activity which was dependent on pyridoxal phosphate and inhibitable by 0.1 mM benserazide or 0.1 mM alpha-monofluoromethyldopa. Maximum activity in intact islets was about 330 mmol/kg dry islet per h with an apparent Km of 3.3 mM. Islet homogenates had a Vmax of about 120 mmol/kg per h with a Km of 0.3 mM. L-5-Hydroxytryptophan, m-tyrosine and o-tyrosine interfered with the decarboxylation of L-dopa in a way that suggested a high activity also towards those substrates. L-Phenylalanine, L-tyrosine and D-glucose had no effect. At 0.05 mM L-dopa islet homogenates showed a much higher activity than homogenates of liver, kidney, or spleen. Islet uptake of L-[3H]dopa was well in excess of the decarboxylation rate and thus probably not rate-limiting. It is concluded that mouse pancreatic islets have a high activity of aromatic-L-amino-acid decarboxylase. This is in accordance with previous suggestions of a stimulatory effect of this enzyme on insulin secretion.