Morpholine-induced formation of l-alanine dehydrogenase activity in Mycobacterium strain HE5

An NAD-dependent, morpholine-stimulated l-alanine dehydrogenase activity was detected in crude extracts from morpholine-, pyrrolidine-, and piperidine-grown cells of Mycobacterium strain HE5. Addition of morpholine to the assay mixture resulted in an up to 4.6-fold increase of l-alanine dehydrogenase activity when l-alanine was supplied at suboptimal concentration. l-Alanine dehydrogenase was purified to near homogeneity using a four-step purification procedure. The native enzyme had a molecular mass of 160 kDa and contained one type of subunit with a molecular mass of 41 kDa, indicating a tetrameric structure. The sequence of 30 N-terminal amino acids was determined and showed a similarity of up to 81% to that of various alanine dehydrogenases. The pH optimum for the oxidative deamination of l-alanine, the only amino acid converted by the enzyme, was determined to be pH 10.1, and apparent K _m values for l-alanine and NAD were 1.0 and 0.2 mM, respectively. K _m values of 0.6, 0.02, and 72 mM for pyruvate, NADH, and NH₄ ⁺, respectively, were estimated at pH 8.7 for the reductive amination reaction. Received: 25 September 1998 / Accepted: 11 March 1999     

Purification and characterization of glutamine synthetase from the commerical mushroom Agaricus bisporus

Agaricus bisporus glutamine synthetase, a key enzyme in nitrogen metabolism, was purified to apparent homogeneity. The native enzyme appeared to be a GS-II type enzyme. It has a molecular weight of 325 kDa and consists of eight 46-kDa subunits. Its pI was found at 4.9. Optimal activity was found at 30°C. The enzyme had low thermostability. Stability declined rapidly at temperatures above 20°C. The enzyme exhibits a K _m for glutamate, ammonium, and ATP of 22mm, 0.16mm and 1.25mm respectively in the biosynthetic reaction, with optimal activity at pH 7. The enzyme is slightly inhibited by 10mm concentrations of l-alanine, l-histidine, l-tryptophan, anthranilic acid, and 5-AMP and was strongly inhibited by methionine sulfoximine and phosphinothricine. For the transferase reaction K _i-values were 890 m and 240 m for methionine sulfoximine and phosphinothricine respectively. For the biosynthetic reaction K _i was 17 m for both methionine sulfoximine and phosphinothricine.     

Thermostable phenylalanine dehydrogenase from a mesophilic Microbacterium sp. strain DM 86-1

Bacteria that produced NAD⁺-dependent phenylalanine dehydrogenase (EC 1.4.1.20) were selected among l-methionine utilizers isolated from soil. A bacterial strain showing phenylalanine dehydrogenase activity was chosen and classified in the genus Microbacterium. Phenylalanine dehydrogenase was purified from the crude extract of Microbacterium sp. strain DM 86-1 (TPU 3592) to homogeneity as judged by SDS-polyacrylamide disc gel electrophoresis. The enzyme has an isoelectric point of 5.8 and a relative molecular weight (M _r) of approximately 330,000. The enzyme is composed of eight identical subunits with an M _r of approximately 41,000. The apparent K _m values for l-phenylalanine and NAD⁺ were calculated to be 0.10 mM and 0.20 mM, respectively. No loss of the enzyme activity was observed upon incubation at 55° C for 10 min. Received: 30 July 1997 / Accepted: 4 November 1997     

Methionine-repressible homoserine dehydrogenase of serratia marcescens: Purification and properties

Summary Serratia marcescens Sa-3 possesses two homoserine dehydrogenases and neither has any aspartokinase activity unlike the case ofEs-cherichia coli enzymes. The two enzymes have been separated. One of them is active with either NAD^– or NADP⁺ and has been purified about 180-fold to homogeneity. This enzyme is completely repressed by the presence of 1mm methionine or homoserine in the growth medium, but its activity is unaffected by any amino acid of the aspartate family either singly or together. In many of its properties (such as pH optimum, K_m for substrate and cofactors), it resembles its counterpart inE. coli K₁₂. Potassium ions stabilize the enzyme but are not essential for activity. Its molecular weight is around 155,000 as determined by gel filtration and approximately 76,000 by SDS-polyacrylamide gel electrophoresis. This suggests that the enzyme has two subunits (polypeptide chains) in the molecule: 8m urea has no effect on enzyme activity. This enzyme represents approximately 30% of the total homoserine dehydrogenase activity ofS. marcescens unlike inSalmonella typhimurium andE. coli K₁₂ where it is a minor or a negligible component.     

Okadaic acid-induced transcriptional downregulation of type I collagen gene expression is mediated by protein phosphatase 2A

Summary In Saccharomyces cerevisiae, a small proportion of the glucose-6-P dehydrogenase activity is firmly associated with the mitochondrial fraction and is not removed by repeated washing or density-gradient centrifugation. However, the enzyme is released by sonic disruption. Mitochondrial glucose-6-P dehydrogenase that is released by sonication and partially purified has been found to be similar to cytosol glucose-6-P dehydrogenase with respect to electrophoretic mobility, isoelectric point, pH optimum, molecular size, and apparent K _m 's for NADP⁺ and glucose-6-P. These results indicate that a single species of glucose-6-P dehydrogenase is synthesized in S. cerevisiae and that the enzyme has more than one intracellular location. Mitochondrial glucose-6-P dehydrogenase may be a source of intramitochondrial NADPH and may function with hexokinase and transhydrogenase to provide a pathway for glucose oxidation that is coupled to the synthesis of mitochondrial ATP. A constant proportion of total glucose-6-P dehydrogenase activity remains compartmented in the mitochondrial fraction throughout the growth cycle.     

The effect of some glycolytic intermediates and long-chain acyl-CoA esters on rat skeletal muscle mitochondrial α-glycerophosphate dehydrogenase

Summary -Glycerophosphate dehydrogenase (sn-glycerol-3-phosphate: acceptor oxidoreductase, EC 1.1.99.5) activity in mitochondria isolated from rat skeletal muscle has been studied. The pH optimum of the enzyme activity was about 7.4 and the apparent K_m value for DL--glycerophosphate was approxinately 1.6mm. The activity of this enzyme was found to be inhibited by DL-glyceraldehyde-3-phosphate, phosphoenolpyruvate and 3-phosphoglycerate in a competitive manner: the apparent K_i values at pH 7.4 being 0.3mm, 1.5mm and 4.0mm respectively. The enzyme was found to be more sensitive to phosphoenolpyruvate at pH 7.0 than 7.6.The activity of -glycerophosphate dehydrogenase in rat skeletal muscle was also inhibited by palmitoyl-CoA and stearoyl-CoA in a competitive manner. The K_i values being about 9.0 m for both metabolites. This inhibition was partly reversed by Ca²⁺ and Mg²⁺ ions. Palmitoylcarnitine also exerted inhibitory effect on -glycerophosphate dehydrogenase activity but palmitate, carnitine and CoA added alone was without effect. It is proposed that the activity of -glycerophosphate dehydrogenase in rat skeletal muscle mitochondria may be controlled by changes of the cytosolic levels of some glycolytic intermediates and long-chain acyl-CoA esters. These results are discussed with respect to the regulation of -glycerophosphate shuttle activity in skeletal muscle.     

A novel <Emphasis Type="SmallCaps">l</Emphasis>-aspartate dehydrogenase from the mesophilic bacterium <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1: molecular characterization and application for <Emphasis Type="SmallCaps">l</Emphasis>-aspartate production

l-aspartate dehydrogenase (EC 1.4.1.21; l-AspDH) is a rare member of amino acid dehydrogenase superfamily and so far, two thermophilic enzymes have been reported. In our study, an ORF PA3505 encoding for a putative l-AspDH in the mesophilic bacterium Pseudomonas aeruginosa PAO1 was identified, cloned, and overexpressed in Escherichia coli. The homogeneously purified enzyme (PaeAspDH) was a dimeric protein with a molecular mass of about 28 kDa exhibiting a very high specific activity for l-aspartate (l-Asp) and oxaloacetate (OAA) of 127 and 147 U mg⁻¹, respectively. The enzyme was capable of utilizing both nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) as coenzyme. PaeAspDH showed a T _m value of 48°C for 20 min that was improved to approximately 60°C by the addition of 0.4 M NaCl or 30% glycerol. The apparent K _m values for OAA, NADH, and ammonia were 2.12, 0.045, and 10.1 mM, respectively; comparable results were observed with NADPH. The l-Asp production system B consisting of PaeAspDH, Bacillus subtilis malate dehydrogenase and E. coli fumarase, achieved a high level of l-Asp production (625 mM) from fumarate in fed-batch process with a molar conversion yield of 89.4%. Furthermore, the fermentative production system C released 33 mM of l-Asp after 50 h by using succinate as carbon source. This study represented an extensive characterization of the mesophilic AspDH and its potential applicability for efficient and attractive production of l-Asp. Our novel production systems are also hopeful for developing the new processes for other compounds production.     

Modulation of mitochondrial succinate dehydrogenase activity,mechanism and function

Summary The mitochondrial succinate dehydrogenase (E.C. 1.3.3.99) is subjected to apparently complicated regulatory mechanism. Yet, systematic analysis of the mechanism reveals the simplicity of the control. There are two stable forms of the enzyme; the non-active form stabilized as 1:1 complex with oxaloacetate and the active form stabilized by binding of activating ligands. This model quantitatively describes either the equilibrium level of active enzyme or the kinetics of activation-deactivation, in the presence of various concentrations of opposing effectors. The site where the regulatory ligands interact with the enzyme is not the substrate bonding site. The marked differences of dissociation constants of the same ligand from the two sites clearly distinguish between them.This model is fully developed for simple cases where the activating ligands are dicarboxylic acids or monovalent anions. On the other hand with activators such as ATP or CoQH₂, quantitation is still not at hand. This stems from the difficulties in maintaining determined, measurable, concentrations of the ligand in equilibrium with the membranal enzyme.While in active form the histidyl flavin moity of the enzyme is reduced by physiological substrate (succinate; CoQH₂). The non-active form is not reduced by these compounds, only strong reductants with low redox potential reduce the non-active enzyme. It is suggested that deactivation is a simple modulation of the redox potential of the flavin form E 0 mV in the active enzyme to E < –190 mV. The switch from one state to another might be achieved by distortion of the planar form of oxidized flavin to the bend configuration of the reduced flavin. Thus, in the active enzyme such distortion will destabilize the oxidized state of the flavin, shifting the redox potential to the higher value. The binding of oxaloacetate to the regulatory sites releases the distorting forces by relaxing the conformation of the enzyme. Consequently, the flavin assumes its planar form with the low redox potential. This assumption is supported by the spectral shifts of the flavin associated with the activation deactivation transition.The suicidal oxidation of malate to oxaloacetate, carried by the succinate dehydrogenase, plays an important role in modulating the enzyme activity in the mitochondria. This mechanism might supply oxaloacetate for deactivation in spite of the negligible concentration of free oxaloacetate in the matrix. The oxidation of malate by the enzyme is controlled by the redox potential at the immediate vicinity of the enzyme, and is imposed by the redox level of the membranal quinone.Finally, the modulation of succinate dehydrogenase activity is closely associated with regulation of NADH oxidation through the mutual inhibition between oxidases (Gutman, M. in Bioenergetics of Membranes, L. Packer et al., ed. Elsevier 1977, p. 165). The consequence of these interactions is the selection for the main electron donnor for the respiratory chain, during mixed substrate respiration, according to the metabolic demands from the mitochondria.Abbreviations SDH succinate dehydrogenase (succinate: acceptor oxidoreductase (E.C. 1.3.99.1)); - OAA oxaloacetate - Act activator - E_A, E_A active and non active forms of the enzyme, respectively - K'eq apparent equilibrium constant - K'd apparent dissociation constant - K_Act, K_OAA dissociation constant of the respective ligand from the enzyme - K'a, k'd the apparent rate constants of activation and deactivation, respectively - ka, kd the true rate constant of activation and deactivation respectively - ETP, ETP_II non phosphorylating and phosphorylating submitochondrial particles - PMS phenazine methosulfate - DCIP dichlorophenol indophenol - CoQ ubiquinone - TIFA Thenotriflouvoacetone - NEM N methyl Maleimide     

Metabolic characterisation of a novel vanillylmandelate-degrading bacterium

A newly isolated gram-negative bacterium, possibly Brevundimonas diminuta, utilised d,l-vanillylmandelate (d,l-VMA) as a sole carbon and energy source. The organism converted d,l-VMA to vanillylglyoxylate using a soluble NAD-dependent dehydrogenase specific for d-VMA and a dye-linked, membrane-associated l-VMA dehydrogenase. Vanillylglyoxylate was further metabolised by decarboxylation, dehydrogenation and demethylation to protocatechuate. A 4,5-dioxygenase cleaved protocatechuate to 2-hydroxy-4-carboxymuconic semialdehyde. Partially purified d-VMA dehydrogenase exhibited optimal activity at 30° C and pH 9.5 and had an apparent K _m for d-VMA of 470 μM. Although induced by several substituted mandelates, the enzyme had a narrow substrate specificity range with virtually no activity towards d-mandelate. Such properties render the enzyme of potential use in both diagnostic and biosynthetic applications. Received: 23 January 1996 / Accepted: 9 April 1996     

Mechanism of action ofVibrio cholerae enterotoxin

Summary The characteristics of the cholera toxin-stimulated adenylate cyclase of toad (Bufus marinus) and rat erythrocyte plasma membranes have been examined, with special emphasis on the response to purine nucleotides, fluoride, magnesium and catecholamine hormones. Toad erythrocytes briefly exposed to low concentrations of cholera toxin (40,000 to 60,000 molecules per cell) and incubated 2 to 4 hr at 30°C exhibit dramatic alterations in the kinetic and regulatory properties of adenylate cyclase. The approximateK _m for ATP, Mg⁺⁺ increases from about 1.8 to 3.4mm in the toxinstimulated enzyme. The stimulation by cholera toxin increases with increasing ATP, Mg⁺⁺ concentrations, from 20% at low levels (0.2mm) to 500% at high concentrations (greater than 3mm). Addition of GTP, Mg⁺⁺ (0.2mm) restores normal kinetic properties to the toxin-modified enzyme, such that stimulation is most simply explained by an elevation ofV _max. GTP enhances the toxin-treated enzyme activity two-to fourfold at low ATP concentrations, but this effect disappears at high levels of the substrate. At 0.6mm ATP and 5mm MgCl₂ the apparentK _a for GTP, Mg⁺⁺ is 5 to 10m. The control (unstimulated) enzyme demonstrates a very small response to the guanyl nucleotide. 5-ITP also stimulates the toxin-treated enzyme but cGMP, guanine, and the pyrimidine nucleotides have no effect. Cholera toxin also alters the activation of adenylate cyclase by free Mg⁺⁺, decreasing the apparentK _a from about 25 to 5mm. (–)-Epinephrine sensitizes the toad erythrocyte adenylate cyclase to GTP and also decreases the apparentK _a for free metal. Sodium fluoride, which cause a 70- to 100-fold activation of enzyme activity, has little effect on sensitivity to GTP, and does not change the apparentK _a for Mg⁺⁺; moreover, it prevents modulation of these parameters by cholera toxin. Conversely, cholera toxin severely inhibits NaF activation, and in the presence of fluoride ion the usual three- to fivefold stimulation by toxin becomes a 30 to 60% inhibition of activity. The toxin-stimulated enzyme can be further activated by catecholamines; in the presence of GTP the (–)-epinephrine stimulation is enhanced by two- to threefold. The increased catecholamine stimulation of toad erythrocyte adenylate cyclase induced by cholera toxin is explained primarily by an increase in the maximal extent of activation by the hormones. Rat erythrocyte adenylate cyclase is also modified by cholera toxin. In the mammalian system the apparent affinity for the hormone appears to be increased. Cholera toxin thus induces profound and nearly permanent changes in adenylate cyclase by a unique process which mimics the stimulation by hormones in important ways, and which also accentuates the normal hormonal response. The relevance of these findings to the mechanism of action of cholera toxin is considered.Part of this work was reported at the 1974 meeting of the Federation of American Societies for Experimental Biology (Bennett & Cuatrecasas, 1974).     

Pathway of propionate formation in Desulfobulbus propionicus

Whole cells of Desulfobulbus propionicus fermented [1-¹³C]ethanol to [2-¹³C] and [3-¹³C]propionate and [1-¹³C]-acetate, which indicates the involvement of a randomizing pathway in the formation of propionate. Cell-free extracts prepared from cells grown on lactate (without sulfate) contained high activities of methylmalonyl-CoA: pyruvate transacetylase, acetase kinase and reasonably high activities of NAD(P)-independent L(+)-lactate dehydrogenase NAD(P)-independent pyruvate dehydrogenase, phosphotransacetylase, acetate kinase and reasonably high activity of NAD(P)-independent L(+)-lactate dehydrogenase, fumarate reductase and succinate dehydrogenase. Cell-free extracts catalyzed the conversion of succinate to propionate in the presence of pyruvate, CoA and ATP and the oxaloacetate-dependent conversion of propionate to succinate. After growth on lactate or propionate in the presence of sulfate similar enzyme levels were found except for fumarate reductase which was considerably lower. Fermentative growth on lactate led to higher cytochrome b contents than growth with sulfate as electron acceptor.The labeling studies and the enzyme measurements demonstrate that in Desulfobulbus propionate is formed via a succinate pathway involving a transcarboxylase like in Propionibacterium. The same pathway may be used for the degradation of propionate to acetate in the presence of sulfate.Abbreviations DCPIP 2,6-dichlorophenolindophenol - PEP phosphoenolpyruvate     

Interactions of lanthanum with purified and intact cell acetylcholinesterase of electrophorus electricus

Summary The effects of lanthanum on the activity of purified preparations of acetylcholinesterase (AChE) from the electric organ ofE. electricus and on the activity of AChE in intact electro-plaques from the same species were studied. 0.1mm LaCl₃ produced an initial inhibition of purified AChE which was followed by a delayed activation of the enzyme. Upon pretreatment of purified enzyme with LaCl₃, initial activity was markedly increased. LaCl₃ exerted a marked, concentration-dependent inhibition of intact cell AChE.La³⁺ and Ca²⁺ appear to interact competitively. In the presence of both 10mm CaCl₂ and 0.1mm LaCl₃, the initial activity of purified AChE was increased at lower ACh concentrations and inhibited at ACh concentrations greater than 3 × 10^–4 m. Inhibition of intact cell enzyme by 0.1mm LaCl₃ was relieved by increasing the CaCl₂ concentration to 10mm at ACh concentrations less than 2 × 10^–4 m.The data were analyzed assuming Michaelis-Menten kinetics and interpreted with reference to the differential binding of divalent and trivalent cations to regulatory anionic sites which are separate and distinct from the anionic site of the active center of the enzyme.     

Purification and characterization of extracellular β-glucosidase from Myceliophthora thermophila

The extracellular -glucosidase has been purified from culture broth of Myceliophthora thermophila ATCC 48104 grown on crystalline cellulose. The enzyme was purified approximately 30-fold by (NH₄)₂SO₄ precipitation and column chromatography on DEAE-Sephadex A-50, Sephadex G-200 and DEAE-Sephadex A-50. The molecular mass of the enzyme was estimated to be about 120 kD by both sodium dodecyl sulphate gel electrophoresis and gel filtration chromatography. It displayed optimal activity at pH 4.8 and 60°C. The purified enzyme in the absence of substrate was stable up to 60°C and pH between 4.5 and 5.5. The enzyme hydrolysed p-nitrophenyl--d-glucoside, cellobiose and salicin but not carboxymethyl cellulose or crystalline cellulose. The K _m of the enzyme was 1.6mm for p-nitrophenyl--d-glucoside and 8.0mm for cellobiose. d-Glucose was a competitive inhibitor of the enzyme with a K of 22.5mm. Enzyme K activity was inhibited by HgCl₂, FeSO₄, CuSO₄, EDTA, sodium dodecyl sulphate, p-chloromercurobenzoate and iodoacetamide and was stimulated by 2-mercaptoethanol, dithiothreitol and glutathione. Ethanol up to 1.7 m had no effect on the enzyme activity.The authors are with the Department of Microbiology, Bose Institute, 93/1, A.P.C. Road, Calcutta 700 009, India. S.K. Raha is presently with the Department of Medicine, University of Saskatchewan, Saskatoon, Canada S7N OXO.     

Purification and properties of an NADP+-specific isocitrate dehydrogenase from Rhizobium meliloti

An NADP⁺-specific isocitrate dehydrogenase has been purified and characterized from Rhizobium meliloti. The enzyme showed Mn⁺⁺ or Mg⁺⁺ requirement. The apparent K_m values were 2.00×10^-5 m and 1.51×10^-5 m for dl-isocitrate and NADP⁺, respectively. The enzyme was inhibited by ATP, to a lesser extent by ADP and AMP. -Ketoglutarate also inhibited the enzyme activity. Oxalacetate and glyoxylate together inhibited the enzyme activity. The inhibition was competitive. Studies with thiol inhibitors suggested that the enzyme contained a sulfhydryl group at or near the active site. The enzyme has an approximate molecular weight of 60 000. Fluorescence studies suggested that the enzyme contained tryptophan     

Purification and partial characterization of alanine dehydrogenase from Streptomyces aureofaciens

Alanine dehydrogenase was purified to near homogeneity from cell-free extract of Streptomyces aureofaciens, which produces tetracycline. The molecular weight of the enzyme determined by size-exclusion high-performance liquid chromatography was 395 000. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis was 48 000, indicating that the enzyme consists of eight subunits with similar molecular weight. The isoelectric point of alanine dehydrogenase is 6.7. The pH optimum is 10.0 for oxidative deamination of L-alanine and 8.5 for reductive amination of pyruvate. K _M values were 5.0 mM for L-alanine and 0.11 mM for NAD⁺. K _M values for reductive amination were 0.56 mM for pyruvate, 0.029 mM for NADH and 6.67 mM for NH₄Cl.Abbreviation AlaDH alanine dehydrogenase     

Purification and characterization of mouse alcohol dehydrogenase from two inbred strains that differ in total liver enzyme activity

Alcohol dehydrogenase activity in mouse liver homogenate-supernatants is 1.7 times greater in the C57BL/10 strain than in the BALB/c strain, regardless of whether activity is expressed in units per gram liver, total liver, or milligram DNA. The K _m values for ethanol and NAD⁺, approximately 0.4 and 0.03mm, respectively, of enzyme purified from both strains are similar. Moreover, the K _i for NADH, 1 µm, the pH optimum for ethanol oxidation, 10.5, and the V _max for ethanol oxidation, 160 min^–1, for ADH from the C57BL/10 and BALB/c strains are similar. Therefore, the difference in ADH activity in the two strains cannot be due to differences in the catalytic properties of the enzyme. The electrophoretic and isoelectric focusing patterns and two-dimensional tryptic peptide maps of the purified enzyme from both strains are identical. Thus the amino acid sequences of enzyme from C57BL/10 and BALB/c mice must also be identical or very similar. The difference in ADH activity in the two strains is most likely the result of genetic differences in the content of ADH protein in liver.Supported by NIAAA Grant AA 04307.     

Thermostimulation of conidiation and succinic oxidative metabolism of Neurospora crassa

Summary The conidiogenic effect of temperature was investigated for its manifestation on the succinic oxidative system of Neurospora crassa. It was found that the initiation of conidiation was associated with a peak of succinate dehydrogenase activity. The succinate dehydrogenase activity decreased after the peak period of conidial differentiation. The strongest succinate dehydrogenase activity was measured in 37° C cultures.A second wave increase of succinate dehydrogenase was present in femalefertile (25° C) but was absent in the female-sterile (37° and 40° C) cultures.The cytochrome oxidase activity steadily decreased with the aging of the cultures.Malonate (10^-1 m) was found to stimulate conidiation. This stimulation was associated with higher succinate dehydrogenase activity.Riboflavine was found to accumulate with aging and was higher in 37° and 40° C cultures respectively as compared to 25° C cultures.The ultrathin sections of the conidia revealed that increase in conidial size at 37° C was accompanied with the swelling of the mitochondria and prominence of the endoplasmic reticulum. In conidia from 40° C cultures the mitochondria were shrunken and the endoplasmic reticulum broken.Dedicated with gratitude to Prof. Dr. A. Rippel-Baldes.     

Isoforms of Na,K-ATPase inArtemia salina: II. Tissue distribution and kinetic characterization

Summary To characterize the molecular properties conveyed by the isoforms of the subunit of Na,K-ATPase, the two major transepithelial transporting organs in the brine shrimp (Artemia salina), the salt glands and intestines, were isolated in pure form. The isoforms were quantified by ATP-sensitive fluorescein isothiocyanate (FITC) labeling. The salt gland enzyme exhibits only the 1 isoform, whereas the intestinal enzyme exhibits both the 1 and the 2 isoforms. After 32 hours of development, Na,K-ATPase activity [in mol P_i/mg protein/hr (1u)] in whole homogenates was 32±6 in the salt glands and 12±3 in the intestinal preparations (mean±sem). The apparent half-maximal activation constants (K _1/2) of the salt gland enzyme as compared to the intestinal enzyme were 3.7±0.6mm vs. 23.5±4mm (P<0.01) for Na⁺, 16.6±2.2mm vs. 8.29±1.5mm for K⁺ (P<0.01), and 0.87±0.8mm vs. 0.79±1.1mm for ATP (NS). The apparentK _i's for ouabain inhibition were 1.1×10^–4 m vs. 2×10^–5 m, respectively. Treatment of whole homogenates with deoxycholic acid (DOC) produced a maximal Na,K-ATPase activation of 46% in the salt gland as compared to 23% in the intestinal enzyme. Similar differences were found with sodium dodecyl sulfate (SDS). The two distinct forms of Na,K-ATPase isolated from the brine shrimp differed markedly in three kinetic parameters as well as in detergent sensitivity. The differences inK _1/2 for Na⁺ and K⁺ are more marked than those reported for the mammalian Na,K-ATPase isoforms. These differences may be attributed to the relative abundances of the subunit isoforms; other potential determinants (e.g. differences in membrane lipids), however, have not been investigated.During the tenure of an Educational Commission For Foreign Medical Graduates Visiting Associate Professorship.     

Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z

Actinobacillus sp. 130Z fermented glucose to the major products succinate, acetate, and formate. Ethanol was formed as a minor fermentation product. Under CO₂-limiting conditions, less succinate and more ethanol were formed. The fermentation product ratio remained constant at pH values from 6.0 to 7.4. More succinate was produced when hydrogen was present in the gas phase. Actinobacillus sp. 130Z grew at the expense of fumarate and l-malate reduction, with hydrogen as an electron donor. Other substrates such as more-reduced carbohydrates (e.g., d-sorbitol) resulted in higher succinate and/or ethanol production. Actinobacillus sp. 130Z contained the key enzymes involved in the Embden-Meyerhof-Parnas and the pentose-phosphate pathways and contained high levels of phosphoenolpyruvate (PEP) carboxykinase, malate dehydrogenase, fumarase, fumarate reductase, pyruvate kinase, pyruvate formate-lyase, phosphotransacetylase, acetate kinase, malic enzyme, and oxaloacetate decarboxylase. The levels of PEP carboxykinase, malate dehydrogenase, and fumarase were significantly higher in Actinobacillus sp. 130Z than in Escherichia coli K-12 and accounted for the differences in succinate production. Key enzymes in end product formation in Actinobacillus sp. 130Z were regulated by the energy substrates. Received: 2 September 1996 / Accepted: 10 January 1997     

Membranous localization and properties of ATPase of rat liver lysosomes

Summary Lysosomes isolated from rat liver were found to have ATPase activity (EC No. 3.6.1.3). Subfractionation of the lysosomes revealed a membranous localization of ATPase activity. The enzyme has half maximal activity at 0.2mm ATP and is inhibited by high concentrations of ATP. The apparentK _m for divalent metal is 0.2mm, and either ca²⁺ or Mg²⁺ give maximal activity.The ATPase activity has latency when lysosomes are isolated from rats treated with Triton WR-1339. This latency may be due to the presence of internalized sucrose because the activity ofL fraction lysosomes is much less latent and Triton WR-1339 itself is not inhibitory. The latency of glucosamindase, a marker enzyme for lysosomes, contrasts with the low latency of the ATPase and points to an ATPase with an exposed active site in intact lysosomes.