Mole Of Substrate Research Articles

Anaerobic degradation of isovalerate by a defined methanogenic coculture

Isovalerate-oxidizing strictly aneerobic bacteria were isolated from marine sediment and sewage sludge in coculture with Desulfovibrio sp. Cells stained Gram positive and behaved Gram positive also in Gram classification with KOH. Isovalerate degradation depended on interspecies hydrogen transfer to syntrophic hydrogen-oxidizing sulfate reducers or methanogens. Isovalerate was the only substrate utilized and was fermented to 3 mol acetate and 1 mol hydrogen per mol substrate. The degradation pathway was studied by enzyme assays in crude cell extracts, and included acetyl-CoA dependent activation of isovalerate, oxidation to methylcrotonyl-CoA and carboxylation to methylgluta-conyl-CoA which is hydrated and cleaved to acetoacetate and acetyl-CoA. Studies with inhibitors and ionophores suggest that energy conservation with this organism depends on either acetate efflux-driven proton symport or on an ion-gradient driven carboxylation mechanism.

Fuels and pathways as designed systems for support of muscle work.

Muscle in all animals relies upon four potential sources of energy: ATP hydrolysis, phosphagen hydrolysis, fermentations or oxidative metabolism. Although the relative contributions of different fuels varies greatly in different organisms, in none is there a simple reliance on stored ATP. Muscle work therefore requires a balance between rates of utilization and formation of ATP, a provision supplied by one of the three remaining fuels and metabolic pathways. Useful endogenous fuels must be storable at high level, and rapidly mobilizable with minimal perturbation of [ATP] and with minimal end-product effects on pH, charge or osmotic balance. In addition to displaying these properties, good exogenous fuels must be transferable at high rates between depot sites and muscle; actual flux rates of exogenous fuels depend upon respective ATP yields and are lowest for fuels which most amplify the yield of ATP per mol substrate oxidized. Substrate flux rates must be matched with O2 flux rates and with rates of endogenous substrate mobilization in order that the right energy-yielding pathways are activated at the right times. Of various control possibilities, an effective competition for ADP (and possibly Pi) seems at this time to be the dominant strategy for assuring integration of aerobic and anaerobic ATP-yielding pathways.

Large lanthanide‐induced chemical shifts in cobalt complexes of enethials (α,β‐unsaturated thioaldehydes)

AbstractEu(fod)3‐, Yb(fod)3‐ and Pr(fod)3‐induced chemical shifts of the ‘thioaldehydic’ protons in enethial ligands complexed to a cobalt cyclopentadienyl group are unusually large and in the same direction (10–30 ppm downfield per mole of shift reagent per mole of substrate). The shifts of the protons induced by Eu(fod)3 and Pr(fod)3 in the enethial ligands show an alternation in sign on proceeding away from the sulfur atom. In contrast to the results with the fod reagents, the ytterbium and lanthanum shift reagents Yb(thd)3 and La(thd)3 caused only small shifts of protons in the 2‐phenylpropenethial ligand. No induced shifts with the Eu or Pr reagents were observed for a cyclopentadienyl cobalt complex of dithioglyoxal. The induced shifts in these enethial complexes may be caused by varying blends of complex formation, contact and pseudocontact shifts. Caution is advised in assigning origins to lanthanide induced shifts in such organometallic systems.

Lentil seedlings amine oxidase: Preparation and properties of the copper-free enzyme

The reaction of copper-free lentil seedlings amine oxidase with substrates has been studied. While devoid of catalytic activity, this enzyme preparation is still able to oxidize two moles of substrate and to release two moles of aldehyde and two moles of ammonia per mole of dimeric protein. The same stoichiometry has been determined on the native enzyme in the absence of oxygen. Although copper is essential for the reoxidation of the reduced enzyme, a binding of oxygen to the copper-free protein has been demonstrated.

The static head method for determining the charge stoichiometry of coupled transport systems Applications to the sodium-coupled d-glucose transporters of the renal proximal tubule

The static head method for determining the charge stoichiometry (the number of moles of charge translocated per mole of substrate) of a coupled transport system is presented. The method involves establishing experimental conditions under which a membrane potential exactly balances the thermodynamic driving force of a known substrate gradient. The charge stoichiometry can then be calculated from thermodynamic principles. In contrast to the usual steady-state method for determining charge stoichiometry in cell suspensions and vesicle preparations, the static head method is applicable to systems which are not capable of maintaining a constant membrane potential over time. The charge stoichiometries of two renal sodium coupled d-glucose transporters previously identified in brush-border membrane vesicle preparations from the outer cortex (early proximal tubule) and outer medulla (late proximal tubule) are determined. The charge stoichiometries of these transporters are in good agreement with their sodium/glucose coupling ratios arguing against the possibility that glucose transport is coupled to ions other than sodium in these membranes.

Studies on the Purification of Chitin Synthase from Coprinus cinereus

Chitin synthase has been characterized from the stipes of Coprinus cinereus by a number of techniques. In the absence of digitonin, on gel filtration columns and during electrophoresis the enzyme showed properties consistent with having molecular weights from 1.5 105 to several million, suggesting its reversible aggregation into large multimolecular units. Gel chromatography in buffers containing digitonin gave highly reproducible results, and when followed by anion-exchange chromatography gave preparations with very high activity [e.g. 3.4 mol substrate incorporated min-1 (mg protein)-1] and apparent molecular weight 8.0 105. The best purification (140-fold) was achieved by gel chromatography followed by copper chelate affinity chromatography, giving a nearly pure enzyme preparation of activity 4.7 mol substrate incorporated min-1 (mg protein)-1, which showed only one band of molecular weight 6.7 104 on SDS-polyacrylamide electrophoresis. These purified preparations were free of the nucleoside diphosphatase and protease activities that were present in the early stages of purification.

Effect of lipid composition of liposomes on their sensitivity to peroxidation.

The effect of lipid composition of liposomes on peroxidation induced by ferrous ion and ascorbate was examined. Temperature affects the sensitivity of liposomes; the peroxidation rate was increased with increase of the incubation temperature. With liposomes consisting of 1-palmitoyl-2-arachidonyl phosphatidylcholine (substrate) and a peroxidation-insensitive lipid, 1-palmitoyl-2-oleoyl phosphatidylcholine, peroxidation was dependent on the density of the substrate. No appreciable peroxidation was observed with liposomes containing less than 10 mol% of the substrate at 37 degrees C. When 1 mol substrate was mixed with 9 mol dimyristoyl phosphatidylcholine, peroxidation occurred below 10 degrees C, but not above 20 degrees C. Above 20 degrees C, the substrates should be located homogeneously on the membranes, whereas they should be clustered below 10 degrees C, since the gel-liquid crystalline phase transition temperature of matrix membrane of dimyristoylphosphatidylcholine was 17-21 degrees C. Peroxidation of liposomes consisting of 1-palmitoyl-2-arachidonyl phosphatidylcholine was also suppressed by cholesterol. These findings indicate that the lateral distribution as well as the density of the substrate on membranes affects the sensitivity of the substrate to peroxidation. It was also found that alpha-tocopherol is preferentially located in the 1-palmitoyl-2-arachidonyl phosphatidylcholine-rich regions of membranes consisting of mixed phospholipids, and efficiently suppresses peroxidation of liposomal lipids.

Purification and properties of NADP‐dependent glutamate dehydrogenase from Sphaerostilbe repens

NADP‐dependent glutamate dehydrogenase (EC 1.4.1.4) extracted from Sphaerostilbe repens was purified to homogeneity by using ammonium sullate fractionation hydroxyapatite and DEAE‐cellulose column chromatography and, finally, preparative polyacrylamide gel electrophoresis. The turnover number of the enzyme for the amination reaction was about 66000 mol substrate transformed min‐1 (molecule of GDH)‐1. Molecular weight of the native enzyme was estimated to be 280000 dalton by polyacrylamide gradient gel electrophoresis. The same technique in the presence of sodium dodecyl sulfatc gave a single protein band that corresponded to the subunit molecular weight of 48000 dalton. Thus, it is concluded that NADP‐GDH is composed of six identical polypeptidic chains.The pH optimums were 6.9 and 8.4 for the forward and reverse reactions respectively. The NADP‐GDH lost practically none of its activity for ten days at 4°C and for 15 h at room temperature, but was inactivated by higher temperatures. Thiol compounds such as 2‐mercaptoethanol and dithiolhrcitol protected the enzyme from rapid inactivation. The Michaelis constants for GDH were 0.64, 0.049. 0.043 and 5.5 mM for α‐ketoglutaratc. NADPH, NADP and glutamate, respectively. The enzyme had a negative cooperativity for ammonium (Hill number of 0.66), and its Km value increased from 2.6 to 21.2 mM when the ammonium concentration exceeded 16 mM. The deamination reaction was highly sensitive to inhibition by ammonium, while the amination reaction was only slightly inhibited by glutamate. These results, considered together with the Km values, indicate that the NADP‐GDH in Sphaerostilbe repens is primarily concerned with glutamate biosynthesis.

The Utilization of 5-Oxoproline, Ammonia and Glutamine by Rhizobium leguminosarum in Chemostat Culture

The growth in continuous culture of Rhizobium leguminosarum on the nitrogen sources glutamine, ammonia, or ammonia and 5-oxoproline (formed when aqueous glutamine solutions are autoclaved) is described. When growth was nitrogen-limited with autoclaved glutamine the specific rate of 5-oxoproline utilization was independent of dilution rate, whereas the specific rate of ammonia utilization increased with dilution rate. Although the culture was nitrogen-limited, excess nitrogen (as 5-oxoproline) was still present in the supernatant, especially at high dilution rates. Values obtained for Y max glucose and Y max oxgen were 72·2 and 32·5 g dry wt bacteria (mol substrate)−1, respectively, under ammonia/5-oxoproline limitation and 95·7 and 30·2 g dry wt bacteria (mol substrate)−1, respectively, under glutamine limitation.

The metabolism of 3-cyclohexenecarboxylic acid by Alcaligenes faecalis

Alcaligenes faecalis, grown on 3-cyclohexenecarboxylic acid supplemented with gluconate, was induced to metabolize cyclohexanecarboxylic acid, 3-cyclohexenecarboxylic acid, 1, 4-cyclohexadienecarboxylic acid, benzoic acid, and catechol. During growth, 1, 4-cyclohexadienecarboxylic acid, 3-heptene-1, 7-dioic acid, and benzoic acid were shown to be present in the culture supernatants. The results suggest that 3-cyclohexenecarboxylic acid may be metabolized by two pathways having 1, 4-cyclohexadienecarboxylic acid as a common intermediate. One pathway involved β-oxidation of coenzyme A intermediates; the other pathway involved benzoic acid as an intermediate. Studies on the consumption of oxygen per mole of substrate by cell suspensions indicated that the major portion of 3-cyclohexenecarboxylic acid was metabolized by the pathway involving benzoic acid. When grown on 3-cyclohexenecarboxylic acid as sole carbon and energy source, the organism was additionally induced to metabolize salicylic acid and gentisic acid, and growth medium contained salicylic acid. Thus, under the latter growth conditions a pathway involving salicylic acid as an intermediate was also induced.

The isolation and characterization of catalytically competent porphobilinogen deaminase—intermediate complexes

Porphobilinogen deaminase catalyses the homopolymerization of 4 molecules of porphobilinogen (1) to give preuroporphyrinogen (2) [ 1,2]. Preuroporphyrinogen is the natural substrate for uroporphyrinogen III cosynthetase leading to the formation of uroporphyrinogen III (3) [3] in quantitative yield. In the absence of cosynthetase, preuroporphyrinogen spontaneously converts to uroporphyrinogen I (4). The order in which the deaminase catalyses the assembly of the 4 porphobilinogen molecules has been determined in [4-61 and it appears that addition of the 4 pyrrole rings to the enzyme follows the sequence ring a followed by ring b and c and finally ring d (see structure (2)). Observations that the addition of stoichiometric amounts of porphobilinogen to deaminase give tightly bound species in which 1,2 or 3 mol substrate (or equivalent) are bound to the enzyme have suggested that discrete enzyme intermediate complexes exist [S]. Elegant experiments [7] involving excess [3H]porphobilinogen and human deaminase have shown the existence of 4 labelled protein bands with deaminase enzyme activity again suggesting the existence of discrete enzyme intermediate complexes. Related work [8] with the deaminase from Rhodopseudomonas spheroides has largely confirmed these latter findings although only 3 discrete labelled protein bands are formed with the bacterial enzyme when incubated with [ 14C] porphobilinogen. In addition we have established that the enzyme forms a relatively stable covalent link with the bound pyrrole residues [9]. Although the appearance of 3-intermediate complexes in the case of the bacterial deaminase suggests

Mixotrophic Growth of Thiobacillus A2 in Chemostat Culture on Formate and Glucose

In aerobic chemostat culture Thiobacillus A2-GFI grew autotrophically on formate and heterotrophically on glucose with maximum specific growth rates (μmax) of 0·21 and 0·33 h−1, respectively. At dilution rates of 0·1 and 0·18 h−1, it grew mixotrophically on formate + glucose mixtures, completely consuming both substrates. Ribulose-1,5-bisphosphate carboxylase and formate dehydrogenase were present at high specific activity in autotrophic and mixotrophic cultures, but were repressed in cultures on glucose alone. A greater proportion of added glucose was assimilated in mixotrophic culture than in heterotrophic culture. Raising the dilution rate of a mixotrophic culture from 0·1 or 0·18 to 0·3 h−1 resulted in washout (with an apparent μ max for mixotrophic growth of 0·25 h−1) and the establishment of a culture dependent on glucose for growth. Growth yields on formate and glucose were, respectively, 3·3 and 100 g dry wt (mol substrate consumed)−1. Steady state biomass production in mixotrophic culture indicated additive growth yields. The biomass produced in cultures on formate + glucose at a dilution rate of 0·3 h−1 suggested that growth only occurred on glucose, but organisms still contained high activities of ribulose-1,5-bisphosphate carboxylase and formate dehydrogenase. At a formate: glucose ratio (mm) of 100:1, some formate was oxidized and CO2 was fixed, but formate was not used when this ratio was 50:5. Formate-glucose mixotrophy benefits Thiobacillus A2-GFI when substrates are limited at low growth rates (<μ max for formate), but is characterized by a μ max below that possible on glucose. Physiological behaviour at high growth rates was influenced by the formate:glucose ratio, resulting under some conditions, at least, in loss of mixotrophy and the establishment of heterotrophic growth.

The venom from the snakes Agkistrodon bilineatus taylori and Crotalus durissus totonacus: Lethality, biochemical and immunological properties

IN THIS communication we report the lethality, enzymatic activities, number of electrophoretically different proteins and immunogenic capacities of two snake venoms . Thevenomfrom the Mexican snakes Agkistrodon bilineatus taylori and Crotalus durissus totonacus was collected at the Gladys Porter Zoo, Brownsville, Texas in cooled vessels, centrifuged at 5000 x g for 5 min and the supernatant immediately lyophilized . Hyaluronic acid was obtained from Nutritional Biochemical Co., N-benzoyl-L-arginine-ethyl ester (BAEE) and N-benzoyl-L-tyrosine-ethyl ester (BTEE) from Sigma Chemical Co., and hemoglobin from Worthington. Horse serum directed against the venom of snakes from the genusCrotalus(Anticrotalico, lot No. 1344), from the genus Bothrops (Antibotropico, lot No . 1212) and against the venom from both groups of snakes (Antiviperino, lot No. 1420) were purchased from the Instituto National de Higiene (Secretaria de Salubridad y Asistencia, M6xico 17 D. F. M6xico). Lethality was tested by i.p . injections into albino mice using ten different doses (ten mice for each dose), as previously reported (POSSANI et al., 1977). The direct haemolytic effect and the indirect haemolytic effect (after incubation with phospholipids) upon human red cells were measured following a technique originally described by Tu et al. (1970), with some modifications (SOSA et al., 1979). Hyaluronidase activity was measured by a turbidimetric method (TOLKsDoRF et al., 1949). One unit of activity is defined as that amount of protein required to hydrolyze 1 Rg of hyaluronic acid per min at 25°C in 200 PI of a 200 Vg/ml solution of hyaluronic acid at pH 5-3 . Hydrolase (esterase) activity was measured spectrophotometrically using benzoyl esters (BAEE and BTEE) as substrates . These assays were carried out at 25°C by adding the venom to a 1 cm pathway quartz cell containing 50 mM CaC12, 1 mM of BAEE or 0-5 mM of BTEE in 40 mM Tris-HCI buffer (pH 7-8) and measuring the absorbance at 255 nm at 15 sec intervals for 3-5 min periods, in 1-5 ml total volume . One unit of enzymatic activity is defined as the amount of protein needed to hydrolyze 1 mole of substrate in I min under the above conditions . The phospholipase activity was measured titrimetrically by the technique of SmLoAH et al. (1973), using egg

Preparation of homogeneous crystals of myo-inositol 1-phosphate synthase from rat testicles ? further data on the chemical and catalytic properties of the enzyme (studies on the biosynthesis cyclitols XXXIX1)

Pre-purified preparations of myoinositol-1-phosphate synthase (E.C. 5.5.1.4) from rat testes can be purified to homogeneity by first crystallizing the enzyme according to JAKOBY and then recrystallizing it at a pH value close to the isoelectric point while slowly increasing the temperature from 0 to 15 degrees C. This method gives a much yield of homogeneous enzyme than the previously used purification by affinity chromatography. It was further found that the pure enzyme contains close to 2 mol NAD+ per mol enzyme; it does not contain any metal. At substrate saturation the enzyme binds close to 1 mol substrate per mol enzyme, as determined by using radioactively labelled substrate and binding it to the enzyme by reduction with NaBH4. The reaction catalyzed by the enzyme is irreversible.

Active-site titration of pig-plasma benzylamine oxidase.

1. Titration of benzylamine oxidase with benzylamine under anaerobic conditions shows that full reduction of the enzymic 470-nm chromophore is obtained on the addition of one mole of substrate per mole of enzyme. Concomitantly, one mole of benzaldehyde per mole of enzyme is produced. 2. A single prosthetic group interacting with carbonyl reagents can be detected on titration of benzylamine oxidase with phenylhydrazine. Titration data reported to indicate a higher content of prosthetic groups were obtained under conditions where equilibration between enzyme and phenylhydrazine is insufficiently complete. 3. It is concluded that pig-plasma benzylamine oxidase contains a single catalytically active site. This means that the two copper atoms present in the enzyme may be structurally or functionally different.

Multiple dioxygenation by lipoxygenase of lipids containing all- cis-1,4,7-octatriene moieties

Soybean lipoxygenase-1 acting upon polyunsaturated fatty acids containing a suitably positioned all- cis-1,4,7-octatriene moiety generates bishydroperoxy derivatives while consuming approximately 2 mol of O 2 per mole of substrate. The reaction has been separated into two steps: The first is marked by the rapid monohydroperoxidation of the starting material and the second is marked by the much slower hydroperoxidation of the first product. All of the compounds which are successfully converted to bishydroperoxy derivatives contain an ω6,9,12 grouping of cis double bonds. α-Linolenic acid (ω3,6,9) cannot serve as a substrate because its preferred site of oxygenation is in the center of the octatriene moiety. The K m for arachidonic acid in the reaction leading to the singly hydroperoxidized monohydroperoxide is 8.6× 10 −5, m, which is approximately 200 times smaller than the K m for the monohydroperoxide in the second step leading to the bishydroperoxide. All of the substrates which undergo double dioxygenation form conjugated trienes.

Substrate specificity of ascorbate oxidase

Ascorbate oxidase oxidizes leuco 2, 6-dichloroindophenol to the blue quinoid dye and produces spectral changes in the UV spectra of certain substituted polyhydric and amino phenols at pH 5.7. The new peaks produced by the addition of enzyme to the dichlorohydroquinones (2,5 and 2,6) and hydroxyhydroquinone correspond to the respective p-quinones of these substrates. At pH 5.7, the enzyme does not oxidize hydroquinone, barely oxidizes chlorohydroquinone, but oxidizes 2,6- and 2,5-dichlorohydroquinone and hydroxyhydroquinone at a rate about 1 12 that of ascorbic acid, with the uptake of one gram atom of oxygen per mole of substrate. A correlation has been found between the concentration of anion present in solution at pH 5.7 and the rate of oxidation of compounds of the hydroquinone series by the enzyme. The results indicate that an anionic form of the substrate is an important requirement of the enzyme specificity.

Desulfuromonas acetoxidans gen. nov. and sp. nov., a new anaerobic, sulfur-reducing, acetate-oxidizing bacterium.

Anaerobic sea or fresh water media with acetate and elemental sulfur yielded enrichments of a new type of strictly anaerobic, rod-shaped, laterally flagellated, Gram-negative bacterium. Three pure culture-strains from different sulfide-containing sea water sources were characterized in detail and are described as a new genus and species Desulfuromonas acetoxidans. The new bacterium is unable to ferment organic substances; it obtains energy for growth by anaerobic sulfur respiration. Acetate, ethanol or propanol can serve as carbon and energy source for growth; their oxidation to CO2 is stoichiometrically linked to the reduction of elemental sulfur to sulfide. Organic disulfide compounds, malate or fumarate are the only other electron acceptors used. Butanol and pyruvate are used in the presence of malate only; no other organic compounds are utilized. Biotin is required as a growth factor. The following dry weight yields per mole of substrate are obtained: in the presence of sulfur: 4.21 g on acetate, 9.77 g on ethanol; in the presence of malate: 16.5 g on acetate, 34.2 g on ethanol and 46.2 g on pyruvate. Accumulations of cells are pink; cell suspensions exhibit absorption spectra resembling those of c-type cytochromes (abs. max. at 419, 523 and 553 nm). Malate-ethanol grown cells contain a b-type cytochrome in addition. In the presence of acetate, ethanol or propanol, Desulfuromonas strains form robust growing syntrophic mixed cultures with phototrophic green sulfur bacteria.

The reactions of alpha-chymotrypsin and related proteins with ester substrates in non-aqueous solvents.

1. The reactivity of alpha-chymotrypsin toward p-nitrophenylacetate has been studied in dimethylformamide, dimethylsulfoxide, formamide and methylacetamide. p-Nitrophenol is liberated in dimethylsulfoxide only. 2. The reactions of alpha-chymotrypsin in dimethylsulfoxide are characterized by the same kinetic and equilibrium constants with either the p-nitrophenyl esters of straight chain carboxylic acids (from acetic to n-caprylic) or with the "specific substrate", N-carbobenzoxy-DL-phenylalanine p-nitrophenyl ester. This signifies that reactions of alpha-chymotrypsin in dimethylsulfoxide, unlike those in aqueous medium, have no specificity toward su-strate structure. 3. The stoichiometry of alpha-chymotrypsin reactions in dimethylsulfoxide was shown to be about five moles of substrate per mole of enzyme. After attaining this stoichiometry, the reaction is completed. 4. Optical rotatory dispersion spectra indicate that in non-aqueous media alpha-chymotrypsin undergoes a large conformational transition which results in a random coil. 5. Chymotrypsinogen, trypsin, trysinogen, lysozyme and serum albumin react with p-nitrophenylacetate in dimethylsulfoxide at rates which are approximately equal to those of alpha-chymotrypsin. Thus, the "activity" of alpha-chymotrypsin in dimethylsulfoxide toward p-nitrophenylacetate does not differ from the "activity" of other proteins, some of which are not even hydrolytic enzymes.

Effect of uncoupling agents and respiratory inhibitors on the growth of Streptococcus agalactiae.

2,4-Dinitrophenol, dicoumarol, carbonylcyanide, m-chlorophenyl-hydrazone and pentachlorophenol all depressed aerobic molar growth yields of Streptococcus agalactiae to values equal to, or less than, those supported by substrate level phosphorylation. When the only source of energy was from substrate phosphorylation (anaerobic growth conditions), there was also a severe depression of the molar growth yield by the same four uncoupling agents. These results indicate that the effect of these agents is to uncouple both substrate and oxidative phosphorylation in S. agalactiae. Amytal inhibited glucose utilization, reduced the amount of O(2) used per mole of substrate and reduced the molar cell yield to that supported by substrate phosphorylation. Atebrin inhibited the respiration rate, but final O(2) consumed per mole of substrate was unchanged, and the respiration was coupled to biosynthesis. Rotenone had no effect on respiration, substrate utilization, or on molar growth yields.