Factors affecting the reassociation and reactivation of the half-molecular weight nonidentical subunits of pigeon liver fatty acid synthetase.

The pigeon liver fatty acid synthetase complex (14 S) is dissociated in low ionic strength buffer containing dithiothreitol to form a half-molecular weight subunits (9 S) which are completely inactive for the synthesis of saturated fatty acids. The dithiothreitol-protected (reduced) subunits are rapidly reassociated and reactivated to form the active enzyme complex, not only by an increase in salt concentration but also by micromolar concentrations of NADP+ or NADPH. Increases in KCl or NADPH concentration result in an increase in the extent of reactivation (equilibrium) with no change in the over-all rate of the reaction or the half-life ofreactivation of the enzyme. The extent (equilibrium) of reactivation of the enzyme is the same in 0.2 M potassium phosphate buffer, pH 7.0; 0.2 M KCl in 5 mM Tris-35 mM glycine buffer, PH 8.3; and 50 muM NADP+ or NADPH in the Tris-glycine buffer. The extent and rate of reactivation of the enzyme is dependent not only on ionic strength and NADPH concentration, but also on pH and temperature. Reactivation with 0.2 M KCl is optimal between pH 7.3 and 8.5. At higher and lower pH values the rate and extent of reactivation are lowered. The rate and extent of reactivation are also decreased as the temperature is lowered below 10 degrees. At 0 degrees there is little reactivation of enzyme activity. However, in the presence of 0.2 M KCl containing 15 to 40% glycerol at 0 degrees, reactivation of the enzyme is about 50% complete. The rate of reactivation of enzyme in the presence of KCl or NADPH conforms to first order kinetics. This result suggests that the subunits first combine to form an inactive complex which is subsequently transformed to an enzymatically active complex. Evidence for the presence of inactive complex was obtained in experiments carried out in 0.2 M KCl at pH 6.0, and in 0.2 M KCl at pH 8.3, at both 6 and 3 degrees. Under these conditions the amount of complex observed upon ultracentrifugation was greater than expected from determinations of enzyme activity. The above findings suggest that ionic and hydrophobic interactions, and possibly the water structure surrounding the interacting sites, are of prime importance in reassociation and reactivation of enzyme. In addition, NADP+ and NADPH have very specific effects in bringing about reassociation and in maintaining the structural integrity of the multienzyme complex.     

Rat liver cytoplasmic glucose-6-phosphate dehydrogenase. Steady-state kinetic properties and circular dichroism.

Steady-state kinetic studies including initial velocity, NADPH product inhibition, dead-end inhibition, and combined dead-end and product inhibition measurements with purified rat liver glucose-6-phosphate dehydrogenase indicate a sequential and obligatory addition of substrates in the order of NADP+, glucose-6-P for the catalytic pathway at pH 8.0. Although instability of 6-phosphoglucono-delta-lactone precluded product inhibition experiments which might directly exclude an enzyme-6-phosphoglucono-delta-lactone complex, the absence of an enzyme-glucose-6-P complex suggests that the enzyme-lactone product is unlikely and the release of products is also ordered, with NADPH released last. Consideration of the kinetic constants (Ka = 2.0 muM, Kiq = 13 muM) and cellular concentration of the substrates and products suggests extensive inhibition of the enzyme in vivo and control by the NADPH/NADP+ ratios. Circular dichroism spectra of the enzyme in 20 mM phosphate buffer at pH 7.0 and 25 degrees C indicate 51% helix and 33% pleated sheet structures which is considerably different from results (14% helix) with yeast enzymes.     

Kinetic studies of 6-phosphogluconate dehydrogenase from sheep liver

The steady-state kinetics of the oxidative decarboxylation of 6-phosphogluconate catalysed by 6-phosphogluconate dehydrogenase from sheep liver in triethanolamine and phosphate buffers (pH 7.0) have been reinvestigated. In triethanolamine buffer the enzyme is inhibited by high NADP+ concentrations in the presence of low fixed concentrations of 6-phosphogluconate. Data are consistent with an asymmetric sequential mechanism in which NADP+ and 6-phosphogluconate bind randomly and product release is ordered. The pathway through the enzyme--6-phosphogluconate complex appears to be preferred in triethanolamine buffer. Pre-steady-state studies of the oxidative decarboxylation reaction at pH 6.0-8.0 show that hydride transfer is greater than 900 s-1. After the fast formation of NADPH in amounts equivalent to about half of the enzyme-active-centre concentration, the rate of NADPH formation is equal to the steady-state rate. Two possible interpretations are considered. Rapid fluorescence measurements of the displacement of NADPH from its complex with the enzyme at pH 6.0 and 7.0 indicate that the dissociation of NADPH is fast (greater than 800 s-1) and cannot be the rate-limiting step in oxidative decarboxylation. Coenzyme binding studies at equilibrium have been extended to include the determination of the dissociation constants for the binary complexes of enzyme with NADPH and NADP+ at pH 6.0-8.0 and the dissociation constant for NADPH in the ternary enzyme--6-phosphogluconate--NADPH complex in triethanolamine buffer, pH 7.0.     

Kinetic mechanism from steady-state kinetics of the reaction catalysed by baker''s-yeast glucose 6-phosphate dehydrogenase in solution and covalently attached to sepharose.

1. The reaction catalysed by glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate-NADP+ oxidoreductase, EC 1.1.1.49) from baker's yeast was studied in 42mM-glycylglycine buffer, pH7.4 at 25 degrees C, by initial-velocity studies and by the use of NADPH as a product inhibitor. 2. The reactions catalysed by both the soluble enzyme and a stable enzyme covalently attached to CNBr-activated Sepharose 4B probably follow an ordered reaction mechanism with NADP+ and NADPH as the leading reactants. 3. The kinetic constants obtained for the soluble enzyme lere: KNADP+m, 19 muM; KNADP+s, 23 muM; KNADPHs, 15 muM. Similar values were obtained for the immobilized enzyme. 4. The assay of the immobilized enzyme was done by using a micro packed-bed recirculation reactor, and the advantages of this technique are discussed.     

Kinetic characteristics of 6-phosphogluconate dehydrogenase from bull adrenal cortex

The hyperbolic dependence of the initial rate of 6-phosphogluconate dehydrogenase-catalyzed reaction on 6-phosphogluconate and NADP concentrations has been established. The Lineweaver-Burk plots of V0 against concentration of one substrate with constant unsaturating concentrations of another substrate cross left from the ordinate axis. The Km value for 6-phosphogluconate is equal ot 0.035 mM, for NADP--0.018 mM. It has been shown that NADPH inhibits 6-phosphogluconate dehydrogenase by the competitive type with respect to NADP and by the noncompetitive one with respect to 6-phosphogluconate. Ribulose-5-phosphate inhibits the reaction by the mixed type with respect to NADP and by the noncompetitive type with respect to 6-phosphogluconate. Kinetic data are in agreement with the consecutive mechanism of the reaction: the first substrate is NADP, the last product--NADPH. The Arrhenius plot for the reaction shows a break at 27 degrees C.     

Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland.

1. 6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate.     

Prevention of cold inactivation of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase by NADPH.

Solubilized 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) from rat liver microsomes has been reported to be reversibly inactivated by temperatures below 19 degrees C. Cold inactivation has now been found to be completely prevented by NADPH and by NADP+ at a concentration of 3 mM. NADPH, however, was more active than NADP+ at lower concentrations and prevented 50% of the cold inactivation at 0.2 mM, whereas a 1.1 mM NADPH+ without effect and the substrate 3-hydroxy-3-methylglutaryl coenzyme A prevented only 30% of the cold inactivation at a concentration 50 times greater than the Km value.     

Regulation of glucose-6-phosphate dehydrogenase in spinach chloroplasts by ribulose 1,5-diphosphate and NADPH/NADP+ ratios.

The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) FROM SPINACH CHLOROPLASTS IS STRONGLY REGULATED BY THE RATIO OF NADPH/NADP+, with the extent of this regulation controlled by the concentration of ribulose 1,5-diphosphate. Other metabolites of the reductive pentose phosphate cycle are far less effective in mediating the regulation of the enzyme activity by NADPH/NADP+ ratio. With a ratio of NADPH/NADP+ of 2, and a concentration of ribulose 1,5-diphosphate of 0.6 mM, the activity of the enzyme is completely inhibited. This level of ribulose 1,5-diphosphate is well within the concentration range which has been reported for unicellular green algae photosynthesizing in vivo. Ratios of NADPH/NADP+ of 2.0 have been measured for isolated spinach chloroplasts in the light and under physiological conditions. Since ribulose 1,5-diphosphate is a metabolite unique to the reductive pentose phosphate cycle and inhibits glucose-6-phosphate dehydrogenase in the presence of NADPH/NADP+ ratios found in chloroplasts in the light, it is proposed that regulation of the oxidative pentose phosphate cycle is accomplished in vivo by the levels of ribulose 1,5-diphosphate, NADPH, and NADP+. It already has been shown that several key reactions of the reductive pentose phosphate cycle in chloroplasts are regulated by levels of NADPH/NADP+ or other electron-carrying cofactors, and at least one key-regulated step, the carboxylation reaction is strongly affected by 6-phosphogluconate, the metabolic unique to the oxidative pentose phosphate cycle. Thus there is an interesting inverse regulation system in chloroplasts, in which reduced/oxidized coenzymes provide a general regulatory mechanism. The reductive cycle is activated at high NADPH/NADP+ ratios where the oxidative cycle is inhibited, and ribulose 1,5-diphosphate and 6-phosphogluconate provide further control of the cycles, each regulating the cycle in which it is not a metabolite.     

Reduced nicotinamide adenine dinucleotide phosphate, a structural and conformational probe of chicken liver fatty acid synthetase.

Structural and conformational organization of chicken liver fatty acid synthetase has been probed using its fluorescent coenzyme, NADPH. Three NADPH binding sites per mole of the enzyme complex, of apparently identical dissociation constant (KD = 0.6 muM) can be titrated at temperatures above 12 degrees. These results are in disagreement with the earlier studies of Hsu and Wagner (Hsu, R. Y., and Wagner, B. J. (1970) Biochemistry, 9, 245-251) in which four such sites could be titrated. At 12 degrees, the composite sites split into two subsets: a pair of sites with a KD of 0.3 muM and a third site with a Kd of 1.1 muM. At lower temperatures (5 degrees or 2 degrees), the site with weak affinity disappears, leaving a pair of sites with a Kd of 0.5 muM. Similar observations were made when the enzyme was modified with phenylmethylsulfonyl fluoride, a specific and selective inhibitor of fatty acyl-CoA deacylase (s) of the pigeon liver enzyme complex (Kumar, S. (1975) J. Biol. Chem. 250, 5150-5158). Partial modification with phenylmethylsulfonyl fluoride elicits a NADPH binding response similar to the binding observed at 12 degrees, i.e. two sets of binding sites with nonidentical dissociation constants. Further modification corresponding to the complete loss of deacylase function results in a set of two apparently identical binding sites, and the third site is not available for titration. The modified enzyme retains the two reductase functions as measured by the model substrates, acetoacetyl-N-acetylcysteamine and crotonyl-CoA. Furthermore, the addition of acetyl- and malonyl-CoA (100 muM each) to the modified enzyme lowers the NADPH binding affinity by a factor of 3. Other observations show that the quantum yield, as measured by the ratio of fluorescence intensity of bound and free NADPH, changes with temperature and ionic strength. Lowering the temperature from 30 degrees to 2 degrees increases the enhancement ratio by 50%, whereas increase in ionic strength from 0.05 to 0.2 M potassium phosphate lowers it to 50% of the original level. Measurement of NADPH binding in the presence of NADP+, NADH, NAD+ and adenosine-2'-monophospho-5'-diphosphoribose demonstrates that NADP+ shows competitive behavior for NADPH sites (KD = 10.6 muM), whereas NADH and NAD+ show noncompetitive (KD (apparent) = nearly 600 muM) and rather complicated interactions implicating nonspecific conformational alteration of the enzyme complex. The behavior of adenosine 2'-monophospho-5'-diphosphoribose is intermediate between NADP+ and NADH. These data are discussed in terms of substrate-mediated conformational changes and the moles of each of the reductase enzymes per mole of the enzyme complex, the polarity of the NADPH binding region, and the probable structure of the nicotinamide moiety when bound to the enzyme.     

Kinetics and control of bovine adrenal glucose-6-phosphate dehydrogenase.

Michaelis-Menten kinetics are observed in studies of highly purified bovine adrenal glucose-6-phosphate dehydrogenase at pH8.0 in 0.1 M bicine. The Km for NADP+ is 3.8 muM and for glucose-6-phosphate, 61 muM. At pH 6.9 Km for NADP+ increases to 6.5 muM. The enzyme is inhibited by NADPH both at pH 6.8 and at 8.0 with a Kip of 2.36 muM at pH 8.0. Inhibition is competitive with respect to both substrates implying that addition of substrates is random ordered. The data are also interpreted in terms of "reducing charge", the mole fraction of coenzyme in the reduced form. This appears to be the major mechanism for regulation of the pentose shunt. D-glucose, oxidized by the enzyme at a very slow rate, is also a competitive inhibitor for the natural substrate with a Ki of 0.29 M. Phosphate is a competitive inhibitor for glucose-6-phosphate oxidation but both phosphate and sulfate accelerate glucose oxidation suggesting a common binding site for the two anions and the phosphate of the natural substrate. While binding of ACTH to our enzyme preparations has been observed, we have not been able, in spite of repeated attempts, to demonstrate augmentation of the activity of the enzyme by the addition of ACTH.     

Purification and kinetic characterization of 6-phosphogluconate dehydrogenase from Schizosaccharomyces pombe.

6-Phosphogluconate dehydrogenase is the pivotal enzyme that links the gluconate route and the oxidative phase of the pentose phosphate pathway in Schizosaccharomyces pombe. The enzyme differs from the known 6-phosphogluconate dehydrogenases of other sources in that the Schizosaccharomyces enzyme is tetrameric having a subunit mass of 38 kDa, that it requires NADP+ obligatorily for activity, and that it can be activated by divalent metal ions such as Co2+ and Mn2+. Steady-state kinetic studies were undertaken. Initial rate and product inhibition results suggest that 6-phosphogluconate dehydrogenase from Schizosaccharomyces pombe catalyzes NADP(+)-linked oxidative decarboxylation of 6-phosphogluconate by an equilibrium random mechanism with two independent binding sites, namely one site for the nicotinamide coenzyme, NADP+/NADPH, and another site for 6-phosphogluconate-D-ribulose-5-phosphate and for CO2. Studies of pH dependence implicated a basic residue with a pK value of 7.4 in the binding of 6-phosphogluconate and an acidic residue with a pK value of 6.7 in the cation-mediated interaction of NADP+ with the enzyme.     

Purification and regulation of glucose-6-phosphate dehydrogenase from Bacillus licheniformis

d-Glucose-6-phosphate nicotinamide adenine dinucleotide phosphate (NADP) oxidoreductase (EC 1.1.1.49) from Bacillus licheniformis has been purified approximately 600-fold. The enzyme appears to be constitutive and exhibits activity with either oxidized NAD (NAD(+)) or oxidized NADP (NADP(+)) as electron acceptor. The enzyme has a pH optimum of 9.0 and has an absolute requirement for cations, either monovalent or divalent. The enzyme exhibits a K(m) of approximately 5 muM for NADP(+), 3 mM for NAD(+), and 0.2 mM for glucose-6-phosphate. Reduced NADP (NADPH) is a competitive inhibitor with respect to NADP(+) (K(m) = 10 muM). Phosphoenolpyruvate (K(m) = 1.6 mM), adenosine 5'-triphosphate (K(m) = 0.5 mM), adenosine diphosphate (K(m) = 1.5 mM), and adenosine 5'-monophosphate (K(m) = 3.0 mM) are competitive inhibitors with respect to NAD(+). The molecular weight as estimated from sucrose density centrifugation and molecular sieve chromatography is 1.1 x 10(5). Sodium dodecyl sulfate gel electrophoresis indicates that the enzyme is composed of two similar subunits of approximately 6 x 10(4) molecular weight. The intracellular levels of glucose-6-phosphate, NAD(+), and NADP(+) were measured and found to be approximately 1 mM, 0.9 mM, and 0.2 mM, respectively, during logarithmic growth. From a consideration of the substrate pool sizes and types of inhibitors, we conclude that this single constitutive enzyme may function in two roles in the cell-NADH production for energetics and NADPH production for reductive biosynthesis.     

Enzymic synthesis of lignin precursors. Purification and properties of a cinnamoyl-CoA: NADPH reductase from cell suspension cultures of soybean (Glycinemax).

A cinnamoyl-coenzyme A reductase catalyzing the NADPH-dependent reduction of substituted cinnamoyl-CoA thiol esters to the corresponding cinnamaldehydes was isolated from cell suspension cultures of soybean (Glycine max L. var. Mandarin). A 1660-fold purification of the enzyme was achieved by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, hydroxyapatite and Sephadex G-100 and affinity chromatography on 5'-AMP-Sepharose. The apparent molecular weight of the reductase was found to be about 38 000 on the basis of the elution volume from a Sephadex G-100 column. Maximum rate of reaction was observed between pH 6.0 and 6.2 in 0.1-0.2 M citrate buffer at 30 degrees C. The enzyme was markedly inhibited by thiol reagents. The reductase showed a high degree of specificity for cinnamoyl-CoA esters. Feruloyl-CoA was the substrate with the lowest Km value (73 muM) and highest V (230 nkat/mg) followed by 5-hydroxy-feruloyl-CoA, sinapoyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA and cinnamoyl-CoA. No reaction took place with acetyl-CoA. The Km value for NADPH varied with the type of substrate. Km values of 28, 120, and 290 muM were found with feruloyl-CoA, sinapoyl-CoA, and p-coumaroyl-CoA, respectively. The rate of reaction observed with NADH was only about 5% of that found with NADPH. The reaction products CoASH and NADP+ inhibited the reaction. The Ki values were in the range of 0.5-1 mM and the inhibition was of a noncompetitive (mixed) type. The role of the reductase in the biosynthesis of lignin precursors is discussed.     

Glucose-6-phosphate dehydrogenase. Purification and partial characterization.

Glucose-6-phosphate dehydrogenase has been purified 1000-fold from pig liver. This enzyme exists as an active dimer of molecular weight 133,000 and an inactive monomer of molecular weight 67,500. The pH of maximum activity is 8.5 and the ionic strength maximum is 0.1 to 0.5 M. Glucose-6-phosphate dehydrogenase is highly specific for NADP+ and glucose 6-phosphate. Apparent Km values of 3.6 muM and 5.4 muM were obtained for glucose 6-phosphate and NADP+. This enzyme is located almost entirely within the soluble portion of the cellular cytoplasm.     

Human Brain 6-Phosphogluconate Dehydrogenase: Purification and Kinetic Properties

6-Phosphogluconate dehydrogenase has been purified from human brain to a specific activity of 22.8 U/mg protein. The molecular weight was 90,000. At low ionic strengths enzyme activity increased, due to an increase in Vmax and a decrease in Km for 6-phosphogluconate, and activity subsequently decreased as the ionic strength was increased (above 0.12). Both 6-phosphogluconate and NADP+ provided good protection against thermal inactivation, with 6-phosphogluconate also providing considerable protection against loss of activity caused by p-chloromercuribenzoate and iodoacetamide. Initial velocity studies indicated the enzyme mechanism was sequential. NADPH was a competitive inhibitor with respect to NADP+, and the Ki values for this inhibition were dependent on the concentration of 6-phosphogluconate. Product inhibition by NADPH was noncompetitive when 6-phosphogluconate was the variable substrate, whereas inhibition by the products CO2 and ribulose 5-phosphogluconate and NADP+ were varied. In totality these data suggest that binding of substrates to the enzyme is random. CO2 and ribulose 5-phosphate are released from the enzyme in random order with NADPH as the last product released.     

Binding of coenzyme and substrate and coenzyme analogues to 6-phosphogluconate dehydrogenase from sheep liver. An X-ray study at 0.6 nm resolution.

The analogues of the coenzyme NADP+, nicotinamide--8-bromo-adenine dinucleotide phosphate (Nbr8ADP+) and 3-iodopyridine--adenine dinucleotide phosphate (io3PdADP+), were prepared. Nbr8ADP+ was found to be active in the hydrogen transfer adn io3PdADP+ is a coenzyme competitive inhibitor for 6-phosphogluconate dehydrogenase. The binding of NADP+, NADPH and NADPH together with 6-phosphogluconate as well as that of both analogues to crystals of the enzyme 6-phosphogluconate dehydrogenase has been investigated at 0.6-nm resolution using difference electron density maps. The molecules bind in a similar position in a cleft in the enzyme subunit distant from the dimer interface. The orientation of the coenzyme in the site has been determined from the io3PdADP+ -NADP+ difference density. The ternary complex difference density extends beyond that of the nicotinamide moiety of the coenzyme and tentatively indicates substrate binding. No clear identification of the bromine atom of Nbr8ADP+ can be made. However, the analogue is bound more deeply in the cleft than is NADP+. The NADPH density is the most clearly defined and has thus been used to fit a molecular model using an interactive graphics system, checking for preferred geometry. A possible conformation is presented which is significantly different from that of NAD+ in the lactate dehydrogenase ternary complex.     

Oxidized and reduced nicotinamide–adenine dinucleotide phosphate in tissue suspensions of rat liver

1. The concentrations of NADP and NADPH(2) in homogenates of rat liver (expressed as mug./g. wet wt. of tissue homogenized) were compared with values obtained from intact samples of liver taken from the same female rat. With 0.25m-sucrose alone as the suspending medium, or in combination with tris buffer or 0.01-0.1m-nicotinamide, considerable decreases in the sum of the NADP+NADPH(2) concentrations were occasionally observed during 30min. storage of homogenates at 0 degrees . However, addition of 0.5m-nicotinamide+5mm-tris buffer to 0.25m-sucrose for use as a suspending medium maintained the sum of the NADP+NADPH(2) concentrations in homogenates at the level found in intact tissue for at least 30min. at 0 degrees . 2. The effects of freezing intact tissue and homogenates in liquid nitrogen before the extraction of NADP and NADPH(2) were studied. Freezing alone appears to convert a significant amount (approx. 30%) of liver NADPH(2) into an equivalent amount of NADP in intact tissue. This is discussed in terms of the ;bound NADP' reported by Burch, Lowry & Von Dippe (1963). 3. The intracellular distributions of NADP and NADPH(2) in intracellular fractions of rat liver were studied by using a modified centrifuging scheme that allows extraction of the isolated fractions to be performed within 45min. of killing the animal. Approx. 50% of the total NADP+NADPH(2) was found in the large-particle fractions and the remaining 50% was mostly in the soluble fraction of the cell. 4. Further investigations are reported on the nature of ;bound NADP' in rat liver. Most of this material appears associated with the ;nuclear' (containing nuclei, debris, erythrocytes etc.) or large-mitochondrial fractions, or both, obtained by low-speed centrifuging of rat-liver homogenates. 5. Although in some experiments the variations produced in the concentration of NADPH(2) present in large-particle fractions were followed by similar changes in that of ;bound NADP', in other cases no such direct relationship was obtained. Addition of phenazine methosulphate, for example, consistently lowered the concentration of NADPH(2) yet raised the concentration of ;bound NADP' in rat-liver mitochondrial fractions.     

Regulatory characteristics of amino acid transport in newborn rat renal cortex cells.

The uptaken of alpha-aminoisobutyric acid by slices of kidney cortex from newborn rats is enhanced by a preliminary incubation of the tissue in buffer at 37 degrees C. This effect is abolished by anaerobiosis, the presence of dinitrophenol or the removal of Na+ during the preliminary incubation. Cycloheximide (50 muM) and purimycin (1 mM) as well as alpha-aminoisobutyric acid, glycine and proline (5 mM) in the preincubation buffer also abolish the effect, while actinomycin D (0.8 muM) partially the phenomenon indicates that the enhanced uptake is due to an increased entry rate into the cells without a change in effux. There is no alteration in the apparent transport Km but an increase in the V for entry. The effect is dependent on tissue age being observed between birth and 22 days, after which there is a decrease in response to preliminary incubation with no effect seen in adult tissues.     

Purification and characterization of rat lens pyrroline-5-carboxylate reductase

delta 1-Pyrroline-5-carboxylate reductase (L-proline:NAD(P)+ 5-oxidoreductase, EC 1.5.1.2) has been purified from rat lens and biochemically characterized. Purification steps included ammonium sulfate fractionation, affinity chromatography on Amicon Matrex Orange A, and gel filtration with Sephadex G-200. These steps were carried out at ambient temperature (22 degrees C) in 20 mM sodium phosphate/potassium phosphate buffer (pH 7.5) containing 10% glycerol, 7 mM mercaptoethanol and 0.5 mM EDTA. The enzyme, purified to apparent homogeneity, displayed a molecular weight of 240 000 by gel chromatography and 30 000 by SDS-polyacrylamide gel electrophoresis. This suggests that the enzyme is composed of eight subunits. The purified enzyme displays a pH optimum between 6.5 and 7.1 and is inhibited by heavy metal ions and p-chloromercuribenzoate. Kinetic studies indicated Km values of 0.62 mM and 0.051 mM for DL-pyrroline-5-carboxylate as substrate when NADH and NADPH respectively were employed as cofactors. The Km values for the cofactors NADH and NADPH with DL-pyrroline-5-carboxylate as substrate were 0.37 mM and 0.006 mM, respectively. With L-pyrroline-5-carboxylate as substrate, Km values of 0.21 mM and 0.022 mM were obtained for NADH and NADPH, respectively. Enzyme activity is potentially inhibited by NADP+ and ATP, suggesting that delta 1-pyrroline-5-carboxylate reductase may be regulated by the energy level and redox state of the lens.     

Glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from Lactobacillus casei: responses with different modulators

Glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) were separated and partially purified from glucose-grown cells of Lactobacillus casei. The enzymes had similar pH optima, thermosensitivity and molecular weights. They had different net charges and their pI values were 5.38 and 4.52, respectively. Histidine, arginine, lysine and cysteine residues were essential for the activity of G6PD, and all the above amino acids with the exception of lysine were required for 6PGD activity. Mg2+ activated 6PGD up to 15 mM concentration, above which it was inhibitory. It had no effect on G6PD activity. G6PD was specific for NADP+, but 6PGD showed some activity with NAD+ as the cofactor, although it was essentially NADP(+)-preferring. Both the enzymes, were inhibited by NADPH. 6PGD was also inhibited by its product, ribulose 5-phosphate. ATP inhibited 6PGD only at subsaturating concentrations of NADP+. The inhibition was sigmoidal in the absence of Mg2+ and hyperbolic in its presence.