Purification and biochemical studies of lactate dehydrogenase-X from mouse

Summary Lactate dehydrogenase-X from testes of several rodent species was purified to homogeneity by an 8-(6-aminohexyl)-amino-AMP-Sepharose affinity column. In the case of mouse, the testicle extracts was first heated to 60° for fifteen minutes before the passage through the affinity column. A biospecific elution with reduced NAD⁺-pyruvate adduct resulted in a homogeneous preparation of lactate dehydrogenase-X. A similar procedure was also employed for the purification of lactate dehydrogenase-X from hamster, guinea pig and rat. After purification by affinity chromatography, lactate dehydrogenase-X was separated from residual somatic lactate dehydrogenase isozymes by DEAF-Sephadex chromatography. Adenosine, AMP, ADP, and ADP-ribose were shown to be coenzyme-competitive inhibitors of lactate dehydrogenase-X. The effectiveness of binding of these compounds increased with the size of the adenosine derivatives employed. Multiple inhibition analysis suggested that these compounds are interacting with the same region of coenzyme-binding site as shown by the mutual exclusion of one another from binding to the enzyme. The data suggest that the binding of coenzyme to the enzyme occurs through interactions involving the adenosine moiety and pyrophosphate grouping. Fluorescence spectroscopy was employed for the study of the mechanism of action of mouse lactate dehydrogenase-X. Both oxidized and reduced coenzymes induced significant quenching of protein fluorescence. Significant enhancements of NADH fluorescence and protein energy transfer were observed upon the addition of lactate dehydrogenase-X to the coenzyme solution. In the presence of lactate dehydrogenase-X and NAD⁺, the addition of pyruvate or -ketovalerate resulted in a time-dependent quenching of protein fluorescence and an increase in absorbance at 325 nm indicating the formation of a ternary complex. The results of this study suggest a similar molecular mechanism for different lactate dehydrogenase isozymes.To whom inquires should be addressed.NIH visiting fellowThis purification procedure is currently being adopted by Professor Erwin Goldberg at Northwestern University, Evanston, Ill. for large scale preparation of mouse LDH-X.     

Tricyclohexyltin hydroxide effects on cationic and substrate activation kinetics of beta-adrenergic–stimulated cardiac Ca2+-ATPase

Previous studies from this laboratory have indicated that tricyclohexyltin hydroxide (Plictran) is a potent inhibitor of both basal- and isoproterenol-stimulated cardiac sarcoplasmic reticulum (SR) Ca²⁺-ATPase, with an estimated IC-50 of 2.5 × 10^?8M. The present studies were initiated to evaluate the mechanism of inhibition of Ca²⁺-ATPase by Plictran. Data on substrate and cationic activation kinetics of Ca²⁺-ATPase indicated alteration of V_max and K_m by Plictran (1 and 5×10^?8M), suggesting a mixed type of inhibition. The beta-adrenergic agonist isoproterenol increased V_max of both ATP- and Ca²⁺-dependent enzyme activities. However, the K_m of enzyme was decreased only for Ca²⁺ Plictran inhibited isoproterenol-stimulated Ca²⁺-ATPase activity by altering both and V_max and K_m of ATP as well as Ca²⁺-dependent enzyme activities, suggesting that after binding to a single independent site, Plictran inhibits enzyme catalysis by decreasing the affinity of enzyme for ATP as well as for Ca²⁺ Preincubation of enzyme with 15 μM cAMP or the addition of 2mM ATP to the reaction mixture resulted in slight activation of Plictran-inhibited enzyme. Pretreatment of SR with 5 × 10^?7M propranolol and 5 × 10^?8M Plictran resulted in inhibition of basal activity in addition to the loss of stimulated activity. Preincubation of heart SR preparation with 5 × 10^?5M coenzyme A in combination with 5 × 10^?8M Plictran partly restored the beta-adrenergic stimulation. These results suggest that some critical sites common to both basal- and beta-adrenergic-stimulated Ca²⁺-ATPase are sensitive to binding by Plictran, and the resultant conformational change may lead to inhibition of beta-adrenergic stimulation.     

Bilirubin inhibition of enzymes involved in the mitochondrial malate-aspartate shuttle

The effect of increasing bilirubin concentrations upon the catalytic activity of a series of dehydrogenases and aminotransferases was examined. The particular enzymes were chosen to examine the effect of bilirubin upon the activity of enzymes responsible for the indirect transfer of reducing equivalents across the inner mitochondrial membrane. Malate dehydrogenase was inhibited at very low concentrations of bilirubin and showed competitive inhibition with respect to coenzyme of 2 μM, while the cytosolic form of this enzyme exhibited a 15 μM inhibition constant. Cytosolic glycerol-3-phosphate dehydrogenase was not appreciably inhibited by bilirubin. Both the mitochondrial and cytosolic forms of aspartate aminotransferase showed moderate competitive bilirubin inhibition with respect to substrates with a K_i of 30 μM with respect to 2-oxoglutarate and a K_i of 80 μM with respect to aspartate. Preincubation studies indicated that inhibition was reversible for all enzymes examined. These results are interpreted in terms of the inhibition of the malate-aspartate shuttle by relatively low concentrations of bilirubin.     

The Characteristics of Arylamine N-Acetyltransferase in Pseudomonas aeruginosa

N-Acetyltransferase (NAT), responsible for bioactivation and detoxification of arylamines, has been demonstrated to be widely distributed in many organisms ranging from humans to microorganisms. Using high performance liquid chromatography (HPLC) to analyze NAT activity in bacteria, the authors found that Pseudomonas aeruginosa exhibited high NAT activity with 2-aminofluorene (2-AF) as substrate. Characteristics of this bacterial NAT were further investigated. The N-acetylation catalyzed by this enzyme is an acetyl coenzyme A (AcCoA)-dependent reaction. As the concentration of AcCoA in the reaction mixture was increased, the apparent K _m and V _max for 2-AF increased. The K _m and V _max were 0.504 ± 0.056 mM and 31.92 ± 3.23 nmol/min/mg protein, respectively, for the acetylation of 2-AF with 0.5 mM AcCoA. The optimum pH for the enzyme activity was estimated to be around 8.5. It was active at a temperature range from 5°C to 55°C, with maximum activity at 37°C. The enzyme activity was inhibited by divalent metal ions including Cu⁺⁺, Fe⁺⁺, Zn⁺⁺, Ca⁺⁺, Co⁺⁺, Mn⁺⁺, and Mg⁺⁺, suggesting that a sulfhydryl group is involved in the N-acetylation activity. The three chemical modification agents, iodoacetamide, phenylglyoxal, and diethylpyrocarbonate, all exhibited a dose-, time-, and temperature-dependent inhibition effect. Preincubation of the NAT with AcCoA provided significant protection against the inhibition of iodoacetamide and diethylpyrocarbonate, but only partial protection against the inhibition of phenylglyoxal. These results indicate that cysteine, histidine, and arginine residues are essential for this bacterial enzyme activity, and the first two are likely to reside on the AcCoA binding site, but arginine residue may be located only near the AcCoA binding site. Our data demonstrate that P. aeruginosa possesses highly active N-acetyltransferase which shares a similar catalytic mechanism as that of higher organisms. These findings are very helpful for further investigating the role of arylamine NAT in this bacterial species. Received: 29 August 1997 / Accepted: 23 December 1997     

Isolation and properties of lactate dehydrogenase from germinating pea plants

Lactate dehydrogenase (LDH) was isolated from pea seedlings by means of protamine sulphate and (NH₄)₂SO₄ fractionation and chromatography on DEAE-cellulose and Sephadex G-150. The enzyme had a MW of ca 145 500. The kinetic properties studied were the lactate oxidation pH optimum (9·1) and the pyruvate reduction pH optimum (7·1). K_m values were determined for four natural substrates (Lactate, pyruvate, NAD⁺ and NADH) and for other acids (glycollate, α-ketoglutarate and glyoxylate). The K_i value was determined for p-chloromercuribenzoate (PCMB) which is a noncompetitive inhibitor of LDH from pea plants, and the course of irreversible inhibition of the enzyme by iodoacetamide (IA) and n-ethylmaleimide (NEMI) was studied. Preincubation of LDH with the coenzyme protects against PCMB inhibition, indicating the important role of the sulfhydryl group in the active site.     

Inhibition of proton-translocating ATPases of Streptococcus mutans and Lactobacillus casei by fluoride and aluminum

One of the major effects of fluoride on oral bacteria is a reduction in acid tolerance, and presumably also in cariogenicity. The reduction appears to involve transport of protons across the cell membrane by the weak acid HF to dissipate the pH gradient, and also direct inhibition of the F₁F₀, proton-translocating ATPases of the organisms, especially for Streptococcus mutans. This direct inhibition by fluoride was found to be dependent on aluminum. The dependence on aluminum was indicated by the protection against fluoride inhibition afforded by the Al-chelator deferoxamine and by loss of protection after addition of umolar levels of Al³⁺, which were not inhibitory for the enzyme in the absence of fluoride. The F₁ form of the enzyme dissociated from the cell membrane previously had been found to be resistant to fluoride in comparison with the F₁F₀ membrane-associated form. However, this difference appeared to depend on less aluminum in the F₁ preparation in that the sensitivity of the F₁ enzyme to fluoride could be increased by addition of umolar levels of Al³⁺. The effects of Al on fluoride inhibition were apparent when enzyme activity was assayed in terms of phosphate release from ATP or with an ATP-regenerating system containing phosphoenolpyruvate, pyruvate kinase, NADH and lactic dehydrogenase. Also, Be²⁺ but not other metal cations, e.g. Co²⁺, Fe²⁺, Fe³⁺, Mn², Sn²⁺, and Zn²⁺, served to sensitize the enzyme to fluoride inhibition. The differences in sensitivities of enzymes isolated from various oral bacteria found previously appeared also to be related to differences in levels of Al. Even the fluoride-resistant enzyme of isolated membranes of Lactobacillus casei ATCC 4646 could be rendered fluoride-sensitive through addition of Al³⁺. Thus, the F₁F₀ ATPases of oral bacteria were similar to E₁E₂ ATPases of eukaryotes in being inhibited by Al-F complexes, and the inhibition presumably involved formation of ADP-Al-F _inf3 ^sup- complexes during catalysis at the active sites of the enzymes.     

Immunological Studies on Antisera of Mice Immunized with Dried or DEPC-treated Ehrlich Ascites Tumor Cells

Intracellular arylsulfatases from Klebsiella aerogenes W70 cells grown in methionine medium (M enzyme) and inorganic sulfate medium containing tyramine (T enzyme) were purified respectively by fractionation with (NH₄)₂SO₄, followed by successive chromatographies on DEAE cellulose, hydroxylapatite, Sephadex G-100 and DEAE Sephadex A-25. On polyacrylamide gel electrophoresis, the two enzymes gave single bands with the same mobilities. Molecular weights of both, determined by SDS gel electrophoresis and by Sephadex G-100 chromatography, were 47,000 and 45,000, respectively. Their activities were maximal at pH 7.5. The affinities of the enzymes (M and T enzymes) for their substrate (Km) and the maximum velocity of hydrolysis (V_max) were enhanced by addition of electron withdrawing substituents. The enzymes were inhibited by inorganic phosphate, cyanide, hydroxylamine and tyramine. The inhibition by tyramine was competitive (Ki = 1.0 × 10^?4 m). These results show that the two enzymes were identical. This was confirmed by the fact that mutant strains, which were unable to synthesize arylsulfatase when grown with methionine, could also not synthesize the enzyme when grown with tyramine.     

STUDY ON THE INHIBITION OF BRAIN GLUTAMATE DECARBOXYLASE BY PYRIDOXAL PHOSPHATE OXIME-O-ACETIC ACID

The kinetics of the inhibition of mouse brain glutamate decarboxylase by pyri-doxaI-5′-phosphate oxime-O-acetic acid (PLPOAA) was studied. The inhibition was noncompetitive with regard to glutamic acid; it could be partially reversed by pyridoxal phosphate, but only when the concentration of the latter in the incubation medium was higher than that of pyridoxal-5′-phosphate oxime-O-acetic acid. The inhibition produced by aminooxyacetic acid, which is remarkably greater than that produced by PLPOAA, was also partially reversed only when an excess of pyridoxal phosphate was added. Both in the presence and in the absence of a saturating concentration of pyridoxal phosphate, the activity of the enzyme was decreased by PLPOAA at a 10^?4m concentration to a value of about 50 per cent of the control value obtained without added coenzyme. This activity could not be further reduced even when PLPOAA concentration was increased to 5 × 10^?3m . This same minimal activity of glutamate decarboxylase was obtained after dialysis of the enzymic preparation, or after incubation with glutamic acid in the cold followed by filtration through Sephadex G-25. The addition of pyridoxal phosphate to the dialysed or glutamic acid-treated enzyme restored the activity to almost the control values. PLPOAA did not affect the activity of glutamate decarboxylase from E. coli or that of DOPA decarboxylase and GABA transaminase from mouse brain. To account for the results obtained it is postulated that brain glutamate decarboxylase has two types of active site, one with firmly bound, non-dialysable pyridoxal phosphate and the other with loosely bound, dialysable coenzyme; PLPOAA behaves as a weak inhibitor probably because it can combine mainly with the loosely bound coenzyme site, while aminooxyacetic acid is a potent inhibitor probably because it can block both the ‘loosely bound coenzyme’ and the ‘firmly bound coenzyme’ sites.     

Continuous enzymatic transformation in an enzyme membrane reactor with simultaneous NAD(H) regeneration

Multienzyme reaction systems with simultaneous coenzyme regeneration have been investigated in a continuously operated membrane reactor at bench scale. NAD(H) covalently bound to polyethylene glycol with a molecular weight of 10⁴ [PEG-10,000-NAD(H)] was used as coenzyme. It could be retained in the membrane reactor together with the enzymes. L -leucine dehydrogenase (LEUDH) was used as catalyze for the reductive amination of α-ketoisocaproate (2-oxo-4-methylpentanoic acid) to L -leucine. Format dehydrogenease (FDH) was used for the regeneration of NADH. Kinetic experiments were carried out to obtain data which could be used in a kinetic model in order to predict the performance of an enzyme membrane reactor for the continuous production of L -leucine. The kinetic constants V_max and K_m of enzymes are all in the same range regardless of whether native NAD(H) or PEG-10,000-NAD(H) is used as coenzyme. L -leucine was produced continuously out of α-ketoisocaproate for 48 days; a maximal conversion of 99.7% was reached. The space-time yield was 324 mmol/L day (or 42.5 g/L day).     

Alteration of coenzyme specificity in halophilic NAD(P) glucose dehydrogenase by site-directed mutagenesis

Structural analysis of glucose dehydrogenase from Haloferax mediterranei revealed that the adenosine 2′-phosphate of NADP⁺ was stabilized by the side chains of Arg207 and Arg208. To investigate the structural determinants for coenzyme specificity, several mutants involving residues Gly206, Arg207 and Arg208 were engineered and kinetically characterized. The single mutants G206D and R207I were less efficient with NADP⁺ than the wild type, and the double and triple mutants G206D/R207I and G206D/R207I/R208N showed no activity with NADP⁺.In the single mutant G206D, the relation k_cat/K_NAD⁺ was 1.6 times higher than in the wild type, resulting in an enzyme that preferred NAD⁺ over NADP⁺. The single mutation was sufficient to modify coenzyme specificity, whereas other dehydrogenases usually required more than one or two mutations to change coenzyme specificity. However, the highest reaction rates were reached with the double mutant G206D/R207I and with coenzyme NAD⁺, where the k_cat was 1.6 times higher than the k_cat of the wild-type enzyme with NADP⁺. However, catalytic efficiency with NAD⁺ was lower, as the K_m value for coenzyme was 77 times higher than the wild type with NADP⁺.     

Reduction of acetophenone to R(+)-phenylethanol by a new alcohol dehydrogenase from Lactobacillus kefir

Summary A new alcohol dehydrogenase catalysing the enantioselective reduction of acetophenone to R(+)-phenylethanol was found in a strain of Lactobacillus kefir. A 70-fold enrichment of the enzyme with an overall yield of 76% was obtained in two steps. The addition of Mg²⁺ ions was found to be necessary to prevent rapid deactivation. The enzyme depends essentially on NADPH and was inactive when supplied with NADH as the coenzyme. Important enzymological data of the dehydrogenase are: K _m (acetophenone) 0.6 mM, K _m (NADPH) 0.14 mM, and a pH optimum for acetophenone reduction at 7.0. Addition of EDTA leads to complete deactivation of the enzyme activity. Added iodoacetamide or p-hydroxymercuribenzoate cause only slight inhibition, revealing that the active centre of the enzyme contains no essential SH-group. Besides acetophenone several other aromatic and long-chain aliphatic secondary ketones are substrates for this enzyme. Batch production of phenylethanol was examined using three different methods for the regeneration of NADPH: glucose/glucose dehydrogenase, glucose-6-phosphate/glucose-6-phosphate dehydrogenase, and isopropanol.     

Inhibition of secretory phospholipase A2 enzyme by bilirubin: A new role as endogenous anti-inflammatory molecule

Bilirubin is a powerful antioxidant that suppresses the inflammatory process. However its interaction with proinflammatory PLA₂ enzyme is not known. Inhibition of several secretory phospholipase A₂ (sPLA₂) enzyme activities by bilirubin was studied using ¹⁴C-oleate labeled Escherichia coli as substrate. Bilirubin inhibits purified sPLA₂ enzyme from Vipera russellii and Naja naja venom and partially purified sPLA₂ enzymes from human ascitic fluid, pleural fluid and normal serum in a dose dependent manner. IC₅₀ values calculated for these enzymes ranges from 1.75 to 10.5 μM. Inflammatory human sPLA₂ enzymes are more sensitive to inhibition by bilirubin than snake venom sPLA₂s. Inhibition of sPLA₂ activity by bilirubin is independent of calcium concentration. Increasing substrate concentration (upto 180 nmol) did not relieve the inhibition of sPLA₂ by bilirubin and it is irreversible. Bilirubin quenched the relative fluorescence intensity of sPLA₂ in a dose dependent manner in the same concentration range at which in vitro sPLA₂ inhibition was observed. In the presence of bilirubin, apparent shift in the far UV-CD spectra of sPLA₂ was observed, indicating a direct interaction with the enzyme. Inhibition of sPLA₂ induced mouse paw edema by bilirubin confirms its sPLA₂ inhibitory activity in vivo also. These findings indicate that inhibition of sPLA₂ by bilirubin is mediated by direct interaction with the enzyme and bilirubin may act as an endogenous regulator of sPLA₂ enzyme activity.     

The putative coenzyme B12-dependent methylmalonyl-CoA mutase from potatoes is a phosphatase

The reported presence of a coenzyme B₁₂-dependent methylmalonyl-CoA mutase in potatoes has been reexamined. The enzyme converting methylmalonyl-CoA was purified to electrophoretic homogeneity. Examination of the reaction product by ¹H, ³¹P NMR and mass spectrometry revealed that it was methylmalonyl-3′-dephospho-CoA. The phosphatase enzyme needs neither coenzyme B₁₂ nor S-adenosylmethionine as a cofactor.     

MECHANISMS OF LYSOSOMAL ENZYME RELEASE FROM HUMAN LEUKOCYTES : I. Effect of Cyclic Nucleotides and Colchicine

In order to study mechanisms underlying selective enzyme release from human leukocytes during phagocytosis, the effects were studied of compounds which affect microtubule integrity or the accumulation of cyclic nucleotides. Human leukocytes selectively extrude lysosomal enzymes (β-glucuronidase) from viable cells during phagocytosis of zymosan or immune complexes, or upon encounter with immune complexes dispersed along a non-phagocytosable surface such as a millipore filter. In each circumstance, lysosomal enzyme release was reduced by previous treatment of cells with pharmacological doses of drugs which disrupt microtubules (e.g. 10^-3–10^-5 M colchicine) or with agents which affect accumulation of adenosine 3'5'-monophosphate (cAMP) (e.g. 10^-3 M cyclic nucleotides and 2.8 x 10^-4–2.8 x 10^-6 M prostaglandin E (PGE) and A (PGA) compounds). Preincubation of cells with 5 µg/ml cytochalasin B resulted in complete inhibition of zymosan ingestion, but not of adherence of zymosan particles to plasma membranes or selective enzyme release. In this system, in which enzyme release was independent of particle uptake, preincubation of cells with colchicine, vinblastine, dibutyryl cAMP, or PGE₁ also reduced extrusion of lysosomal enzymes. When cell suspensions were incubated with membrane-lytic crystals of monosodium urate (MSU), cytoplasmic as well as lysosomal enzymes were released with subsequent death of the cells. However, enzyme release followed phagocytosis of crystals (as measured by enhanced C-1 oxidation of glucose) and was due to "perforation from within" of the lysosomal membrane, rather than lysis by crystals of the plasma membrane. Enzyme release after MSU ingestion was also reduced when cells were treated with pharmacological doses of the test agents. When cells were killed by Triton X-100, acting on the plasma membrane, C-1 oxidation of glucose was abolished and enzyme release could not be inhibited pharmacologically. These observations suggest that lysosomal enzyme release from human phagocytes can be an active process which accompanies plasma membrane stimulation, is independent of cell death, and may be controlled by cyclic nucleotides and agents which affect microtubules.     

Reductive activation of the corrinoid-containing enzyme involved in methyl group transfer between methyl-tetrahydromethanopterin and coenzyme M inMethanosarcina barkeri

The conversion of methyl-tetrahydromethanopterin to methylcoenzyme M inMethanosarcina barkeri is catalyzed by two enzymes: an enzyme with a bound corrinoid, which becomes methylated during the reaction and an enzyme which tranfers the methyl group from this corrinoid to coenzyme M. As in the similar methyltransfer reaction inMethanobacterium thermoautotrophicum the corrinoid enzyme inM. barkeri needs to be activated by H₂ and ATP. ATP can be replaced by Ti(III)citrate or CO.     

Engineering of a matched pair of xylose reductase and xylitol dehydrogenase for xylose fermentation by Saccharomyces cerevisiae

Metabolic engineering of Saccharomyces cerevisiae for xylose fermentation has often relied on insertion of a heterologous pathway consisting of nicotinamide adenine dinucleotide (phosphate) NAD(P)H-dependent xylose reductase (XR) and NAD⁺-dependent xylitol dehydrogenase (XDH). Low ethanol yield, formation of xylitol and other fermentation by-products are seen for many of the S. cerevisiae strains constructed in this way. This has been ascribed to incomplete coenzyme recycling in the steps catalyzed by XR and XDH. Despite various protein-engineering efforts to alter the coenzyme specificity of XR and XDH individually, a pair of enzymes displaying matched utilization of NAD(H) and NADP(H) was not previously reported. We have introduced multiple site-directed mutations in the coenzyme-binding pocket of Galactocandida mastotermitis XDH to enable activity with NADP⁺, which is lacking in the wild-type enzyme. We describe four enzyme variants showing activity for xylitol oxidation by NADP⁺ and NAD⁺. One of the XDH variants utilized NADP⁺ about 4 times more efficiently than NAD⁺. This is close to the preference for NADPH compared with NADH in mutants of Candida tenuis XR. Compared to an S. cerevisiae-reference strain expressing the genes for the wild-type enzymes, the strains comprising the gene encoding the mutated XDH in combination a matched XR mutant gene showed up to 50% decreased glycerol yield without increase in ethanol during xylose fermentation.     

Pathways of autotrophic CO2 fixation and of dissimilatory nitrate reduction to N2O in Ferroglobus placidus

The strictly anaerobic Archaeon Ferroglobus placidus was grown chemolithoautotrophically on H₂ and nitrate and analyzed for enzymes and coenzymes possibly involved in autotrophic CO₂ fixation. The following enzymes were found [values in parentheses = μmol min^–1 (mg protein)^–1]: formylmethanofuran dehydrogenase (0.2), formylmethanofuran:tetrahydromethanopterin formyltransferase (0.6), methenyltetrahydromethanopterin cyclohydrolase (10), F₄₂₀-dependent methylenetetrahydromethanopterin dehydrogenase (1.5), F₄₂₀-dependent methylenetetrahydromethanopterin reductase (0.4), and carbon monoxide dehydrogenase (0.1). The cells contained coenzyme F₄₂₀ (0.4 nmol/mg protein), tetrahydromethanopterin (0.9 nmol/ mg protein), and cytochrome b (4 nmol/mg membrane protein). From the enzyme and coenzyme composition of the cells, we deduced that autotrophic CO₂ fixation in F. placidus proceeds via the carbon monoxide dehydrogenase pathway as in autotrophically growing Archaeoglobus and Methanoarchaea species. Evidence is also presented that cell extracts of F. placidus catalyze the reduction of two molecules of nitrite to 1 N₂O with NO as intermediate (0.1 μmol N₂O formed per min and mg protein), showing that – at least in principle –F. placidus has a denitrifying capacity. Received: 23 August 1996 / Accepted: 6 November 1996     

The effect of nitrogen deficiency on the growth and metabolism of Lemna minor L.

When Lemna is deprived of nitrogen, growth and respiration decrease and the pattern of ¹⁴CO₂ release from [1-¹⁴C]glucose and [6-¹⁴C]glucose is consistent with a relatively strong inhibition of glycolysis. Protein degradation is enhanced but the concentration of free amino acid decreases. It is argued that the biological significance of the increased protein degradation does not lie in its contribution to respiration but as a mechanism to replace one set of enzymes adapted to a particular environmental condition (high nitrogen) with another set of enzymes adapted for low nitrogen in the environment. The change in enzyme pattern associated with the change from high to zero nitrogen in the growth medium has been examined for nine enzymes. The changes in activity observed are consistent with the observed apparent inhibition of glycolysis during nitrogen starvation, but do not explain the inhibition of the pentose phosphate pathway.     

Alteration of Coenzyme Specificity of Lactate Dehydrogenase from Thermus thermophilus by Introducing the Loop Region of NADP(H)-Dependent Malate Dehydrogenase

Previously we found that replacement of seven amino acid residues in a loop region markedly shifted the coenzyme specificity of malate dehydrogenase from NAD(H) toward NADP(H). In the present study, we replaced the seven amino acid residues in the corresponding region of an NAD(H)-dependent lactate dehydrogenase with those of NADP(H)-dependent malate dehydrogenase, and examined the coenzyme specificity of the resulting mutant enzyme. Coenzyme specificity was significantly shifted by 399-fold toward NADPH when k _cat?K _m ^coenzyme was used as the measure of coenzyme specificity. The effect of the replacements on coenzyme specificity is discussed based on in silico simulation of the three-dimensional structure of the lactate dehydrogenase mutant.     

Ribulose bisphosphate carboxylase and glycollate oxidase from jack pine: Effects of sulphur dioxide fumigation

Ribulose bisphosphate (RuBP) carboxylase and glycollate oxidase were partially purified from jack pine (Pinus banksiana Lamb.) needles. Preincubation of RuBP carboxylase with HCO₃^? and Mg²⁺ markedly stimulated its activity. RuBP carboxylase showed hyperbolic reaction kinetics with respect to HCO₃^?, Mg²⁺, and RuBP. Both SO₃^2- and SO₄^2- inhibited RuBP carboxylase, but SO₃^2- was more inhibitory than SO₄^2-. The SO₃^2- inhibition was competitive with respect to HCO₃^? (whether SO₃^2- was present during activation or was added to the activated enzyme), while the SO₄^2- inhibition was non-competitive with respect to HCO₃^?. Glycollate oxidase was inhibited more severely by low concentrations of SO₃^2- than by SO₄^2-. Fumigation of jack pine seedlings with 0.34 ppm sulphur dioxide for 24 and 48 hr produced a considerable decline in the activities of these enzymes, but 1 hr of fumigation produced no effect. During the longer exposures the sulphur content of the needles increased considerably, although the needles showed no visible injury. It is suggested that the accumulation of SO₃^2- and SO₄^2- in the needles following sulphur dioxide exposure influenced the enzyme activities.