A thermostable shikimate 5-dehydrogenase from the archaeon Archaeoglobus fulgidus

Shikimate 5-dehydrogenase (SKDH; EC 1.1.1.25) catalyzes the reversible reduction of 3-dehydroshikimate to shikimate and is a key enzyme in the aromatic amino acid biosynthesis pathway. The shikimate 5-dehydrogenase gene, aroE, from Archaeoglobus fulgidus was cloned and overexpressed in Escherichia coli. The recombinant enzyme purified as a homodimer and yielded a maximum specific activity of 732 U/mg at 87 degrees C (with NADP+ as coenzyme). Apparent Km values for shikimate, NADP+, and NAD+ were estimated at 0.17+/-0.03 mM, 0.19+/-0.01 mM, and 11.4+/-0.4 mM, respectively. The half-life of the A. fulgidus SKDH is 2 h at the assay temperature (87 degrees C) and 17 days at 60 degrees C. Addition of 1 M NaCl or KCl stabilized the enzyme's half-life to approximately 70 h at 87 degrees C and approximately 50 days at 60 degrees C. This work presents the first kinetic analysis of an archaeal SKDH.     

Some properties of p-coumarate decarboxylase from Cladosporium phlei.

The optimal pH and temperature of p-coumarate decarboxylase were 6.0 and 23 degrees C respectively. The enzyme activity was reduced to three quarters by heat treatment at 35 degrees C for 5 min and by half at 25 degrees C in 24 h, but kept almost unchanged at -20 degrees C at least for 10 days. The activity was not inhibited by potassium cyanide, sodium diethyldithiocarbamate, ethylenediaminetetraacetic acid disodium salt, or sodium citrate at 10 mM concentration, but was inhibited by p-chloromercuribenzoate or iodoacetate at 0.1 mM, the inhibition by the former being prevented to a great extent by the presence of reduced glutathione or dithiothreitol. The activity was inhibited by maleic acid cinnamic acid, or p-methoxycinnamic acid, but not by fumaric acid, acrylic acid, p-hydroxystyrene, furcatin p-hydroxyphenylacetic acid, or phloretic acid. An unsubstituted p-hydroxy group on the benzene ring and an acrylic acid side chain were required for the enzyme activity. Km value for trans-p-coumaric acid was about 6.5 X 10(-4) M.     

Glutamate dehydrogenase from liver of euthermic and hibernating Richardson's ground squirrels: evidence for two distinct enzyme forms.

Glutamate dehydrogenase (GDH) was purified to homogeneity from the liver of euthermic (37 degrees C body temperature) and hibernating (torpid, 5 degrees C body temperature) Richardson's ground squirrels (Spermophilus richardsonii). SDS-PAGE yielded a subunit molecular weight of 59.5+/-2 kDa for both enzymes, but reverse phase and size exclusion HPLC showed native molecular weights of 335+/-5 kDa for euthermic and 320+/-5 kDa for hibernator GDH. Euthermic and hibernator GDH differed substantially in apparent Km values for glutamate, NH4+, and alpha-ketoglutarate, as well as in Ka and IC50 values for nucleotide and ion activators and inhibitors. Kinetic properties of each enzyme were differentially affected by assay temperature (37 versus 5 degrees C). For example, the Km for alpha-ketoglutarate of euthermic GDH was higher at 5 degrees C (3.66+/-0.34 mM) than at 37 degrees C (0.10+/-0.01 mM), whereas hibernator GDH had a higher affinity for alpha-ketoglutarate at 5 degrees C (Km was 0.98+/-0.08 mM at 37 degrees C and 0.43+/-0.02 mM at 5 degrees C). Temperature effects on Ka ADP values of the enzymes followed a similar pattern; GTP inhibition was strongest with the euthermic enzyme at 37 degrees C and weakest with hibernator GDH at 5 degrees C. Entry into hibernation leads to stable changes in the properties of ground squirrel liver GDH that allow the enzyme to function optimally at the prevailing body temperature.     

The thermal stability of a castor bean seed acid phosphatase

The effect of temperature on the activity and structural stability of an acid phosphatase (EC 3.1.3.2.) purified from castor bean (Ricinus communis L.) seeds have been examined. The enzyme showed high activity at 45 degrees C using p-nitrophenylphosphate (p-NPP) as substrate. The activation energy for the catalyzed reaction was 55.2 kJ mol(-1) and the enzyme maintained 50% of its activity even after 30 min at 55 degrees C. Thermal inactivation studies showed an influence of pH in the loss of enzymatic activity at 60 degrees C. A noticeable protective effect from thermal inactivation was observed when the enzyme was preincubated, at 60 degrees C, with the reaction products inorganic phosphate-P (10 mM) and p-nitrophenol-p-NP(10 mM). Denaturation studies showed a relatively high transition temperature (Tm) value of 75 degrees C and an influence of the combination of Pi (10 mM) and p-NP (10 mM) was observed on the conformational behaviour of the macromolecule.     

Biochemical analysis of a native and proteolytic fragment of a high-molecular-weight thermostable lipase from a mesophilic Bacillus sp.

An extracellular lipase was isolated from the cell-free broth of Bacillus sp. GK 8. The enzyme was purified to 53-fold with a specific activity of 75.7 U mg(-1) of protein and a yield of 31% activity. The apparent molecular mass of the monomeric protein was 108 kDa as estimated by molecular sieving and 112 kDa by SDS-PAGE. The proteolysis of the native molecule yields a low molecular weight component of 11.5 kDa that still retains the active site. It was stable at the pH range of 7.0-10.0 with optimum pH 8.0. The enzyme was stable at 50 degrees C for 1 h with a half life of 2 h, 40 min, and 18 min at 60, 65, and 70 degrees C, respectively. With p-nitrophenyl laurate as substrate the enzyme exhibited a K(m) and V(max) of 3.63 mM and 0.26 microM/min/ml, respectively. Activity was stimulated by Mg(2+) (10 mM), Ba(2+) (10 mM), and SDS (0.1 mM), but inhibited by EDTA (10 mM), phenylmethane sulfonyl fluoride (100 mM), diethylphenylcarbonate (10 mM), and eserine (10 mM). It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C(4) and C(18:1). Thermostability of the proteolytic fragment at 60 degrees C was observed to be 37% of the native protein. The native enzyme was completely stable in ethylene glycol and glycerol (30% v/v each) for 60 min at 65 degrees C.     

Highly thermostable, thermophilic, alkaline, SDS and chelator resistant amylase from a thermophilic Bacillus sp. isolate A3-15

A thermostable alkaline alpha-amylase producing Bacillus sp. A3-15 was isolated from compost samples. There was a slight variation in amylase synthesis within the pH range 6.0 and 12.0 with an optimum pH of 8.5 (8mm zone diameter in agar medium) on starch agar medium. Analyses of the enzyme for molecular mass and amylolytic activity were carried out by starch SDS-PAGE electrophoresis, which revealed two independent bands (86,000 and 60,500 Da). Enzyme synthesis occurred at temperatures between 25 and 65 degrees C with an optimum of 60 degrees C on petri dishes. The partial purification enzyme showed optimum activity at pH 11.0 and 70 degrees C. The enzyme was highly active (95%) in alkaline range of pH (10.0-11.5), and it was almost completely active up to 100 degrees C with 96% of the original activity remaining after heat treatment at 100 degrees C for 30 min. Enzyme activity was enhanced in the presence of 5mM CaCl2 (130%) and inhibition with 5mM by ZnCl2, NaCl, Na-sulphide, EDTA, PMSF (3mM), Urea (8M) and SDS (1%) was obtained 18%, 20%, 36%, 5%, 10%, 80% and 18%, respectively. The enzyme was stable approximately 70% at pH 10.0-11.0 and 60 degrees C for 24h. So our result showed that the enzyme was both, highly thermostable-alkaline, thermophile and chelator resistant. The A3-15 amylase enzyme may be suitable in liquefaction of starch in high temperature, in detergent and textile industries and in other industrial applications.     

Yersinia pseudotuberculosis nucleoside-kinase

Enzyme capable of catalyzing the phosphorylation of thymidine and uridine was isolated from Y. pseudotuberculosis cells by fractionation with the use of ammonium sulfate, ion exchange and affinity chromatography. The degree of purification of thymidine- and uridine-kinase was approximately 350 times, and at all stages of isolation the activity of both nucleoside-kinases was detected in the same peaks. The purified enzyme was capable of the phosphorylation of thymidine and uridine at temperatures of 8-10 degrees C to 50 degrees C and exhibited the maximum enzymatic activity at pH 8-8.5 and 45 degrees C in the presence of 0.5-1.0 mM MgCl2 and 2 mM ATP. The enzyme was found to have no strict substrate specificity and transferred the phosphate group from ATP to radiolabeled thymidine, uridine and desoxycytidine with different effectiveness, but did not use thymidine-monophosphate as phosphate acceptor.     

Purification and characterization of thermostable aspartase from Bacillus sp. YM55-1.

A thermostable aspartase was purified from a thermophile Bacillus sp. YM55-1 and characterized in terms of activity and stability. The enzyme was isolated by a 5-min heat treatment at 75 degrees C in the presence of 11% (w/v) ammonium sulfate and 100 mM aspartate, followed by Q-Sepharose anion-exchange and AF-Red Toyopearl chromatographies. The native molecular weight of aspartase determined by gel filtration was about 200,000, and this enzyme was composed of four identical monomers with molecular weights of 51,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Unlike Escherichia coli aspartase, the enzyme was not activated by the presence of magnesium ion at alkaline pH. At the optimum pH, the Km and Vmax were 28.5 mM and 700 units/mg at 30 degrees C and 32.0 mM and 2200 units/mg at 55 degrees C, respectively. The specific activity was four and three times higher than those of E. coli and Pseudomonas fluorescens enzymes at 30 degrees C, respectively. Eighty percent of the activity was retained after a 60-min incubation at 55 degrees C, and the enzyme was also resistant to chemical denaturants; 80% of the initial specific activity was detected in assay mixtures containing 1.0 M guanidine hydrochloride. The purified enzyme shared a high sequence homology in the N-terminal region with aspartases from other organisms.     

Factors stabilizing pyrroline-5-carboxylate synthase of rat intestine mucosa at a physiological temperature

Mammalian pyrroline-5-carboxylate (PC) synthase in the mitochondrial membrane of rat small intestine mucosa possesses marked thermal instability at temperatures of 30 to 37 degrees C [Y. Wakabayashi, J. G. Henslee, and M. E. Jones (1983) J. Biol. Chem. 258, 3873-3882]. Factors stabilizing the enzyme activity at 37 degrees C were extensively examined by incubating the enzyme with various compounds before assay. In the presence of 60% sorbitol, the enzyme retained full activity for 30 min. Xylitol, glycerol, and fructose were also effective, although sucrose, ethylene glycol, polyethylene glycol and dimethyl sulfoxide were ineffective. AMP, GMP, IMP, and UMP (15 mM) were completely protective while ATP and adenosine were not. Phosphate and arsenate at 10 mM maintained 90 and 82%, respectively, of the original activity after 10 min. NADPH and NADP (3 mM) were protective whereas 3 mM NADH was not. The possibility that phosphate and NADPH are stabilizing PC synthase in vivo was discussed. Addition of 0.13 mM p-chloromercuriphenylsulfonic acid or 0.55 mM 5,5'-dithiobis-(2-nitrobenzoic acid) to the enzyme resulted in complete loss of activity, but prior addition of excess dithiothreitol to the enzyme prevented the inactivation, suggesting that a sulfhydryl group is involved in the activity.     

Inhibitory effect of dibutyryl cyclic adenosine monophosphate on the induction of alkaline phosphatase in human fetal liver cell line

When human fetal liver cells (HuL-1-317), cultured continuously in a serum-free medium, were incubated with a combination of prednisolone, butyrate and a hypertonic concentration of NaCl at 37 degrees C, alkaline phosphatase activity increased. However, the addition of dibutyryl adenosine cyclic monophosphate (Bt2cAMP) to these agents inhibited the increase in alkaline phosphatase activity in a dose-dependent manner: the inhibitory effect of Bt2cAMP was significant at 0.05 mM, but disappeared at 0.01 mM. Both cycloheximide and actinomycin D inhibited the increase in alkaline phosphatase activity with the combination described above. Western blotting showed that this enzyme activity increase was a consequence of greater biosynthesis of enzyme molecules in HuL-1-317 cells, and that Bt2cAMP regulated the synthesis of enzyme molecules. We conclude that the changes in alkaline phosphatase activity under various conditions are based on the changes in the number of enzyme molecules in HuL-1-317 cells.     

Effects of ATP on the stability of enzymes against heat treatment in 6-aminonicotinamide treated quail

The stabilities of liver and pectoral muscle enzymes in 6-aminonicotinamide (6-AN) treated quail against heat treatment in the presence and absence of added ATP were investigated. Only ATP level in the brain and pectoral muscle of 6-AN treated group was significantly reduced compared to the control group whereas ADP and AMP levels were not affected. In the thermal stability (55 degrees C) of liver enzymes, the activity of acetylcholinesterase (AChE) was not affected whereas the activities of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) were significantly lowered (P<0.01). The addition of 1mM ATP to liver enzyme extracts of 6-AN group afforded 4- and 1.7-fold more protection for GAPDH and LDH, respectively (P<0.01). In liver, LDH appeared to be more protected by ATP than GAPDH. In muscle, however, GAPDH and AChE activity were significantly affected but not LDH. The addition of 1mM ATP to muscle enzyme extracts of 6-AN group afforded 1.7-fold more protection for GAPDH (P<0.01) but rather inactivated AChE. A marked reduction in ATP levels in muscle did not affect specifically muscle enzyme activities only since liver enzyme activities were also affected to the same degree as muscle.     

Inhibition of aspartate aminotransferase by glycation in vitro under various conditions

Incubation of 50 mM D-glucose with aspartate aminotransferase (AST, EC 2.6.1.1) preparations (purified pig heart enzyme or a rat liver 20,000 x g supernatant) at 25 degrees C had no effect on enzyme activity. 50 mM D-fructose or D-ribose gradually inhibited pig heart AST under the same conditions to zero activity after 14 days. 50 mM DL-glyceraldehyde decreased enzyme activity to zero after 6 days of incubation. The inhibition of pig heart AST by 50 mM D-fructose or D-ribose was marked even at a temperature of 4 degrees C but it was less pronounced than at 25 degrees C. There was no effect of 0.5 mM 2-oxoglutarate on AST activity during incubation, while the presence of 25 mM L-aspartate decreased it rapidly. 0.5 mM 2-oxoglutarate partly prevented inhibition of AST by D-ribose or D-fructose, while an analogous experiment with 25 mM aspartate resulted in a rapid decline similar to that in the absence of sugars.     

Carbonic anhydrase activity of intact carbonic anhydrase II-deficient human erythrocytes

Intact erythrocytes from subjects with deficiency of blood carbonic anhydrase (CA) II and from normal subjects were assayed for enzyme activity by use of an 18O exchange technique in a solution containing 25 mM (CO2 + NaHCO3) plus 125 mM NaCl. At 25 degrees C and pH 7.4, the catalyzed reaction velocity was 0.32 +/- 0.04 M/s for the CA II-deficient and 1.60 +/- 0.12 M/s for the normal cells, a ratio of 1:5. Under the same conditions at 37 degrees C the relative difference between the CA II-deficient and normal cells was much less: the velocity for the CA II-deficient cells was 0.84 +/- 0.07 M/s and for the normal cells 1.60 +/- 0.32 M/s, a ratio of 1:1.9. Results were comparable for the hemolysates with the NaHCO3 reduced to 85 mM (the corresponding intracellular concentration): at 25 degrees C CA II-deficient cells had a velocity of 0.36 +/- 0.01 M/s compared with 1.12 +/- 0.04 M/s for the normal cells, a ratio of 1:3.1. At 37 degrees C again the relative difference between hemolysates from CA II normal and deficient cells was much less: the CA II-deficient cells had a reaction velocity of 1.17 +/- 0.22 M/s vs. 2.60 +/- 0.36 M/s for the normal cells, a ratio of 1:2.2. The greater fractional reduction of enzyme velocity of CA II-deficient cells at 25 degrees C compared with 37 degrees C appears to be explained by a greater chloride inhibition of the presumed CA I at the lower temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification, purification, and characterization of a thermally stable lipase from rice bran. A new member of the (phospho) lipase family.

A thermally stable lipase (EC 3.1.1.3.) was first identified in rice (Oryza sativa) bran, and the enzyme was purified to homogeneity using octyl-Sepharose chromatography. The enzyme was purified to 7.6-fold with the final specific activity of 0.38 micromol min(-1) mg(-1) at 80 degrees C using [9,10-(3)H]triolein as a substrate. The purified enzyme was found to be a glycoprotein of 9.4 kD. Enzyme showed a maximum activity at 80 degrees C and at pH 11.0. The protein was biologically active and retained most of its secondary structure even at 90 degrees C as judged by the enzymatic assays and far-ultraviolet circular dichroism spectroscopy, respectively. Differential scanning calorimetric studies indicated that the transition temperature was 76 degrees C and enthalpy 1.3 x 10(5) Calorie mol(-1) at this temperature. The purified lipase also exhibited phospholipase A(2) activity. Colocalization of both the hydrolytic activities in reverse-phase high-performance liquid chromatography and isoelectric focusing showed that the dual activity was associated with a single protein. Further, a direct interaction between both the substrates and the purified protein was demonstrated by photoaffinity labeling, using chemically synthesized analogs of triolein and phosphatidylcholine (PC). Apparent K(m) for triolein (6.71 mM) was higher than that for PC (1.02 mM). The enzyme preferentially hydrolyzed the sn-2 position of PC, whereas it apparently exhibited no positional specificity toward triacylglycerol. Diisopropyl fluorophosphate inhibited both lipase and phospholipase activities of the purified enzyme. This enzyme is a new member from plants in the family of lipases capable of hydrolyzing phospholipids.     

Biochemical and phylogenetic analyses of a cold-active beta-galactosidase from the lactic acid bacterium Carnobacterium piscicola BA

We are investigating glycosyl hydrolases from new psychrophilic isolates to examine the adaptations of enzymes to low temperatures. A beta-galactosidase from isolate BA, which we have classified as a strain of the lactic acid bacterium Carnobacterium piscicola, was capable of hydrolyzing the chromogen 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-Gal) at 4 degrees C and possessed higher activity in crude cell lysates at 25 than at 37 degrees C. Sequence analysis of a cloned DNA fragment encoding this activity revealed a gene cluster containing three glycosyl hydrolases with homology to an alpha-galactosidase and two beta-galactosidases. The larger of the two beta-galactosidase genes, bgaB, encoded the 76.8-kDa cold-active enzyme. This gene was homologous to family 42 glycosyl hydrolases, a group which contains several thermophilic enzymes but none from lactic acid bacteria. The bgaB gene from isolate BA was subcloned in Escherichia coli, and its enzyme, BgaB, was purified. The purified enzyme was highly unstable and required 10% glycerol to maintain activity. Its optimal temperature for activity was 30 degrees C, and it was inactivated at 40 degrees C in 10 min. The K(m) of freshly purified enzyme at 30 degrees C was 1.7 mM, and the V(max) was 450 micromol. min(-1). mg(-1) with o-nitrophenyl beta-D-galactopyranoside. This cold-active enzyme is interesting because it is homologous to a thermophilic enzyme from Bacillus stearothermophilus, and comparisons could provide information about structural features important for activity at low temperatures.     

Purification and biochemical properties of a galactooligosaccharide producing β-galactosidase from Bullera singularis

A beta-galactosidase, catalyzing lactose hydrolysis and galactooligosaccharide (GalOS) synthesis from lactose, was extracted from the yeast, Bullera singularis KCTC 7534. The crude enzyme had a high transgalactosylation activity resulting in the oligosaccharide conversion of over 34% using pure lactose and cheese whey permeate as substrates. The enzyme was purified by two chromatographic steps giving 96-fold purification with a yield of 16%. The molecular weight of the purified enzyme (specific activity of 56 U mg(-1)) was approx. 53 000 Da. The hydrolytic activity was the highest at pH 5 and 50 degrees C, and was stable to 45 degrees C for 2 h. Enzyme activity was inhibited by 10 mM Ag3+ and 10 mM SDS. The Km for lactose hydrolysis was 0.58 M and the maximum reaction velocity (V(max)) was 4 mM min(-1). GalOS, including tri- and tetra-saccharides were produced with a conversion yield of 50%, corresponding to 90 g GalOS l(-1) from 180 g lactose l(-1) by the purified enzyme.     

Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay

We describe an assay to measure the extent of enzymatic unwinding of DNA by a DNA helicase. This assay takes advantage of the quenching of the intrinsic protein fluorescence of Escherichia coli SSB protein upon binding to ssDNA and is used to characterize the DNA unwinding activity of recBCD enzyme. Unwinding in this assay is dependent on the presence of recBCD enzyme and linear dsDNA, is consistent with the known properties of recBCD enzyme, and closely parallels other methods for measuring recBCD enzyme helicase activity. The effects of varying temperature, substrate concentrations, enzyme concentration, and mono- and divalent salt concentrations on the helicase activity of recBCD enzyme were characterized. The apparent Km values for recBCD enzyme helicase activity on linear M13 dsDNA molecules at 25 degrees C are 0.6 nM dsDNA molecules and 130 microM ATP, respectively. The apparent turnover number for unwinding is approximately 15 microM base pairs s-1 (microM recBCD enzyme)-1. When this rate is corrected for the observed stoichiometry of recBCD enzyme binding to dsDNA, kcat for helicase activity corresponds to an unwinding rate of approximately 250 base pairs of DNA s-1 (functional recBCD complex)-1 at 25 degrees C. At 37 degrees C, the apparent Km value for dsDNA molecules was the same as that at 25 degrees C, but the apparent turnover number became 56 microM base pairs s-1 (microM recBCD enzyme)-1 [or 930 base pairs s-1 (functional recBCD complex)-1 when corrected for observed stoichiometry]. With increasing NaCl concentration, kcat peaks at 100 mM, and the apparent Km value for dsDNA increases by 3-fold at 200 mM NaCl. In the presence of 5 mM calcium acetate, the apparent Km value is increased by 3-fold, and kcat decreased by 20-30%. We have also shown that recBCD enzyme molecules are able to catalytically unwind additional dsDNA substrates subsequent to initiation, unwinding, and dissociation from a previous dsDNA molecule.     

Characteristics and application of immobilized papain in a continuous-flow reactor

Papain was immobilized on an anion-exchange resin by physical adsorption followed by cross-linking with glutaraldehyde. The activity and rate of heat inactivation of the enzyme was determined at 37 to 95 degrees C. Activity increased with an increase in temperature up to 85 degrees C, then decreased slightly at 95 degrees C. Activation of the enzyme by cysteine in 2 mM EDTA increased proportionally with an increase in concentration from 0.004 to 0.12 M. Levels of dissolved oxygen in solutions of EDTA containing cysteine in a closed system and with nitrogen purging in the absence of cysteine in an open system were monitored at 51 degrees C. Both the presence of cysteine and purging with nitrogen were effective in depleting dissolved oxygen. The final oxygen level was essentially independent of cysteine concentrations of 0.01, 0.02, and 0.10 M, and was reached in 95, 85, and 55 min, respectively. However, final levels of oxygen were dependent upon the flow rate of nitrogen from 1 to 10 SLPM and were reached within 15 min. Immobilized papain in a continuous-flow stirred-tank (300 rpm) reactor with a 4-liter reaction volume accompanied by nitrogen purging of 4 SPLM was used to hydrolyze a 1.5% casein solution in the presence of 0.01 M cysteine in 2 mM EDTA at 51 degrees C. The reaction rate was 1 liter/h and activity was measured based on the content of trichloroacetic acid-soluble protein in the outflow. Papain activity was not observed to decrease during a 30-h reaction period.     

Activation of alkaline phosphatase with Mg2+ and Zn2+ in rat hepatoma cells. Accumulation of apoenzyme

In Reuber rat hepatoma cells (R-Y121B), alkaline phosphatase activity increased without de novo enzyme synthesis (Sorimachi, K., and Yasumura, Y. (1986) Biochim. Biophys. Acta 885, 272-281). The enzyme was partially purified by butanol extraction from the particulate fractions. The incubation of the extracted alkaline phosphatase with the cytosol fraction induced a large increase in enzyme activity (5-10-fold of control). The dialyzed cytosol was more effective than the undialyzed cytosol during an early period of incubation at 37 degrees C. This difference between the dialyzed and the undialyzed cytosol fractions was due to endogenous Na+. For maximal activation of the enzyme, both Mg2+ above 1 mM and Zn2+ at low concentrations (below 0.01 mM) were needed, although Zn2+ at high concentrations (above 0.1 mM) showed an inhibitory effect. Zn2+ and Mg2+ alone slightly increased alkaline phosphatase activity. This activation of the enzyme was temperature dependent and was not observed at 0 or 4 degrees C. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the increase in alkaline phosphatase activity did not involve the fragmentation of the enzyme and that 65Zn2+ bound to it during enzyme activation with 65Zn2+ and Mg2+. The cytosol fraction not only supplied Zn2+ to the nascent enzyme but also increased the maximal enzyme activity more than did direct addition of metal ions. Ferritin and metallothionein contributed to the activation of alkaline phosphatase with the metal ions. Since the binding of Zn2+ and Mg2+ to the nascent alkaline phosphatase is disturbed in Reuber rat hepatoma cells (R-Y121B), the apoenzyme is accumulated inside the cells. The binding of Zn2+ and Mg2+ to the apoenzyme readily takes place in the cell homogenates accompanied by an increase in catalytic activity without new enzyme synthesis.     

Purification and characterization of the NAD-dependent glutamate dehydrogenase in the ectomycorrhizal fungus laccaria bicolor (Maire) orton

The NAD-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.2) from Laccaria bicolor was purified 410-fold to apparent electrophoretic homogeneity with a 40% recovery through a three-step procedure involving ammonium sulfate precipitation, anion-exchange chromatography on DEAE-Trisacryl, and gel filtration. The molecular weight of the native enzyme determined by gel filtration was 470 kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave rise to a single band of 116 kDa, suggesting that the enzyme is composed of four identical subunits. The enzyme was specific for NAD(H). The pH optima were 7.4 and 8.8 for the amination and deamination reactions, respectively. The enzyme was found to be highly unstable, with virtually no activity after 20 days at -75 degrees C, 4 days at 4 degrees C, and 1 h at 50 degrees C. The addition of ammonium sulfate improved greatly the stability of the enzyme and full activity was still observed after several months at -75 degrees C. NAD-GDH activity was stimulated by Ca2+ and Mg2+ but strongly inhibited by Cu2+ and slightly by the nucleotides AMP, ADP, and ATP. The Michaelis constants for NAD, NADH, 2-oxoglutarate, and ammonium were 282 &mgr;M, 89 &mgr;M, 1.35 mM, and 37 mM, respectively. The enzyme had a negative cooperativity for glutamate (Hill number of 0.3), and its Km value increased from 0.24 to 3.6 mM when the glutamate concentration exceeded 1 mM. These affinity constants of the substrates, compared with those of the NADP-GDH of the fungus, suggest that the NAD-GDH is mainly involved in the catabolism of glutamate, while the NADP-GDH is involved in the catalysis of this amino acid. Copyright 1997 Academic Press. Copyright 1997 Academic Press