The Antarctic Psychrobacter sp. TAD1 has two cold-active glutamate dehydrogenases with different cofactor specificities. Characterisation of the NAD+-dependent enzyme

Psychrobacter sp. TAD1 is a psychrotolerant bacterium from Antarctic frozen continental water that grows from 2 to 25 degrees C with optimal growth rate at 20 degrees C. The new isolate contains two glutamate dehydrogenases (GDH), differing in their cofactor specificities, subunit sizes and arrangements, and thermal properties. NADP+-dependent GDH is a hexamer of 47 kDa subunits and it is comparable to other hexameric GDHs of family-I from bacteria and lower eukaria. The NAD+-dependent enzyme, described in this communication, has a subunit weight of 160 kDa and belongs to the novel class of GDHs with large size subunits. The enzyme is a dimer; this oligomeric arrangement has not been reported previously for GDH. Both enzymes have an apparent optimum temperature for activity of approximately 20 degrees C, but their cold activities and thermal labilities are different. The NAD+-dependent enzyme is more cold active: at 10 C it retains 50% of its maximal activity, compared with 10% for the NADP+-dependent enzyme. The NADP+-dependent enzyme is more heat stable, losing only 10% activity after heating for 30 min, compared with 95% for the NAD+-dependent enzyme. It is concluded that in Psychrobacter sp. TAD1 not only does NAD+-dependent GDH have a novel subunit molecular weight and arrangement, but that its polypeptide chains are folded differently from those of NADP+-dependent GDH, providing different cold-active properties to the two enzymes.     

Purification and characterization of D-glyceraldehyde-3-phosphate dehydrogenase from the thermophilic archaebacterium Methanothermus fervidus

The D-glyceraldehyde-3-phosphate dehydrogenase from the extremely thermophilic archaebacterium Methanothermus fervidus was purified and crystallized. The enzyme is a homomeric tetramer (molecular mass of subunits 45 kDa). Partial sequence analysis shows homology to the enzymes from eubacteria and from the cytoplasm of eukaryotes. Unlike these enzymes, the D-glyceraldehyde-3-phosphate dehydrogenase from Methanothermus fervidus reacts with both NAD+ and NADP+ and is not inhibited by pentalenolactone. The enzyme is intrinsically stable up to 75 degrees C. It is stabilized by the coenzyme NADP+ and at high ionic strength up to about 90 degrees C. Breaks in the Arrhenius and Van't Hoff plots indicate conformational changes of the enzyme at around 52 degrees C.     

Interaction of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase with pyridoxal 5'-diphospho-5'-adenosine. Affinity labeling of Lys-21 and Lys-343

Pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) inhibits glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides competitively with respect to glucose 6-phosphate and noncompetitively with respect to NAD+ or NADP+, with Ki = 40 microM in the NADP-linked and 34 microM in the NAD-linked reaction. Incubation of glucose-6-phosphate dehydrogenase with [3H]PLP-AMP followed by borohydride reduction shows that incorporation of 0.85 mol of PLP-AMP per mol of enzyme subunit is required for complete inactivation. Both glucose 6-phosphate and NAD+ protect against this covalent modification. The proteolysis of the modified enzyme and isolation and sequencing of the labeled peptides revealed that Lys-21 and Lys-343 are the sites of PLP-AMP interaction and that glucose 6-phosphate and NAD+ protect both lysyl residues against modification. Pyridoxal 5'-phosphate (PLP) also modifies Lys-21 and probably Lys-343. Lys-21 is part of a highly conserved region that is present in all glucose-6-phosphate dehydrogenases that have been sequenced. Lys-343 corresponds to an arginyl residue in other glucose-6-phosphate dehydrogenases and is in a region that is less homologous with those enzymes. PLP-AMP and PLP are believed to interact with L. mesenteroides glucose-6-phosphate dehydrogenase at the glucose 6-phosphate binding site. Simultaneous binding of NAD+ induces conformational changes (Kurlandsky, S. B., Hilburger, A. C., and Levy, H. R. (1988) Arch. Biochem. Biophys. 264, 93-102) that are postulated to interfere with Schiff's-base formation with PLP or PLP-AMP. One or both of the lysyl residues covalently modified by PLP or PLP-AMP may be located in regions of the enzyme undergoing the NAD(+)-induced conformational changes.     

Purification and characterization of a thermostable glutamate dehydrogenase from a thermophilic bacterium isolated from a sterilization drying oven

Glutamate dehydrogenase from axenic bacterial cultures of a new microorganism, called GWE1, isolated from the interior of a sterilization drying oven, was purified by anion-exchange and molecular-exclusion liquid chromatography. The apparent molecular mass of the native enzyme was 250.5 kDa and was shown to be an hexamer with similar subunits of molecular mass 40.5 kDa. For glutamate oxidation, the enzyme showed an optimal pH and temperature of 8.0 and 70 degrees C, respectively. In contrast to other glutamate dehydrogenases isolated from bacteria, the enzyme isolated in this study can use both NAD(+) and NADP(+) as electron acceptors, displaying more affinity for NADP(+) than for NAD(+). No activity was detected with NADH or NADPH, 2-oxoglutarate and ammonia. The enzyme was exceptionally thermostable, maintaining more than 70% of activity after incubating at 100(o)C for more than five hours suggesting being one of the most thermoestable enzymes reported in the family of dehydrogenases.     

Properties and primary structure of the L-malate dehydrogenase from the extremely thermophilic archaebacterium Methanothermus fervidus

L-Malate dehydrogenase from the extremely thermophilic mathanogen Methanothermus fervidus was isolated and its phenotypic properties were characterized. The primary structure of the protein was deducted from the coding gene. The enzyme is a homomeric dimer with a molecular mass of 70 kDa, possesses low specificity for NAD+ or NADP+ and catalyzes preferentially the reduction of oxalacetate. The temperature dependence of the activity as depicted in the Arrhenius and van't Hoff plots shows discontinuities near 52 degrees C, as was found for glyceraldehyde-3-phosphate dehydrogenase from the same organism. With respect to the primary structure, the archaebacterial L-malate dehydrogenase deviates strikingly from the eubacterial and eukaryotic enzymes. The sequence similarity is even lower than that between the L-malate dehydrogenases and L-lactate dehydrogenases of eubacteria and eukaryotes. The phylogenetic meaning of this relationship is discussed.     

Coenzymic activity of NADP derivatives alkylated at 2'-phosphate and 6-amino groups

Coenzymic activities of the following NADP derivatives were investigated: 2'-O-(2-carboxyethyl)phosphono-NAD (I), N6-(2-carboxyethyl)-NADP (II), 2'-O-(2-carboxyethyl)phosphono-N6-(2-carboxyethyl)-NAD (III), 2'-O-[N-(2-aminoethyl)carbamoylethyl]phosphono-NAD (IV), N6-[N-(2-aminoethyl)carbamoylethyl]-NADP (Va), 2',3'-cyclic NADP, and 3'-NADP. Derivatives I and IV show the effects of modification at the 2'-phosphate group, and derivatives II and Va show those at the 6-amino group of NADP. As for enzymes, alcohol, isocitrate, 6-phosphogluconate, glucose, glucose-6-phosphate, and glutamate dehydrogenases were used. These enzymes were grouped on the basis of the ratio of the activities for NAD and NADP into NADP-specific enzymes (ratio less than 0.01), NAD(P)-specific enzymes (0.01 less than ratio less than 100), and NAD-specific enzymes (ratio greater than 100). For NADP-specific enzymes, modifications at the 2'-phosphate group of NADP caused great loss of cofactor activity. The relative cofactor activities (NADP = 100%) of derivatives I and IV for these enzymes were 0.5-20 and 0.01-0.5%, respectively. On the other hand, NAD(P)-specific enzymes showed several types of responses to the NADP derivatives. The relative cofactor activities of I and IV for Leuconostoc mesenteroides and Bacillus stearothermophilus glucose-6-phosphate dehydrogenases and beef liver glutamate dehydrogenase were 60-200%; whereas, for B. megaterium glucose dehydrogenase and L. mesenteroides alcohol dehydrogenase, the values were 0.8-8%. For NAD-specific enzymes, these values were 20-50%. The relative cofactor activities of 2',3'-cyclic NADP and 3'-NADP were very low (less than 0.2%) except for beef liver glutamate dehydrogenase, B. stearothermophilus glucose-6-phosphate dehydrogenase, and horse liver alcohol dehydrogenase. Kinetic studies showed that the losses of the cofactor activity of NADP by these modifications were mainly due to the increase of the Km value. The mechanisms of coenzyme specificity of dehydrogenases are discussed. Unlike the 2'-phosphate group, the 6-amino group is common to NAD and NADP, and the effects of modification at the 6-amino group were independent of the coenzyme specificity of enzymes used for the assay. Derivatives II and Va had high relative cofactor activities (65-130%) for most of the enzymes except for isocitrate and glucose dehydrogenases (less than 1%) and L. mesenteroides alcohol dehydrogenase (20-60%). The cofactor activity of derivative III was generally lower than those of I and II.     

Synthesis of poly(ethylene glycol)-bound NADP by selective modification at the 6-amino group of NADP

The N-1 position of the adenine ring of NADP was selectively alkylated by the reaction of 2',3'-cyclic NADP with 3-propiolactone to yield 2',3'-cyclic 1-(2-carboxyethyl)-NADP (I). Derivative I was converted to a mixture of the isomers of N6-(2-carboxyethyl)-NADP with their phosphate groups at the 2' or 3' position (IIa and IIb) by chemical reduction, alkaline rearrangement and chemical reoxidation. Carbodiimide coupling of the mixture of IIa and IIb to alpha, omega-diaminopoly(ethylene glycol) gave the 2', 3'-cyclic derivative of poly(ethylene glycol)-bound NADP (III), which was enzymically hydrolyzed to yield poly(ethylene glycol)-bound NADP (PEG-NADP). PEG-NADP has good cofactor activity (16-100% of that of NADP) for NADP-specific and NAD(P)-specific dehydrogenases except isocitrate and glucose dehydrogenases. For NAD-specific enzymes, PEG-NADP has higher cofactor activity than NADP: for horse liver alcohol dehydrogenase, the cofactor activity of PEG-NADP is 40 times that of NADP and 14% of that of NAD. Kinetic studies show that for most of enzymes tested, Km values for PEG-NADP are larger than those for NADP and V values for PEG-NADP are similar to those for NADP. PEG-NADP proved to be applicable in a continuous enzyme reactor, in which reactions of glutamate dehydrogenase and glucose-6-phosphate dehydrogenase were coupled by the recycling of PEG-NADP.     

Dehydrogenases of the pentose phosphate pathway in rat liver peroxisomes

Subcellular distribution of pentose-phosphate cycle enzymes in rat liver was investigated, using differential and isopycnic centrifugation. The activities of the NADP+-dependent dehydrogenases of the pentose-phosphate pathway (glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase) were detected in the purified peroxisomal fraction as well as in the cytosol. Both dehydrogenases were localized in the peroxisomal matrix. Chronic administration of the hypolipidemic drug clofibrate (ethyl-alpha-p-chlorophenoxyisobutyrate) caused a 1.5-2.5-fold increase in the amount of glucose-6-phosphate and phosphogluconate dehydrogenases in the purified peroxisomes. Clofibrate decreased the phosphogluconate dehydrogenase, but did not alter glucose-6-phosphate dehydrogenase activity in the cytosolic fraction. The results obtained indicate that the enzymes of the non-oxidative segment of the pentose cycle (transketolase, transaldolase, triosephosphate isomerase and glucose-phosphate isomerase) are present only in a soluble form in the cytosol, but not in the peroxisomes or other particles, and that ionogenic interaction of the enzymes with the mitochondrial and other membranes takes place during homogenization of the tissue in 0.25 M sucrose. Similar to catalase, glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase are present in the intact peroxisomes in a latent form. The enzymes have Km values for their substrates in the millimolar range (0.2 mM for glucose-6-phosphate and 0.10-0.12 mM for 6-phosphogluconate). NADP+, but not NAD+, serves as a coenzyme for both enzymes. Glucose-6-phosphate dehydrogenase was inhibited by palmitoyl-CoA, and to a lesser extent by NADPH. Peroxisomal glucose-6-phosphate and phosphogluconate dehydrogenases have molecular mass of 280 kDa and 96 kDa, respectively. The putative functional role of pentose-phosphate cycle dehydrogenases in rat liver peroxisomes is discussed.     

Chemically Defined Medium for the Accumulation of Intracellular Malate Dehydrogenase by Streptomyces aureofaciens

A chemically defined medium was developed for the production of intracellular malate dehydrogenases by Streptomyces aureofaciens NRRL-B 1286. The composition of the medium (per liter) was as follows: 50 g of starch, 4 g of ammonium sulfate, 7.32 g of l-aspartic acid, 13.8 g of MgSO(4) . 7H(2)O, 1.7 g of K(2)HPO(4), 0.01 g of ZnSO(4) . 7H(2)O, 0.01 g of FeSO(4) . 7H(2)O, 0.01 g of MnSO(4) . H(2)O, and 0.005 g of CoSO(4) . 7H(2)O. The pH of the medium was adjusted to 6.7 to 7.0 after sterilization. The activity of the intracellular malate dehydrogenases of the crude cell extract was greatest after 40 h of mycelium growth in a rotary shaker at 30 degrees C. The best temperature for the enzyme reactions was approximately 35 degrees C for NAD activity at pH 9.7 and 40 degrees C for NADP -linked enzyme at pH 9.0. The NAD activity required Mg, and both activities were sensitive to SH-group reagents. The NADP -dependent activity remained completely stable, and the NAD -dependent activity decreased to a very low residual level after 30 min at 60 degrees C.     

A novel dehydrogenase reaction mechanism for hexose-6-phosphate dehydrogenase isolated from the teleost Fundulus heteroclitus

Hexose-6-phosphate dehydrogenase (refers to hexose-6-phosphate dehydrogenase from any species in general) has been purified to apparent homogeneity from the teleost fish Fundulus heteroclitus. The enzyme was characterized for native (210 kDa) and subunit molecular mass (54 kDa), isoelectric point (6.65), amino acid composition, substrate specificity, and metal dependence. Glucose 6-phosphate, galactose 6-phosphate, 2-deoxyglucose 6-phosphate, glucose 6-sulfate, glucosamine 6-phosphate, and glucose were found to be substrates in the reaction with NADP+, but only glucose was a substrate when NAD+ was used as coenzyme. A unique reaction mechanism for the forward direction was found for this enzyme when glucose 6-phosphate and NADP+ were used as substrates; ordered with glucose 6-phosphate binding first. NAD+ was found to be a competitive inhibitor toward NADP+ and an uncompetitive inhibitor with regard to glucose 6-phosphate in this reaction; Vmax = 7.56 mumol/min/mg, Km(NADP+) = 1.62 microM, Km(glucose 6-phosphate) = 7.29 microM, Kia(glucose 6-phosphate) = 8.66 microM, and Ki(NAD+) = 0.49 microM. The use of alternative substrates confirmed this result. This type of reaction mechanism has not been previously reported for a dehydrogenase.     

NADP+ production using thermostable NAD+ kinase of corynebacterium flaccumfaciens AHU-1622

A sonicate of Corynebacterium flaccumfaciens AHU-1622 had the highest NAD+ kinase activity (1.22 mU/mL culture broth) of the strains of bacteria we investigated. This enzyme was thermostable, with activity maintained at 50 degrees C for 1 h. This treatment inactivated phosphatase activity. Resting cells of the bacterium also had NAD+ kinase activity when treated at 60 degrees C for 30 min with 0.2% Triton X-100. NADP+ production was achieved using 8 mumol NAD+, 8 mumol ATP, 16 mumol MgCl2, 1.6 mumol NaN3, and 12 mU NAD+ kinase (0.1 g of permeabilized wet cells) in 2 mL of 0.1 M phosphate buffer, pH 7.5. The conversion ratio of NADP+ from NAD+ was 75% after 10 h of incubation at 50 degrees C, and the amount of accumulated NADP+ was 3 mumol/mL of reaction mixture. The NAD+ kinase activity of the permeabilized cells was stable and did not decrease after repeated use.     

Pilot scale production and isolation of recombinant NAD+- and NADP+-specific formate dehydrogenases.

The expression of the recombinant wild-type NAD+- and mutant NADP+-dependent formate dehydrogenases (EC 1.2.1.2., FDH) from the methanol-utilizing bacterium Pseudomonas sp. 101 in Escherichia coli cells has been improved to produce active and soluble enzyme up to the level of 50% of total soluble proteins. The cultivation process for E. coli/pFDH8a and E. coli/pFDH8aNP cells was optimized and scaled up to a volume of 100 L. A downstream purification process has been developed to produce technical grade NAD+- and NADP+-specific formate dehydrogenases in pilot scale, utilizing extraction in aqueous two-phase systems.     

Long-chain alcohol and aldehyde dehydrogenase activities in Acinetobacter calcoaceticus strain HO1-N.

Three alcohol dehydrogenases have been identified in Acinetobacter calcoaceticus sp. strain HO1-N: an NAD(+)-dependent enzyme and two NADP(+)-dependent enzymes. One of the NADP(+)-dependent alcohol dehydrogenases was partially purified and was specific for long-chain substrates. With tetradecanol as substrate an apparent Km value of 5.2 microM was calculated. This enzyme has a pI of 4.5 and a molecular mass of 144 kDa. All three alcohol dehydrogenases were constitutively expressed. Three aldehyde dehydrogenases were also identified: an NAD(+)-dependent enzyme, an NADP(+)-dependent enzyme and one which was nucleotide independent. The NAD(+)-dependent enzyme represented only 2% of the total activity and was not studied further. The NADP(+)-dependent enzyme was strongly induced by growth of cells on alkanes and was associated with hydrocarbon vesicles. With tetradecanal as substrate an apparent Km value of 0.2 microM was calculated. The nucleotide-independent aldehyde dehydrogenase could use either Würster's Blue or phenazine methosulphate (PMS) as an artificial electron acceptor. This enzyme represents approximately 80% of the total long-chain aldehyde oxidizing activity within the cell when the enzymes were induced by growing the cells on hexadecane. It is particulate but can be solubilized using Triton X-100. The enzyme has an apparent Km of 0.36 mM for decanal.     

嗜热共生杆菌内消旋-2,6-二氨基庚二酸脱氢酶中Y76对辅酶偏好性影响

辅酶NAD(H)相比NADP(H)有稳定性好、价格低廉及更广的辅酶循环方法等优势,因此在实际应用中常需将NADP(H)依赖型的脱氢酶改造成为NAD(H)依赖型的。来源于嗜热共生杆菌Symbiobacterium thermophilum的NADP(H)依赖型内消旋-2,6-二氨基庚二酸脱氢酶(meso-2,6-diaminopimelate dehydrogenase,St DAPDH)及其突变体酶是催化还原氨化合成D-氨基酸的优良催化剂,本研究试图改变其辅酶偏好性,增强其应用优势。对其晶体结构分析可知,氨基酸残基Y76距离腺嘌呤较近,R35及R36和辅酶上磷酸基团有直接相互作用。依氨基酸侧链基团性质对Y76进行了定点突变,发现不同突变子对两种辅酶的偏好性都发生了变化;对与磷酸基团直接作用的R35、R36进行的双突变R35S/R36V,导致酶对NADP+的催化活力降低;将R35S/R36V和部分Y76突变进行了组合,发现三突变组合以NAD+为辅酶时的活力均大于以NADP+为辅酶的活力,实现了辅酶偏好性转变。这些研究工作为进一步实现St DAPDH的辅酶偏好性完全转变提供依据。     

Kinetic study of sn-glycerol-1-phosphate dehydrogenase from the aerobic hyperthermophilic archaeon, Aeropyrum pernix K1.

A gene having high sequence homology (45-49%) with the glycerol-1-phosphate dehydrogenase gene from Methanobacterium thermoautotrophicum was cloned from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820). This gene expressed in Escherichia coli with the pET vector system consists of 1113 nucleotides with an ATG initiation codon and a TAG termination codon. The molecular mass of the purified enzyme was estimated to be 38 kDa by SDS/PAGE and 72.4 kDa by gel column chromatography, indicating presence as a dimer. The optimum reaction temperature of this enzyme was observed to be 94-96 degrees C at near neutral pH. This enzyme was subjected to two-substrate kinetic analysis. The enzyme showed substrate specificity for NAD(P)H-dependent dihydroxyacetone phosphate reduction and NAD(+)-dependent glycerol-1-phosphate (Gro1P) oxidation. NADP(+)-dependent Gro1P oxidation was not observed with this enzyme. For the production of Gro1P in A. pernix cells, NADPH is the preferred coenzyme rather than NADH. Gro1P acted as a noncompetitive inhibitor against dihydroxyacetone phosphate and NAD(P)H. However, NAD(P)(+) acted as a competitive inhibitor against NAD(P)H and as a noncompetitive inhibitor against dihydroxyacetone phosphate. This kinetic data indicates that the catalytic reaction by glycerol- 1-phosphate dehydrogenase from A. pernix follows a ordered bi-bi mechanism.     

Steroidogenesis despite a variant metabolism in primary cultures of pig granulosa cells

Enzymic changes in primary cultures of granulosa cells over 9 days were measured and compared with changes occurring during follicular development in vivo. Characteristic of in vivo development of granulosa cells was a large increase in activities of the NADP+-dependent isocitrate, glucose-6-phosphate dehydrogenases and malic enzyme, and smaller increases in the activities of the NAD+-dependent lactate and malate dehydrogenases. In vitro, the NAD+-dependent dehydrogenases increased in activity, while the NADP+-dependent enzymes showed transient or no changes. Despite the uncharacteristic metabolism, granulosa cells in culture could synthesize steroids. Our results suggest that the cells in vitro and in vivo use different metabolic pathways to support syntheses dependent on reducing equivalents.     

Purification and comparative studies of dihydrolipoamide dehydrogenases from the anaerobic, glycine-utilizing bacteria Peptostreptococcus glycinophilus, Clostridium cylindrosporum, and Clostridium sporogenes.

Three different dihydrolipoamide dehydrogenases were purified to homogenity from the anaerobic glycine-utilizing bacteria Clostridium cylindrosporum, Clostridium sporogenes, and Peptostreptococcus glycinophilus, and their basic properties were determined. The enzyme isolated from P. glycinophilus showed the properties typical of dihydrolipoamide dehydrogenases: it was a dimer with a subunit molecular mass of 53,000 and contained 1 mol of flavin adenine dinucleotide and 2 redox-active sulfhydryl groups per subunit. Only NADH was active as a coenzyme for reduction of lipoamide. Spectra of the oxidized enzyme exhibited maxima at 230, 270, 353, and 453 nm, with shoulders at 370, 425, and 485 nm. The dihydrolipoamide dehydrogenases of C. cylindrosporum and C. sporogenes were very similar in their structural properties to the enzyme of P. glycinophilus except for their coenzyme specificity. The enzyme of C. cylindrosporum used NAD(H) as well as NADP(H), whereas the enzyme of C. sporogenes reacted only with NADP(H), and no reaction could be detected with NAD(H). Antibodies raised against the dihydrolipoamide dehydrogenase of C. cylindrosporum reacted with extracts of Clostridium acidiurici, Clostridium purinolyticum, and Eubacterium angustum, whereas antibodies raised against the enzymes of P. glycinophilus and C. sporogenes showed no cross-reaction with extracts from 42 organisms tested.     

Glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei: characterization of the enzyme, cloning and sequencing of the gene, and expression in Escherichia coli.

The glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei (optimal growth temperature, 100 to 103 degrees C) was purified to homogeneity. This enzyme was strictly phosphate dependent, utilized either NAD+ or NADP+, and was insensitive to pentalenolactone like the enzyme from the methanogenic archaebacterium Methanothermus fervidus. The enzyme exhibited a considerable thermostability, with a 44-min half-life at 100 degrees C. The amino acid sequence of the glyceraldehyde-3-phosphate dehydrogenase from P. woesei was deduced from the nucleotide sequence of the coding gene. Compared with the enzyme homologs from mesophilic archaebacteria (Methanobacterium bryantii, Methanobacterium formicicum) and an extremely thermophilic archaebacterium (Methanothermus fervidus), the primary structure of the P. woesei enzyme exhibited a strikingly high proportion of aromatic amino acid residues and a low proportion of sulfur-containing residues. The coding gene of P. woesei was expressed at a high level in Escherichia coli, thus providing an ideal basis for detailed structural and functional studies of that enzyme.     

Effects of nicotinamide coenzymes on the stability of enzyme activities and proteins in niacin-deficient quail tissues against trypsin treatment

The stability of liver and muscle enzymes and proteins in niacin-deficient quail towards trypsin treatment in the presence and absence of coenzymes, NAD or NADP, was characterized. The protection of liver dehydrogenases by coenzymes was low when they are subjected to trypsin digestion for 60 min. In contrast, in the muscle there was substantial protection against trypsin inactivation of glyceraldehyde-3-phosphate dehydrogenase by NAD and of 6-phosphogluconate dehydrogenase by NADP. Among all enzymes tested, glyceraldehyde-3-phosphate dehydrogenase showed the greatest protection against trypsin inactivation by NAD. SDS-polyacrylamide gel electrophoresis demonstrated that muscle proteins from the niacin-deficient group were more substantially protected compared to control and pair-fed groups when liver and muscle extracts were spiked with NAD and subjected to trypsin digestion. Overall results suggest that niacin deficiency exerted specific destabilizing effects on the stability of enzymes and proteins in muscle.