Genotype and allele frequency of the 27-bp tandem repeat polymorphism in the endothelial nitric oxide synthase gene in Chinese population.

Genotype and allele frequency of the polymorphic 27-bp repeat, a variable number of tandem repeats (VNTR) located in intron 4 of the endothelial nitric oxide synthase gene, were analyzed in 316 healthy Chinese individuals. Four genotypes, namely 6/5-repeats heterozygous, 5/5-repeats homozygous, 5/4-repeats heterozygous and 4/4-repeats homozygous, were identified. Both observed genotype and allele frequencies of this VNTR in Chinese were similar to those of Japanese, while the 4/4-repeats genotype differed significantly from that of Caucasians in Spain, and all ones did from those of African-American in United States.

Characterization of amino acid aminotransferases of Methanococcus aeolicus.

Four aminotransferases were identified and characterized from Methanococcus aeolicus. Branched-chain aminotransferase (BcAT, EC 2.6.1.42), aspartate aminotransferase (AspAT, EC 2.6.1.1), and two aromatic aminotransferases (EC 2.6.1.57) were partially purified 175-, 84-, 600-, and 30-fold, respectively. The apparent molecular weight, substrate specificity, and kinetic properties of the BcAT were similar to those of other microbial BcATs. The AspAT had an apparent molecular weight of 162,000, which was unusually high. It had also a broad substrate specificity, which included activity towards alanine, a property which resembled the enzyme from Sulfolobus solfataricus. An additional alanine aminotransferase was not found in M. aeolicus, and this activity of AspAT could be physiologically significant. The apparent molecular weights of the aromatic aminotransferases (ArAT-I and ArAT-II) were 150,000 and 90,000, respectively. The methanococcal ArATs also had different pIs and kinetic constants. ArAT-I may be the major ArAT in methanococci. High concentrations of 2-ketoglutarate strongly inhibited valine, isoleucine, and alanine transaminations but were less inhibitory for leucine and aspartate transaminations. Aromatic amino acid transaminations were not inhibited by 2-ketoglutarate. 2-Ketoglutarate may play an important role in the regulation of amino acid biosynthesis in methanococci.

Characterization of enzymes of the branched-chain amino acid biosynthetic pathway in Methanococcus spp.

Methanococcus aeolicus, Methanococcus maripaludis, and Methanococcus voltae contain similar levels of four enzymes of branched-chain amino acid biosynthesis: acetohydroxy acid synthase, acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, and transaminase B. Following growth at low partial pressures of H2-CO2, the levels of these enzymes in extracts of M. voltae are reduced three- to fivefold, which suggests that their synthesis is regulated. The enzymes from M. aeolicus were found to be similar to the eubacterial and eucaryotic enzymes with respect to molecular weights, pH optima, kinetic properties, and sensitivities to O2. The acetohydroxy acid isomeroreductase has a specific requirement for Mg2+, and other divalent cations were inhibitory. It was stimulated threefold by K+ and NH4+ ions and was able to utilize NADH as well as NADPH. The partially purified enzyme was not sensitive to O2. The dihydroxy acid dehydratase is extremely sensitive to O2, and it has a half-life under 5% O2 of 6 min at 25 degrees C. Divalent cations were required for activity, and Mg2+, Mn2+, Ni2+, Co2+, and Fe2+ were nearly equally effective. In conclusion, the archaebacterial enzymes are functionally homologous to the eubacterial and eucaryotic enzymes, which implies that this pathway is very ancient.

Sulfometuron methyl-sensitive and -resistant acetolactate synthases of the archaebacteria Methanococcus spp.

The herbicide sulfometuron methyl (SM) inhibited growth of some methanococci. Of 28 strains tested, the growth of 7 was completely inhibited by 0.55 mM SM. Growth of an additional 14 strains was partially inhibited, and the growth of 7 strains was unaffected by this concentration of SM. In some cases, the branched-chain amino acids protected growth. Growth inhibition was correlated with the Ki for SM of acetolactate synthase (ALS). For the enzymes from bacteria representative of the sensitive, partially resistant, and resistant methanococci (Methanococcus aeolicus, Methanococcus maripaludis, and Methanococcus voltae, respectively), the Ki for SM was 0.0012, 0.34, and greater than 1.0 mM, respectively. Inhibition was uncompetitive with respect to pyruvate. Based on these observations, ALS appeared to be the major if not the sole site of action of SM in the methanococci. The sensitivity of the ALS from these three methanococci to feedback inhibition by branched-chain amino acids was also quite different. Although all three were sensitive to feedback inhibition by valine, the Ki varied 20-fold, from 0.01 to 0.22 mM. Moreover, only the ALS from M. maripaludis was sensitive to inhibition by leucine, and the Ki was 1.8 mM. The Ki for isoleucine for the ALS from both M. maripaludis and M. voltae was about 0.1 mM. The ALS from M. aeolicus was not inhibited by isoleucine. In other respects, the ALSs from the methanococci were very similar. After dialysis, thiamine pyrophosphate but not FAD and Mg2+ was required for maximal activity, and they were all rapidly inactivated by oxygen. Although the methanococcal ALSs exhibited diverse properties, the range of catalytic and regulatory properties closely resembled those of the eubacterial enzymes.