Fructose is a good substrate for rat liver ''glucokinase'' (hexokinase D).

Rat liver 'glucokinase' (hexokinase D) catalyses the phosphorylation of fructose with a maximal velocity about 2.5-fold higher than that for the phosphorylation of glucose. The saturation function is hyperbolic and the half-saturation concentration is about 300 mM. Fructose is a competitive inhibitor of the phosphorylation of glucose with a Ki of 107 mM. Fructose protects hexokinase D against inactivation by 5,5'-dithiobis-(2-nitrobenzoic acid), and the apparent dissociation constants are about 300 mM in the presence of different concentrations of the inhibitor. The co-operativity of the enzyme in the phosphorylation of glucose can be abolished by addition of fructose to the reaction medium. Fructose appears to be no better as a substrate for the other mammalian hexokinases than it is for hexokinase D. It is proposed that the name 'glucokinase' ought to be reserved for enzymes that are truly specific for glucose, such as those of micro-organisms and invertebrates, and that liver glucokinase must be called hexokinase D (or hexokinase IV) within the classification EC 2.7.1.1.     

The taxonomic status of "Halobacterium marismortui" from the Dead Sea: a comparison with Halobacterium vallismortis

A Halobacterium strain, isolated by Ginzburg et al. from the Dead Sea in the late 1960's, often referred to as "Halobacterium marismortui" or "Halobacterium of the Dead Sea" (deposited in the American Type Culture Collection as ATCC 43049) was compared with Halobacterium (Haloarcula) vallismortis ATCC 29715. The strains appeared to be very closely related, as shown by the near identity of their 5S and 16S ribosomal RNA's, and a large number of other common properties. Distinct differences exist, however, in cell morphology, and in their potency to utilize different sugars and other compounds.     

Helicase-defective RuvB(D113E) promotes RuvAB-mediated branch migration in vitro.

In Escherichia coli, the RuvA and RuvB proteins interact at Holliday junctions to promote branch migration leading to the formation of heteroduplex DNA. RuvA provides junction-binding specificity and RuvB drives ATP-dependent branch migration. Since RuvB contains sequence motifs characteristic of a DNA helicase and RuvAB exhibit helicase activity in vitro, we have analysed the role of DNA unwinding in relation to branch migration. A mutant RuvB protein, RuvB(D113E), mutated in helicase motif II (the DExx box), has been purified to homogeneity. The mutant protein forms hexameric rings on DNA similar to those formed by wild-type protein and promotes branch migration in the presence of RuvA. However, RuvB(D113E) exhibits reduced ATPase activity and is severely compromised in its DNA helicase activity. Models for RuvAB-mediated branch migration that invoke only limited DNA unwinding activity are proposed.     

Cl(-) concentration dependence of photovoltage generation by halorhodopsin from Halobacterium salinarum

The photovoltage generation by halorhodopsin from Halobacterium salinarum (shR) was examined by adsorbing shR-containing membranes onto a thin polymer film. The photovoltage consisted of two major components: one with a sub-millisecond range time constant and the other with a millisecond range time constant with different amplitudes, as previously reported. These components exhibited different Cl(-) concentration dependencies (0.1-9 M). We found that the time constant for the fast component was relatively independent of the Cl(-) concentration, whereas the time constant for the slow component increased sigmoidally at higher Cl(-) concentrations. The fast and the slow processes were attributed to charge (Cl(-)) movements within the protein and related to Cl(-) ejection, respectively. The laser photolysis studies of shR-membrane suspensions revealed that they corresponded to the formation and the decay of the N intermediate. The photovoltage amplitude of the slow component exhibited a distorted bell-shaped Cl(-) concentration dependence, and the Cl(-) concentration dependence of its time constant suggested a weak and highly cooperative Cl(-)-binding site(s) on the cytoplasmic side (apparent K(D) of approximately 5 M and Hill coefficient > or =5). The Cl(-) concentration dependence of the photovoltage amplitude and the time constant for the slow process suggested a competition between spontaneous relaxation and ion translocation. The time constant for the relaxation was estimated to be >100 ms.     

Evidence for a role of high Km aldehyde reductase in the degradation of endogenous gamma-hydroxybutyrate from rat brain

gamma-Hydroxybutyrate (GHB) is a putative neurotransmitter in brain. We have already demonstrated that it is transformed into gamma-aminobutyrate (GABA) by rat brain slices incubated under physiological conditions. This conversion occurs via a GABA-transaminase reaction. Therefore, succinic semialdehyde, the oxidative derivative of GHB, appears to be the primary catabolite of GHB degradation. Apparently, the kinetic characteristics and pH optimum of GHB dehydrogenase (high Km aldehyde reductase) in vitro do not favor a role for this enzyme in endogenous brain GHB oxidation. However, in the presence of glucuronate, glutamate, NADP and pyridoxal phosphate, pure GHB dehydrogenase, coupled to purified GABA-transaminase does produce GABA from GHB at an optimum pH close to the physiological value and with a low Km for GHB.     

Brief communication: immunoglobulin (Gm and Km) allotypes in two populations of Catalonia (Spain)

Serum samples from two populations of Catalonia, Spain, 208 from Olot (Gerona) and 209 from Tortosa (Tarragona), were typed for G1m (1, 2, 3, 17), G3m (5, 10, 11, 13, 14, 15, 16, 26), and Km (1). The Gm patterns of the Catalonian populations are characterized by the presence of four haplotypes, Gm 1,17;21,26 Gm 1,2,17;21,26 Gm 1,3;5,10,11,13,14,26 and Gm 3;5,10,11,13,14,26. The homogeneity for haplotype Gm 1,17;21,26 among our data and other European populations suggests the existence of an isofrequency line which starts from the Mediterranean zone of Iberian Peninsula and continues through the northwestern part of Europe. From this line a decreasing cline towards the south can be observed. For the haplotype Gm 1,2;17,21,26, affinities are observed between Catalonian populations and other populations from central Europe. This confirms the existence of a gradient towards low values from NW to SE. The presence of the typical Mongoloid haplotype Gm 1,3;5,10,11,13,14,26 is discussed in this paper. No significant differences in the frequencies of the Km1 allele were observed among the European populations.     

Purification of a manganese-containing superoxide dismutase from Halobacterium halobium

An oxygen-induced superoxide dismutase was purified from the halophilic bacterium, Halobacterium halobium, strain NRL. Due to the high salt requirement for enzyme stability, the purification had to be performed in the presence of 2 M NaCl. The pI of the protein was 4.95. The approximate Mr was 38,500. The subunit size as determined by sodium dodecyl sulfate-electrophoresis was approximately 19,000. Metal analysis showed 1.5 atoms of manganese per dimer, 0.5 atom zinc, and 1.54 atoms copper. The N-terminal sequence of amino acids was determined, and based upon the first 26 amino acids significant homology to other manganese- and iron-containing superoxide dismutases was revealed.     

Defluorination of 1,1,1,2-tetrafluoroethane (R-134a) by rat hepatocytes

As part of its toxicological evaluation we assessed the in vitro metabolism of 1,1,1,2-tetrafluoroethane (R-134a), a non-ozone-depleting chemical likely to replace dichlorodifluoromethane (R-12) as an air-conditioning refrigerant. Hepatocyte suspensions in sealed flasks produced increasing quantities of F- (detected in the liquid media) as the headspace concentration of R-134a increased from 1% to 50% (balance of atmosphere 95% O2-5% CO2); the kinetics of defluorination suggested substrate-saturation. Little F- was detected in cultures without R-134a or in cell suspensions heated prior to addition of R-134a. Halothane (1,1,1-trichloro-2-bromo-2-chloro-ethane), although not defluorinated by hepatocytes maintained with 95% O2, inhibited defluorination of R-134a. Hepatocytes from phenobarbital-treated rats dehalogenated high (greater than or equal to 25%) concentrations of R-134a at greater rates than cells from untreated rats. These findings are consistent with the hypothesis that oxidative metabolism of R-134a by cytochrome P-450 can occur in vivo.