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21.
Purification and characterization of the Danish (skive) variant of mouse liver alcohol dehydrogenase
Douglas K. Rex William F. Bosron Francis Dwulet Ting-Kai Li 《Biochemical genetics》1987,25(1-2):111-121
The partially inbred Danish (Skive) strain of mice exhibits a form of liver alcohol dehydrogenase (ADH) which differs in electrophoretic mobility from that of all other inbred mouse strains thus far examined, e.g., C57BL/10, DBA/2J, and BALB/c. In order to compare the catalytic and molecular properties of the variant and normal enzyme forms, they were purified to homogeneity by ion-exchange and affinity chromatography. Tryptic peptides of reduced and carboxymethylated subunits of the normal and variant ADH forms were mapped by thin-layer two-dimensional electrophoresis and chromatography and by reversed-phase high-performance liquid chromatography. A unique nonapeptide in the Danish mouse liver ADH which did not appear in enzymes from C57BL/10, DBA/2J, or BALB/c mice was identified by both methods. Amino acid sequencing of this peptide revealed that the Arg residue at position 124, as predicted from the cDNA sequence of ADH in DBA/2J mice, has been replaced by Leu in the Danish variant. The Leu for Arg substitution in the variant form appears to account for its decreased cathodic mobility with electrophoresis in starch gels at pH 7.2. The K
m
and V
max
of ADH from the Danish strain for three primary alcohols and three aldehydes were similar in value to those of ADH from the C57BL/10, DBA/2J, and BALB/c strains. Based on the X-ray structure of horse liver ADH, position 124 is on the solvent-exposed surface of the catalytic domain. The finding that the kinetic constants are similar for the normal and variant forms is consistent with the observation that this residue is not in the active site and that there is no known role for it in the ADH catalytic mechanism.This work was supported by NIAAA Grant AA-04307. 相似文献
22.
Summary The complete nucleotide sequence of the Salmonella strain LT2 gnd gene for 6-phosphogluconate dehydrogenase was determined. The gene contains 1404 bases and encodes a 468 amino acid polypeptide, which is the same as for Escherichia coli K12. The DNA sequence shows 14.8% difference between the two and the amino acid sequence 3.6% difference. Changes are mostly in the third codon base and most of the amino acid changes are conservative. 相似文献
23.
Cell extracts (27000xg supernatant) of acetate grown Methanosarcina barkeri were found to have carbonic anhydrase activity (0.41 U/mg protein), which was lost upon heating or incubation with proteinase K. The activity was inhibited by Diamox (apparent K
i=0.5 mM), by azide (apparent K
i=1 mM), and by cyanide (apparent K
i=0.02 mM). These and other properties indicate that the archaebacterium contains the enzyme carbonic anhydrase (EC 4.2.1.1). Evidence is presented that the protein is probably located in the cytoplasm. Methanol or H2/CO2 grown cells of M. barkeri showed no or only very little carbonic anhydrase activity. After transfer of these cells to acetate medium the activity was induced suggesting a function of this enzyme in acetate fermentation to CO2 and CH4. Interestingly, Desulfobacter postgatei and Desulfotomaculum acetoxidans, which oxidize acetate to 2 CO2 with sulfate as electron acceptor, were also found to exhibit carbonic anhydrase activity (0.2 U/mg protein). 相似文献
24.
N. Arfman E. M. Watling W. Clement R. J. van Oosterwijk G. E. de Vries W. Harder M. M. Attwood L. Dijkhuizen 《Archives of microbiology》1989,152(3):280-288
The enzymology of methanol utilization in thermotolerant methylotrophic Bacillus strains was investigated. In all strains an immunologically related NAD-dependent methanol dehydrogenase was involved in the initial oxidation of methanol. In cells of Bacillus sp. C1 grown under methanol-limiting conditions this enzyme constituted a high percentage of total soluble protein. The methanol dehydrogenase from this organism was purified to homogeneity and characterized. In cell-free extracts the enzyme displayed biphasic kinetics towards methanol, with apparent K
m values of 3.8 and 166 mM. Carbon assimilation was by way of the fructose-1,6-bisphosphate aldolase cleavage and transketolase/transaldolase rearrangement variant of the RuMP cycle of formaldehyde fixation. The key enzymes of the RuMP cycle, hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), were present at very high levels of activity. Failure of whole cells to oxidize formate, and the absence of formaldehyde-and formate dehydrogenases indicated the operation of a non-linear oxidation sequence for formaldehyde via HPS. A comparison of the levels of methanol dehydrogenase and HPS in cells of Bacillus sp. C1 grown on methanol and glucose suggested that the synthesis of these enzymes is not under coordinate control.Abbreviations RuMP
ribulose monophosphate
- HPS
hexulose-6-phosphate synthase
- HPI
hexulose-6-phosphate isomerase
- MDH
methanol dehydrogenase
- ADH
acohol dehydrogenase
- PQQ
pyrroloquinoline, quinone
- DTT
dithiothreitol
- NBT
nitrobluetetrazolium
- PMS
phenazine methosulphate
- DCPIP
dichlorophenol indophenol 相似文献
25.
26.
Michel R. Pâquet Robert J. Romanek Robert J. Sargeant 《Molecular and cellular biochemistry》1992,109(2):149-155
Summary In rat adipocytes, the insulin stimulation of the rate of glucose uptake is due, at least partially, to the recruitment of glucose transporter proteins from an intracellular compartment to the plasma membrane.Vanadate is a known insulin mimetic agent and causes an increase in the rate of glucose transport in rat adipocytes similar to that seen with insulin. The objective of the present study was to determine whether vanadate exerts its effect through the recruitment of glucose transporters to the plasma membrane.We report that under conditions where vanadate stimulates the rate of 2-deoxyglucose uptake to the same extent as insulin, the concentration of GLUT-4 in the plasma membrane was increased similarly by both insulin and vanadate, and its concentration was decreased in the low density microsomal fraction. These results suggest that vanadate induces the recruitment of GLUT-4 to the plasma membrane. The effects of vanadate and insulin on the stimulation of 2-deoxyglucose uptake and recruitment of GLUT-4 were not additive.This is the first report of an effect of vanadate on the intracellular distribution of the glucose transporter. 相似文献
27.
Thomas L. Pazdernik Matthew Layton Stanley R. Nelson Fred E. Samson 《Neurochemical research》1992,17(1):11-21
This overview presents data showing that glucose use increases and that excitatory amino acids (i.e., glutamate, aspartate), taurine and ascorbate increase in the extracellular fluid during seizures. During the cellular hyperactive state taurine appears to serve as an osmoregulator and ascorbate may serve as either an antioxidant or as a pro-oxidant. Finally, a unifying hypothesis is given for seizure-induced brain damage. This unifying hypothesis states that during seizures there is a release of excitatory amino acids which act on glutamatergic receptors, increasing neuronal activity and thereby increasing glucose use. This hyperactivity of cells causes an influx, of calcium (i.e. calcium stress) and water movements (i.e., osmotic stress) into the cells that culminate in brain damage mediated by reactive oxygen species.Special issue dedicated to Dr. Frederick E. Samson 相似文献
28.
Energy metabolism and selective neuronal vulnerability following global cerebral ischemia 总被引:9,自引:0,他引:9
Neil R. Sims 《Neurochemical research》1992,17(9):923-931
A short period of global ischemia results in the death of selected subpopulations of neurons. Some advances have been made in understanding events which might contribute to the selectivity of this damage but the cellular changes which culminate in neuronal death remain poorly defined. This overview examines the metabolic state of tissue in the post-ischemic period and the relationship of changes to the development of damage in areas containing ischemia-susceptible neurons. During early recirculation there is substantial recovery of ATP, phosphocreatine and related metabolites in all brain regions. However, this recovery does not signal restitution of normal energy metabolism as reductions of the oxidative metabolism of glucose are seen in many areas and may persist for several days. Furthermore, decreases in pyruvate-supported respiration develop in mitochondria from at least one ischemia-susceptible region at times coincident with the earliest histological evidence of ischemia-induced degeneration. These mitochondrial changes could simply be an early marker of irreversible damage but the available evidence is equally consistent with these contributing to the degenerative process and offering a potential site for therapeutic intervention.Submitted as an Overview article for the volume of Neurochemical Research in honor of Alan N. Davison. 相似文献
29.
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