Biomedical subjects
Publications and source records attributed to R Wallin.
The subcellular localization of acarboxyprothrombin and the vitamin K-dependent carboxylation system has been studied using livers from vitamin K-deficient rats and found to be mainly in the membranes of the rough microsomal fraction. Bovine acarboxyprothrombin or fragment 1 thereof were inactive as substrates in the rat carboxylating system.
NAD(P)H dehydrogenase ('DT-diaphorase', EC 1.6.99.2) and vitamin K epoxidase were removed by affinity chromatography from detergent-solubilized microsomal fractions. Thereby the microsomal fractions normally carrying out vitamin K1-dependent carboxylation of the microsomal precursor proteins of the prothrombin complex were inactivated. Purified NAD(P)H dehydrogenase added to this system restored carboxylation in the presence of vitamin K1 (2-methyl-3-phytyl-1,4-naphthoquinone) plus NADH. Vitamin K1 hydroquinone (2-methyl-3-phytyl-1,4-naphthoquinol) had no effect, in contrast with its effect in the intact system, where it can substitute for vitamin K1 plus NADH. The ability of NAD(P)H dehydrogenase to restore carboxylation in a system without vitamin K epoxidase activity shows that there is no obligatory coupling of the vitamin K1-dependent carboxylation with vitamin K1 epoxidation. These results suggest that the form of vitamin K1 that is active in the carboxylation reaction can be produced independently in two reactions: by NAD(P)H dehydrogenase in the reduction of the quinone and by vitamin K epoxidase in the epoxidation of the hydroquinone.
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A fibrinolysis inhibitor was purified in serum from post-traumatic patients by the use of flat bed electrofocusing of serum desalted by gel chromatography followed by affinity chromatography on a column of matrix-linked plasminogen. Disc gel electrophoresis yielded one protein band. The inhibitor protein was also found in normal serum, but in a lower concentration.
A simple three-step method was established for the purification of NAD(P)H dehydrogenase (quinone) ('DT-diaphorase', EC 1.6.99.2) from rat liver by affinity chromatography with a recovery of above 50%. The final enzyme preparation was purified about 750-fold and was electrophoretically homogeneous. Gel filtration showed that the enzyme had a mol.wt. of about 55 000, and one molecule of FAD was found per 55 000 mol.wt. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave a mol.wt. of about 27 000. Two N-terminal amino acids, asparagine/aspartic acid and glutamine/glutamic acid, were found in about equal yield, suggesting the presence of two non-identical polypeptide chains in the enzyme. NAD(P)H dehydrogenase was selectively removed by this affinity-chromatographic method from a microsomal carboxylation system. The system, which was solubilized by detergent and is dependent on vitamin K (2-methyl-3-phytyl-1,4-naphthaquinone or analogues with other side chains), lost its activity on the removal of the enzyme. The activity can be completely restored to the system by adding purified cytoplasmic NAD(P)H dehydrogenase or by using the quinol form of vitamin K1 (2-methyl-3-phytyl-1,4-naphthaquinol).
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Some immunological studies on prothrombin fragment 1 from bovine prothrombin and its warfarin-induced precursor acarboxyprothrombin are reported. Based on the results, a rapid and simple immunoadsorption method for the isolation of prothrombin fragment 1 in good yield has been established. The method exploits the conformational change induced in the fragment by removal of Ca2+. The principle may be applicable to other gamma-carboxyglutamyl-containing proteins or fragments therof.
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The flavoprotein DT-diaphorase (EC 1.6.99.2) is believed to play an important role in the body's defense system. This enzyme has been purified 13,000-fold with a recovery of 58% from a cytosolic fraction of abdominal fat obtained from an obese patient undergoing elective surgery. Purification of the enzyme to electrophoretic homogeneity was achieved after two chromatographic steps: (1) affinity chromatography on azodicumarol Sepharose 6B; (2) anion exchange chromatography on DEAE Sephacel. The enzyme exhibits a monomer molecular mass of 32 kDa in SDS-PAGE and has 1 FAD prosthetic group per 32 kDa monomer. The FAD prosthetic group appears to be firmly attached to the apoproprotein. The enzyme reduces azodyes and quinones and demonstrates a broad substrate specificity. The enzyme has characteristics that are similar to DT-diaphorase purified from rodent liver, especially the rat liver enzyme. Estimated Km values for NADH, NADPH and menadione are 200, 140 and 3.3 microM, respectively. Vmax values for these substrates in the same order are 762, 667 and 294 mumol/mg.min. Dicumarol and warfarin exhibited competitive inhibition with pyridine nucleotides. The inhibition constants (Ki) for the drugs were estimated to be 10 nM and 2.2 microM, respectively. When compared to several other tissues, abdominal fat has one of the highest DT-diaphorase activities (Martin, L.F., Patrick, S.D. and Wallin, R. (1987) DT-diaphorase in morbidly obese patients. Cancer Lett., 36, 341-347), but the specific role of the enzyme in human fat is unknown.