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Biomedical subjects

K Mosbach

Publications and source records attributed to K Mosbach.

At least 127 records · Page 7Linked to original sources
PubMed1980

The immobilization technique or solid-phase approach as an aid in investigating the interaction of subunits in oligomeric enzymes, notably of dehydrogenases.

The use of immobilization techniques or a solid-phase approach, allows several questions concerning interactions between sub-units of oligomeric enzymes, particularly dehydrogenases to be, answered are the monomers of ADH and LDH active per se, or are interactions necessary for their activity? Are the sub-units (such as those of ADH) identical from a kinetic point of view? Is the increase in activity due to the addition of ligands (as for GDH), the result of the formation of large aggregates, which appear simultaneously, or is this association a secondary effect? A technique using affinity chromatography, with the help of bifunctional nucleotide derivatives, is presented. This technique could prove useful for topographical studies, or provide new information on the spatial arrangements of the sub-units.

Enzymes, Immobilized↗
PubMed1980

p-Toluenesulfonyl chloride as an activating agent of agarose for the preparation of immobilized affinity ligands and proteins.

A number of biomolecules were coupled covalently by nucleophilic displacement to agarose preparations substituted with tosyl groups. In one series of experiments N6-(6-aminohexyl)-adenosine 5'-monophosphate and N6-(6-aminohexyl)adenosine 2',5'-bisphosphate were bound by their terminal amino groups to the polysaccharide support. It could be shown that from a mixture of lactate and 6-phosphogluconate dehydrogenase the immobilized monophosphate showed bio-affinity only for NAD+-dependent lactate dehydrogenase, whereas the immobilized bisphosphate showed affinity only for the NADP+-dependent 6-phosphogluconate dehydrogenase. Furthermore, the immobilized monophosphate (5 mumol/g wet gel) was applied for the single-step purification of lactate dehydrogenase from crude beef heart extract. To demonstrate the immobilization of proteins, soybean trypsin inhibitor (75 mg/g dry support) was immobilized to tosylated agarose, tested as affinity chromatography material and shown to bind 60 mg trypsin/g dry gel. Horseradish peroxidase and horse liver alcohol dehydrogenase were used as model enzymes. Although no optimization had been attempted, the former (approximately 70 mg/g dry support) had a coupling yield of approximately 18% with a specific activity (relative to soluble enzyme) of approximately 10%, whereas approximately 60% of alcohol dehydrogenase was coupled (approximately 100 mg/g dry support) with a specific activity of approximately 25%.

Chromatography, Affinity↗
PubMed1979

N6-[N-(6-Aminohexyl)carbamoylmethyl]-coenzyme A. Synthesis and application in affinity chromatography and as an immobilized active coenzyme.

The synthesis of a new coenzyme A analogue, N6-[N-(6-aminohexyl)carbamoylmethyl]-CoA, suitable for immobilisation through its terminal amino group to support matrices, is described. The synthetic route starts with bis(CoA) and involves the following steps: alkylation with iodoacetic acid and rearrangement yielding bis(N6-carboxymethyl-CoA), elongation of the carboxymethyl terminal with 1,6-diaminohexane using carbodiimide to yield bis(N6-[N-(6-aminohexyl)-carbamoylmethyl]-CoA) and finally the splitting of this bis[CoA analogue) through reduction with dithiothreitol to give the final product in approximately 10% overall yield. This CoA analogue showed 'coenzymic activity' with the enzymes acetyl-CoA synthetase, phosphotransacetylase and succinic thiokinase. Covalent binding of the CoA analogue to Sepharose 4B was normally carried out using its S-(5-thio-2-nitrobenzoic acid) derivative as this allows a convenient way for determining the amount of ligand coupled, based on the amount of 5-thio-2-nitrobenzoic acid liberated from the gel after reduction with dithiothreitol. After covalent binding of the CoA analogue to water-soluble activated dextran 70, the analogue was recycled while present in an ultrafiltration cell using the enzymes phosphotransacetylase and citrate synthase. The reaction was followed by measuring the citrate formed on addition of acetylphosphate and oxaloacetate. In affinity chromatographic studies it was shown that the CoA-Sepharose preparation could bind the CoA-dependent enzymes citrate synthase and succinic thiokinase and these could be biospecifically eluted using soluble CoA.

Animals↗
PubMed1979

The use of biochemical solid-phase techniques in the study of alcohol dehydrogenase. 1. Some studies on subunit interactions in alcohol dehydrogenase with subunits covalently bound to agarose.

The EE and SS isozymes of horse liver alcohol dehydrogenase have been immobilized separately to weakly CNBr-activated Sepharose 4B. The resulting immobilized dimeric preparations lost practically all of their activity after treatment with 6 M urea. However, enzyme activity was regenerated by allowing the urea-treated Sepharose-bound alcohol dehydrogenase to interact specifically with either soluble subunits of dissociated horse liver alcohol dehydrogenase or soluble dimeric enzyme. The regeneration of steroid activity in the immobilized preparations after treatment of the bound S subunits with soluble E subunits seems to show that true reassociation of the enzyme had taken place on the solid phase, since only isozymes with an S-polypeptide chain are active when using 5 beta-dihydrotestosterone as substrate. The results presented in this paper indicate that immobilized single subunits of horse liver alcohol dehydrogenase are inactive and that dimer formation is a prerequisite for the enzymic activity.

Alcohol Oxidoreductases↗