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Biomedical subjects

J Everse

Publications and source records attributed to J Everse.

At least 55 records · Page 3Linked to original sources
PubMed1975

New instrument for rapid determination of activities of lactate dehydrogenase isoenzymes.

Lactate dehydrogenase isoenzymes can be distinguished kinetically by the fact that isoenzyme H is strongly inhibited a few seconds after the reaction is started if high concentrations of pyruvate are present, in contrast to the M isoenzyme. A new instrument that exploits this fact can measure both the total activity and the proportion of H isoenzyme in serum or plasma in 8 to 10 s. The instrument consists of a simplified stopped-flow apparatus in which the plasma is assayed for lactate dehydrogenase activity, and an electronic device that measures the rate of the reaction at two pre-set time intervals. The first rate is taken between 0.2 and 0.4 s after the reaction is started, a time at which both isoenzymes are fully active, and at which the rate obtained thus reflects total lactate dehydrogenase activity in the plasma sample. The second rate is measured 4 to 6 s after the start of the reaction, at which time the H isoenzyme has become inhibited and the observed rate compared to the initial rate is therefore proportional to the percentage of H isoenzyme activity in the serum. These two rates are electronically displayed on two three-digit voltmeters, the first display being the total activity, the second a number proportional to the inhibited slope. The percentage of M isoenzyme can then be calculated from the initial and final rate. A total of five to six repeat assays may be done within a minute on 1 ml of plasma or serum. This instrument may be of significant value in following the progress of myocardial infarctions and other diseases.

Autoanalysis↗
PubMed1974

Purification and separation of pyridine nucleotide-linked dehydrogenases by affinity chromatography techniques.

A number of different dehydrogenases have been shown to bind to Sepharose-bound N(6)-(6-aminohexyl)-AMP. These dehydrogenases can be specifically eluted by binary adducts of NAD(+) or with cofactor gradients. In such manner pure enzymes can be obtained from crude extracts, as demonstrated in the purification on a preparative scale of lactate dehydrogenase from dogfish muscle. The data presented indicate the usefulness of general ligands as affinity agents. The techniques are particularly adaptable for the isolation of human mutant enzymes in blood or in the purification and concentration of enzymes present at low levels in fluids or tissues, as shown in the extensive purification of serum lactate dehydrogenase and glucose 6-phosphate dehydrogenase from hemolysate. lsoenzymes with different affinities for co-enzymes can be separated by affinity techniques. Application of affinity techniques may lead to the separation of isoenzymes or mutant enzymes that are not separable by electrophoretic methods.

Adenosine Monophosphate↗
PubMed1973

Structure-function relationships in lactate dehydrogenase.

The binding of coenzyme and substrate are considered in relation to the known primary and tertiary structure of lactate dehydrogenase (EC 1.1.1.27). The adenine binds in a hydrophobic crevice, and the two coenzyme phosphates are oriented by interactions with the protein. The positively charged guanidinium group of arginine 101 then folds over the negatively charged phosphates, collapsing the loop region over the active center and positioning the unreactive B side of the nicotinamide in a hydrophobic protein environment. Collapse of the loop also introduces various charged groups into the vicinity of the substrate binding site. The substrate is situated between histidine 195 and the C4 position on the nicotinamide ring, and is partially oriented by interactions between its carboxyl group and arginine 171. The spatial arrangements of these groups may provide the specificity for the L-isomer of lactate.

Amino Acid Sequence↗
PubMed1972

Identification of lactate dehydrogenase isoenzymes by rapid kinetics.

Stopped-flow kinetics indicate that human and chicken heart-type(4) lactate dehydrogenases (LDH) become inhibited as DPNH is oxidized in the presence of high concentrations of pyruvate. This inhibition is much less marked with the human and chicken muscle-type(4) enzymes. The initial rates and the difference in inhibition between the two types of enzyme have made it possible to determine the amount of, as well as the ratio between, the two types of LDH that are present in a given sample by a single kinetic assay. The stopped-flow kinetic method has been used to analyze amounts of LDH isoenzyme in different tissues, as well as in serum.

Acetates↗
PubMed1970

Physiological concentrations of lactate dehydrogenases and substrate inhibition.

Lactate dehydrogenases at physiological concentrations are inhibited by high concentrations of pyruvate when the enzyme and the pyruvate are incubated in the presence of oxidized nicotinamide-adenine dinucleotide before assay. The inhibition is much more pronounced with the H-type than with the M-type lactate dehydrogenase. These results suggest that substrate inhibition may be operative in vivo.

Animals↗