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Biomedical subjects

H M Lenhoff

Publications and source records attributed to H M Lenhoff.

At least 19 recordsLinked to original sources
PubMed1987

Spectrophotometric assay for ornithine decarboxylase.

A rapid and sensitive spectrophotometric assay for ornithine decarboxylase is described. It is based on the observation that the product of ornithine decarboxylase, putrescine, reacts with 2,4,6-trinitrobenzenesulfonic acid to give a colored product soluble in 1-pentanol whereas ornithine does not. The amount of putrescine produced by the enzyme was determined by measuring the absorbance of the 1-pentanol extract of the reaction mixture at 420 nm, and by comparing the results to those obtained by the trapping of 14CO2 and by HPLC assays. The three assays were found to be equivalent in sensitivity, with the spectrophotometric assay having the advantages of being relatively rapid, requiring only common laboratory equipment, and not requiring the use of radioactive isotopes.

Ornithine Decarboxylase↗
PubMed1983

Antibody-induced conformational restriction as basis for new separation-free enzyme immunoassay.

Under acid denaturing conditions, hologlucose oxidase labeled with 2,4-dinitrophenyl (DNP) was dissociated into flavin adenine dinucleotide (FAD) and DNP-labeled apoglucose oxidase (DNP-AG). Both lacked catalytic activity. The activity was restored by combining FAD and DNP-AG at about pH 7. If, on the other hand, anti-DNP serum was preincubated with the DNP-AG prior to the addition of FAD, activity was not restored. Furthermore, added DNP-aminocaproic acid counteracted the effects of the antibody in inhibiting the recombining of DNP-AG and FAD to form active enzyme. The anti-DNP serum probably prevented the DNP-AG from combining with FAD to form an active holoenzyme by restricting the mobility of the polypeptide chain of DNP-AG from folding into a catalytically active conformation. Based on such an antibody-induced conformational restriction of the DNP-AG, we developed a separation-free (homogeneous) enzyme immunoassay called AICREIA.

Apoenzymes↗
PubMed1983

Tyrosine decarboxylase. Spectrophotometric assay and application in determining pyridoxal-5'-phosphate.

We have developed a highly sensitive and rapid spectrophotometric assay for tyrosine decarboxylase that can be applied to determining pyridoxal-5'-phosphate. In the assay, tyramine, a product of tyrosine decarboxylation, reacts with 2,4,6-trinitrobenzenesulfonic acid to give a product soluble in toluene whereas tyrosine does not. We determined the amount of tyramine produced enzymatically by reading the absorbance at 340 nm of a toluene extract of the reaction mixture. This method is capable of detecting as low as 2.9 micrograms/mL of the enzyme. Using this method, we find the Km for tyrosine decarboxylase from Streptococcus faecalis to be 3.55 X 10(-4)M. We have also developed a specific and extremely sensitive method for determining pyridoxal-5'-phosphate, a cofactor of the enzyme, by using this spectrophotometric assay with apotyrosine decarboxylase.

Enterococcus faecalis↗
PubMed1983

Amperometric assay for collagenase. Amplification by the use of glucose oxidase conjugated to insoluble collagen.

A sensitive and rapid amperometric assay for collagenase has been developed. The substrate for the assay is glucose oxidase covalently linked to insoluble collagen with dimethylsuberimidate. The collagenase cleaves the insoluble collagen--glucose oxidase conjugate into smaller, soluble fragments that have glucose oxidase activity. That activity is proportional to the collagenase activity hydrolyzing the insoluble conjugate. In the absence of collagenase, no glucose oxidase activity is found in the soluble phase. Glucose oxidase activity was assayed by measuring amperometrically the rate at which hydrogen peroxide is produced. The kinetics follow that proposed for a soluble enzyme acting on an insoluble substrate.

Animals↗
PubMed1982

Enzymes as versatile labels and signal amplifiers for monitoring immunochemical reactions.

Enzymes have proven to be sensitive and versatile labels for immunochemical assays. The sensitivity of an enzyme label stems from its extraordinary catalytic power which in turn provides a great amplification of signals. Its versatility, however, stems from the fact that enzyme activity can be modulated by a number of substances. Enzyme labeled immunoassays can be divided into two categories: (a) heterogeneous and (b) homogeneous (non-separation). In the heterogeneous systems, the quantitation of the antibody bound and unbound fractions requires a physical separation of these two fractions, whereas the homogeneous or non-separation systems do not require such a separation. In the homogeneous systems, the unbound and antibody bound fractions can be distinguished functionally. A total of 11 unique principles used in the development of enzyme labeled immunoassays are described. The advantages, disadvantages and limitations of them are considered, as well as the future paths for research and developments.

Antigens↗
PubMed1982

Structural and conformational properties of peptides interacting with the glutathione receptor of hydra.

We have made a detailed investigation of structure-activity relationships of the glutathione-induced feeding response in the fresh water coelenterate, Hydra attenuata, to map structural and conformational properties of feeding response agonists. We find that the gamma-glutamyl residue of glutathione contains essential binding sites for receptor interaction, without which antagonistic as well as agonistic properties are lost. Any structural alteration which perturbs either the alpha-amino or the alpha-carboxyl group or their relative spatial orientations within the peptide has yielded an inactive derivative. An absolute requirement for activation of the receptor is a second-residue side chain of the appropriate size; analogues with second-residue side chains too large or too small are inhibitory. On the basis of the activity of conformational analogues of glutathione, torsional angles for the second residue equal to those of a right-handed alpha-helix are compatible with stimulus generation.

Animals↗
PubMed1982

Optimizing the o-phenylenediamine assay for horseradish peroxidase: effects of phosphate and pH, substrate and enzyme concentrations, and stopping reagents.

Horseradish peroxidase, assayed with o-phenylenediamine, is irreversibly inactivated when incubated in phosphate buffer, 100 mmol/L, at pH 5. The inactivation depends on both duration and incubation and phosphate concentration. Phosphate was the most potent inactivator and citrate the least potent of a series of buffers tested. The inactivation is not attributable to ionic strength per se or to Na+ or K+. The observed inactivation did not occur at high concentrations (2500 nmol/L, 0.1 g/L) of enzyme; however, this "protective" effect could not be reproduced by adding bovine serum albumin or a surfactant (Tween 20) to lower concentrations of enzyme. The inactivation was independent of commercial source of the enzyme or the kind of chromogenic assay used. On the basis of this information, we optimized the assay so that it gave eightfold greater absorbance values than those reported by others. The improved assay was sensitive to as little as 0.4 pmol/L (16 ng/L) of peroxidase, and was linear over the range of 0.4 to 5 pmol/L (16-200 ng/L).

Buffers↗
PubMed1981

p-Azidophenylglyoxal. A heterobifunctional photoactivable cross-linking reagent selective for arginyl residues.

1. The synthesis described is of p-azidophenylglyoxal (p-APG) by diazotization of p-aminoacetophenone to an intermediate which when reacted with sodium azide gives p-azidoacetophenone; oxidation of the latter with selenium dioxide gives rise to p-APG (corrected melting point, 103-105 degrees C). The phenylglyoxal moiety was designed to react with arginine residues, whereas the p-azidoaryl function generates a reactive nitrene when activated with UV light; p-APG reacts most selectively with arginine and to a lesser extent with cystine and histidine. 2. p-APG has absorption peaks at 205 and 280 nm which decrease on photolysis. 3. Bovine heart lactic dehydrogenase, egg white lysozyme, horse liver alcohol dehydrogenase, and yeast alcohol dehydrogenase, all enzymes having arginyl residues at their active sites, are inhibited by p-APG in the dark. 4. Gel electrophoresis of oligomeric enzymes having arginyl residues at their active sites and exposed to p-APG and to UV irradiation gave varying proportions of monomers and photocross-linked dimers, trimers, tetramers, and larger molecular weight aggregates.

Alcohol Oxidoreductases↗
PubMed1981

Hydra mesoglea: a model for investigating epithelial cell--basement membrane interactions.

Isolated hydra mesoglea served as a suitable substrate for the attachment and spreading of hydra cells in vitro, irrespective of the species tested. Hydra cells did not attach and spread on substrates typically used for culturing mammalian cells. Mammalian and Drosophila cells attached and spread on plastic culture dishes but not on isolated mesoglea. Xenopus epithelial cells spread on both plastic and mesoglea. Because of the similarities of hydra mesoglea to vertebrate basement membranes, suggestions are offered for using mesoglea to study the interactions of epithelial cells with their basement membranes.

Animals↗
PubMed1978

Interaction of glutathione analogues with Hydra attenuata gamma-glutamyltransferase.

gamma-Glutamyltransferase activity was studied in extracts of the cnidarian Hydra attenuata. The binding of gamma-glutamyl peptide analogues to the enzyme was studied by observing their effects on heat denaturation and their inhibition of p-nitroaniline release from gamma-glutamyl p-nitroanilide. Neither position-1 analogues, in which the gamma-glutamyl moiety was changed to a beta-aspartyl (beta-Asp-Abu-Gly) or an alpha-glutamyl (Glu-Abu-Gly) linkage, nor glutamate protected the enzyme against inactivation at 58 degrees C. GSH (reduced glutathione), gamma-Glu-Abu-Gly and gamma-Glu-Met on the other hand did prevent heat denaturation. GSH and analogues of GSH were competitive inhibitors of p-nitroaniline release, but those analogues in which glycine was replaced by 2-aminoisobutyrate, phenylalanine, leucine or tyrosine had Ki values that were approximately five times those of analogues with the cysteine residue replaced.

Amino Acids↗