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R R Bowsher

Publications and source records attributed to R R Bowsher.

61 records · Page 4Linked to original sources
PubMed1985

Synthesis of N pi-methylhistamine and N alpha-methylhistamine by purified rabbit lung indolethylamine N-methyltransferase.

N tau-Methylhistamine is a major inactive metabolite of histamine and is formed by histamine N-methyltransferase (EC 2.1.1.8). However, a controversy exists concerning the presence of other N-methylated histamines, such as N pi- and N alpha-methylhistamine in mammalian tissues. Indolethylamine N-methyltransferase is present in mammalian tissues with the rabbit lung containing the highest concentration, but the physiologic function of this enzyme remains unclear. Using rabbit lung as a tissue source, we purified indolethylamine N-methyltransferase 260-fold and separated it completely from histamine N-methyltransferase. Histamine was a substrate for purified indolethylamine N-methyltransferase and unlike histamine N-methyltransferase which exclusively formed N tau-methylhistamine, indolethylamine N-methyltransferase catalyzed the in vitro formation of both N pi-methylhistamine and N alpha-methylhistamine. In contrast to histamine N-methyltransferase, indolethylamine N-methyltransferase activity was not inhibited by either high histamine concentrations or by quinacrine. Thus, mammalian tissues contain an enzyme capable of forming N pi-methylhistamine and N alpha-methylhistamine. This supports the concept of the existence of these compounds and suggests they may serve a physiologic function in mammals.

Animals↗
PubMed1984

Kinetic analysis of the histamine N-methyltransferase reaction as used in the histamine radioenzymatic assay: optimization of assay specificity.

The specificity of the histamine N-methyltransferase (HNMT) based radioenzymatic assay for histamine has been questioned since N-alpha-methylhistamine is also a substrate for this enzyme. Purification of HNMT for use in the radioenzymatic assay improves sensitivity and specificity of this procedure. In this investigation, further improvements in specificity, with respect to other HNMT substrates, were attained by optimization of reaction conditions based on the evaluation of HNMT kinetic parameters. These studies demonstrate that appropriate control of reaction temperature and concentration of both the enzyme and the radiolabeled methyl donor improve the specificity of this assay for histamine.

Animals↗
PubMed1983

Rat histamine N-methyltransferase. Quantification, tissue distribution, purification, and immunologic properties.

Histamine N-methyltransferase is a major enzyme for inactivating histamine in mammalian tissues. Development of a sensitive and specific assay for histamine N-methyltransferase has permitted quantification of this activity in 25 rat tissues. Additionally, renal histamine N-methyltransferase was purified to electrophoretic homogeneity. The pivotal step in the purification scheme was an affinity column procedure which involved the specific elution of histamine N-methyltransferase from DEAE-Sephacel by 1 mM histamine. The purified enzyme has a molecular weight of 33,400 and a narrow pH dependency with an optimum at 8.0-8.25. Specific antibody produced to renal histamine N-methyltransferase also immunoprecipitated the brain enzyme completely, therefore indicating neuronal and non-neuronal histamine N-methyltransferase share common antigenic determinants.

Animals↗
PubMed1983

A new radioenzymatic assay for histamine using purified histamine N-methyltransferase.

Radioenzymatic assays for histamine (Hm) have found wide application. However, these procedures may lack the sensitivity necessary to quantify Hm in certain biological samples, such as human plasma. Purification of histamine N-methyltransferase (HNMT) has permitted the development of a new and highly sensitive radioenzymatic assay for Hm. HNMT was purified by sequential ion exchange, hydrophobic and molecular exclusion chromatography. The use of purified HNMT in the Hm assay has allowed the inclusion of high specific activity tritiated S-adenosyl-L-methionine ([3H]SAME) and the development of a simplified solvent extraction product isolation procedure. This assay has a sensitivity of approximately 2 picograms and is specific for Hm. Hm was easily quantified in human plasma and was found to be 303 +/- 81 pg/ml (mean +/- SD) in 8 male subjects. Substantial blank reduction and increased product conversion occur when purified HNMT is utilized in the Hm radioenzymatic assay, thus, increasing the sensitivity and possibly improving the specificity of this procedure.

Animals↗
PubMed1983

Decarboxylation of p-tyrosine: a potential source of p-tyramine in mammalian tissues.

The question of the existence of a p-tyrosine decarboxylase pathway for the formation of p-tyramine in mammalian tissues remains unresolved. Development of a sensitive and specific assay for p-tyrosine decarboxylase has permitted demonstration of this activity in rat tissues and human kidney. Tyrosine decarboxylase was purified to electrophoretic homogeneity by pH 5.0 precipitation, ammonium sulfate precipitation, gel filtration, phenyl-Sepharose chromatography, DEAE-Sephacel chromatography, and preparative isoelectric focusing. A specific rabbit antiserum to tyrosine decarboxylase was also obtained. Purified tyrosine decarboxylase possessed a narrow pH dependency with an optimum at 8.0. Benzene and certain other organic solvents dramatically stimulated tyrosine decarboxylase activity of purified enzyme. Purified tyrosine decarboxylase activity also decarboxylated L-DOPA, 5-hydroxytryptophan, 3,4-dihydroxyphenylserine, o-tyrosine, m-tyrosine, phenylalanine, histidine, and tryptophan, which suggested that the purified enzyme was aromatic L-amino acid decarboxylase. This conclusion was supported by a constant ratio of 5-hydroxytryptophan decarboxylase to tyrosine decarboxylase throughout the purification scheme and by parallel immunoprecipitation of decarboxylase activities by the specific antityrosine decarboxylase antisera. Thus, we report that p-tyrosine is decarboxylated by aromatic L-amino acid decarboxylase and that this metabolic transformation may be an important source of p-tyramine in mammalian tissues. In conclusion, neuronal tissues that synthesize catecholamines or serotonin should now be considered capable of synthesizing p-tyramine and other biogenic amines.

Animals↗
PubMedDate not supplied

Urinary desmosine as a biomarker in acute lung injury.

Acute lung injury (ALI) is a complex disorder associated with an acute inflammatory response thought to contribute to tissue injury. Desmosine, a cross-linking amino acid present in elastin, is released during matrix degradation and cleared by the kidney. Results from animal models and human disease studies have suggested that ALI is associated with the release of desmosine, resulting in increased urinary desmosine. A radioimmunoassay was used to monitor urinary desmosine levels over 10 days in ten patients with ALI. The concentration of desmosine was measured with and without acid hydrolysis. Baseline urinary desmosine was increased in two of ten patients. The concentration of desmosine at baseline did not appear to be related to age, gender, neutrophil elastase (NE)/alpha(1)-antiprotease complex concentration or P(a)O(2)/F(i)O(2) ratio. No meaningful changes in desmosine levels were noted after removal from mechanical ventilation. Baseline desmosine concentrations did not appear to correlate with the risk of death. The limited sensitivity, predictive correlations and dynamic modulation would suggest that urine desmosine has a limited role as a biomarker for ALI. Hydrolysis of urine samples appears necessary for optimal measurement of urine desmosine.

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