Michael J. Raxworthy

Fabrication and reorganization of dermal equivalents suitable for skin grafting after major cutaneous injury

The incorporation of fibroblasts into a hydrated collagen lattice results in lattice contraction and collagen reorganization to form a dermal equivalent. Lattices fabricated with 7.7 mg collagen and seeded with 1 X 10(5) cells were found to give the best results in terms of their mechanical properties and ability to maintain cell viability. Newly-cast lattices were found to be completely digested by 0.085 units/ml bacterial collagenase in 3 h, whereas after 30 d in culture, limited digestion took place over 24 h. Electrophoretic analysis showed that the proportion of cross-linked collagen in the 30 d lattice was increased by 2.5-fold compared to the initial collagen preparation. These results indicate that a dermal equivalent better suited for grafting may be produced after 20-30 d in culture.

Benserazide and carbidopa as substrates of catechol-O-methyltransferase: New mechanism of action in Parkinson's disease

Abstract The DOPA-decarboxylase (aromatic- l -amino acid decarboxylase) inhibitors benserazide, its active metabolite 2,3,4-trihydroxybenzylhydrazine and carbidopa are substrates of highly purified catechol- O -methyltransferase. The affinity and maximal velocity of both benserazide and trihydroxy-benzyl hydrazine are extremely favourable implying that these compounds may compete as substrates for catechol- O -methyltransferase with l -DOPA. These results are discussed in the light of the ability of benserazide to cause further elevation of plasma l -DOPA than carbidopa and the use of these compounds in Parkinsons disease.

2-hydroxyethynyloestradiol as a substrate for catechol-O-methyltransferase--implications in the metabolism of ethynyloestradiol.

Highly purified pig catechol-O-methyltransferase catalyses the methylation of 2-hydroxyethynyloestradiol (KM - 11.0 microM, Vmax = 521.2 mU/mg protein, Vmax/KM = 47.4) more efficiently than that of 2-hydroxyoestradiol (KM = 68.5 microM, Vmax = 1056.2 mU/mg protein, Vmax/Km = 15.4), 2-hydroxyoestrone (KM = 38.0 microM, Vmax = 795.0 mU/mg protein, Vmax/KM = 20.9) or 4-hydroxyoestrone (KM = 12.8 microM, Vmax = 159.7, Vmax/KM = 12.5). This efficient methylation of the principal metabolite of ethynyloestradiol substantiates the implications of the studies of Bolt et al.[1] that O-methylation is a major route of ethynyloestradiol metabolism. Furthermore, this also implies that catechol-O-methyltransferase in involved in the protection, by S-adenoysylmethionine, against the impairment of bile secretion by ethynyloestradiol, observed in female rats [2].

THE METABOLISM OF DOPAMINE, NN-DIALKYLATED DOPAMINES AND DERIVATIVES OF THE DOPAMINE AGONIST 2-AMINO-DIHYDROXY-1,2,3,4-TETRAHYDRONAPHTHALENE (ADTN) BY CATECHOL-O-METHYLTRANSFERASE

A variety of dopamine derivatives and analogues were investigated to assess their potential to act as catechol‐O‐methyltransferase (COMT) substrates using purified, homogeneous pig liver enzyme. This enabled accurate kinetic constants to be determined as opposed to previous in‐vivo studies (Rollema et al 1980; Horn et al 1981; Costall et al 1982; Feenstra et al 1983). 2−Amino−6,7−dihydroxy−1,2,3,4−tetrahydronaphthalene (A−6,7−DTN) proved to be a far better substrate (Km = 0·082 mm; Vmax = 300 mu mg−1 protein) than its 5,6−dihydroxy isomer (Km = 2·60 mm; Vmax = 113·9 mu mg−1 protein). This result supports evidence suggesting that differences in brain concentration of these isomers are due to their differential susceptibility to O‐methylation by COMT (Rollema et al 1980). A similar result was obtained with a series of NN‐di‐n‐alkyl substituted ADTN derivatives: the same pattern of preferential O‐methylation of A−6,7−DTN derivatives over the corresponding A−5,6−DTN isomers was observed. However, increasing the length of the alkyl chain increased the susceptibility of both isomers to metabolism by COMT as shown by a decline in Km. An homologous series of NN‐di‐n‐alkylated dopamines showed a similar trend implying that more hydrophobic compounds are better COMT substrates.

The effect of bulk hydrogen ion concentration upon the apparent kinetic parameters of purified pig liver catechol O-methyltransferase

In order to investigate the pH dependence of catechol O-methyltransferase (S-adenosyl-L-methionine:catechol O-methyltransferase, EC 2.1.1.6), kinetic parameters have been determined for the highly purified enzyme from pig liver over the pH range 6.75-8.20 using the substrates S-adenosylmethionine (AdoMet) and 3,4-dihydroxyphenylacetic acid (DOPAC). The Km for AdoMet was found to be invariant with pH while the Km for DOPAC decreased sharply with increasing pH. The group responsible for the latter has a pK of approx. 7.1. The logarithmic (Dixon) plot of Km against pH for both substrates and that of Vmax/Km against pH for DOPAC mirror the kinetic behaviour revealed by linear plots. However, for other parameters, linear graphs indicate peaks too narrow to be explicable by a simple kinetic mechanism, whereas logarithmic plots of these parameters produce graphs apparently not reflecting this behaviour. We conclude that these results are not the products of random error or artefactual data analysis but are too complex to be explicable by a simple model of kinetic behaviour. Possible explanations (adherence of catechol O-methyltransferase to a higher-order mechanism or a dual mode of substrate binding) are advanced.