Isolation, purification and characterization of a novel glucose oxidase from Penicillium sp. CBS 120262 optimally active at neutral pH.

A novel glucose oxidase (GOX), a flavoenzyme, from Penicillium sp. was isolated, purified and partially characterised. Maximum activities of 1.08U mg(-1)dry weight intracellular and 6.9U ml(-1) extracellular GOX were obtained. Isoelectric focussing revealed two isoenzymes present in both intra- and extracellular fractions, having pI's of 4.30 and 4.67. GOX from Penicillium sp. was shown to be dimeric with a molecular weight of 148kDa, consisting of two equal subunits with molecular weight of 70k Da. The enzyme displayed a temperature optimum between 25 and 30 degrees C, and an optimum pH range of 6-8 for the oxidation of beta-d-glucose. The enzyme was stable at 25 degrees C for a minimum of 10h, with a half-life of approximately 30 min at 37 degrees C without any prior stabilisation. The lyophilized enzyme was stable at -20 degrees C for a minimum of 6 months. GOX from Penicillium sp. Tt42 displayed the following kinetic characteristics: Vmax, 240.5U mg(-1); Km, 18.4mM; kcat, 741 s(-1) and kcat/Km, 40 s(-1)mM(-1). Stability at room temperature, good shelf-life without stabilisation and the neutral range for the pH optimum of this GOX contribute to its usefulness in current GOX-based biosensor applications.

Possible involvement of delta-6-desaturase in control of melanoma growth by gamma-linolenic acid.

This study examined the effects of linoleic acid (LA) and gamma-linolenic acid (GLA) on BL6 melanoma growth in cell culture and of safflower oil (SFO) which contains LA and evening primrose oil (EPO) which contains GLA, on melanoma growth when grown in mice. The delta-6-desaturase activity of the melanoma cells in the two systems was also examined and an attempt made to relate the activity of the enzyme to the effects of GLA on cell and tumour growth. LA and GLA were found to be equipotent in inhibiting growth of the in vitro cultured BL6 cells which were found to contain an appreciable level of delta-6-desaturase activity. EPO was however found to be a more potent promoter of in vivo melanoma growth in mice than SFO. Melanomas grown in mice were found to lack delta-6-desaturase activity suggesting that the EPO diet, by providing GLA, was able to compensate for the loss of enzyme activity in the melanomas. The possibility that melanomas in mice have a requirement for GLA for growth while in in vitro cultured cells excess GLA inhibits the growth of the cells through an increase in lipid peroxidation is discussed.

Inhibition of murine melanoma growth by sodium ascorbate.

Ascorbic acid has been reported to play a role in treatment and prevention of cancer. This study was carried out to determine the effect of ascorbate on growth of normal LLCMK cells and transformed BL6 cells in cell culture and on the growth of BL6 melanomas in vivo. Ascorbic acid levels were also measured to determine the effect of tumor growth and supplementary ascorbate on cellular ascorbic acid levels. Ascorbate addition at levels of up to 200 micrograms/ml was found to inhibit the in vitro growth of BL6 cells but not of LLCMK cells. Ascorbic acid levels in both cell types were very similar. The presence of tumors was found to reduce liver ascorbic acid levels in mice. Supplementary dietary ascorbate increased liver and tumor ascorbic acid levels and also reduced the growth of BL6 melanomas transplanted in C57 mice. Ascorbate thus appears to play a role in suppression of BL6 melanoma growth.

Enhanced prostaglandin synthesis as a mechanism for inhibition of melanoma cell growth by ascorbic acid.

Both ascorbic acid and the 1-series prostaglandins have been reported to be important regulators of cell growth and since ascorbic acid also increases the synthesis of the 1-series prostaglandins, it is possible that the effects of ascorbic acid on cell growth might be mediated by changes in 1-series prostaglandin synthesis induced by ascorbic acid. This study attempted to examine this possible relationship. The effects of ascorbic acid, prostaglandin E1 and the essential fatty acid precursors of the prostaglandins, linoleic acid and gamma-linolenic acid on the in vitro growth of transformed BL6 murine melanoma cells and untransformed monkey kidney (LLCMK) cells was determined. The effects of ascorbic acid addition on the growth inhibitory effect of the essential fatty acids and on the activity of delta-6-desaturase, a key enzyme in 1-series prostaglandin synthesis were also examined. Addition of ascorbic acid, prostaglandin E1 and both essential fatty acids was found to reduce BL6 growth while PGE1 and to a lesser extent the essential fatty acids reduced LLCMK cell growth. The growth inhibitory effect of the essential fatty acids was enhanced by ascorbic acid which was also found to stimulate delta-6-desaturase activity in BL6 cells. The growth inhibitory effect of ascorbic acid on BL6 cells may thus be mediated by changes in prostaglandin synthesis through an association with the metabolism of the essential fatty acid precursors of the prostaglandins.

The interaction and inhibition of muscle lactate dehydrogenase by the alkaloid caffeine.

Kinetic analysis showed that the alkaloid caffeine is a competitive inhibitor of the enzyme lactate dehydrogenase with respect to substrate pyruvate, and a non-competitive inhibitor with respect to the coenzyme NADH. The inhibitor constant Ki is 0,54 mM. Scatchard analysis determined the dissociation constant for a single independent binding site of the ternary lactate dehydrogenase - NADH - caffeine complex (KE-NADH-CAFFEINE) and the number of binding sites to be 0,14 mM and 3,83 respectively. Caffeine binds to a hydrophobic domain in the substrate binding site. Alternate nucleophilic - electrophilic functionalities within the inhibitor molecule are proposed to be the fundamental reason for the inhibition.