Photo-oxidation of L-glutamate decarboxylase from Escherichia coli, sensitized by the coenzyme pyridoxal phosphate and by proflavin.

Irradiation of L-glutamate decarboxylase (L-glutamate 1-carboxy-lyase, EC 4.1.1.15) from Escherichia coli by visible light absorbed by the intrinsic chromophore, pyridoxal phosphate, caused the selective modification of two methionines per enzyme monomer. The disulfoxide derivative exhibited modified circular dichroism, chromatographic and kinetic properties, suggesting a conformational role for the two methionine residues. Irradiation of the enzyme in the presence of proflavin revealed the presence of two distinct groups of tryptophan residues with markedly different photooxidation rate constants. No evidence of involvement of tryptophans in the catalytic mechanisms of the enzyme was obtained. The results are compared with those obtained on irradiation of L-glutamate decarboxylase from Clostridium perfringens.

Separation and characterization of NAD- and NADP-specific succinate-semialdehyde dehydrogenase from Escherichia coli K-12 3300.

Two distinct proteins endowed with succinate-semialdehyde dehydrogenase (succinate-semialdehyde:NAD(P)+oxidoreductase, EC 1.2.1.16) activity were separated and partially purified by ammonium sulphate fractionation or Sephadex G-200 gel-filtration or both. They differ by coenzyme specificity (NAD or NADP), molecular weight, temperature and pH resistance, pH-activity curves, beta-mercaptoethanol activation. Moreover, the NADP-specific enzyme catalyzes only the oxidation of succinate-semialdehyde among a number of aldehydes tested, whereas the NAD-specific form is active also towards n-butyraldehyde. The Km for the substrate is also appreciably different according to the coenzyme specificity, while the Km values for NAD and NADP are quite similar. Finally, the growth of the cells on gamma-aminobutyrate as the sole source of nitrogen resulted in enhanced level of the NAD-dependent succinate-semialdehyde dehydrogenase, with concurrent decrease of the alternate enzyme activity. On the basis of the above results, distinct metabolic roles are suggested for the two enzymes forms.

Efficient photosensitization of malignant human cells in vitro by liposome-bound porphyrins.

Comparative kinetics of porphyrin uptake and release by HeLa cells, incubated with equivalent concentrations of either hematoporphyrin (Hp) in aqueous solution or Hp and its dimethylester (HpDME) bound to unilamellar liposomes, show that liposomal porphyrins are bound at a higher rate and in considerably larger amounts. Moreover, the release of cell-bound porphyrins into the medium is remarkably reduced and slowered after cell loading with liposome-bound porphyrins. The presence of 1% bovine or human serum albumin (but not serum globulins) in the medium has no effect on uptake and release of liposome-bound porphyrins by HeLa cells, whereas it remarkably decreases the uptake of aqueous Hp. Parallel studies of cell photodamages under known concentrations of cell-bound porphyrin unequivocally demonstrate that the photodynamic effect is strictly related to the porphyrin load. As a consequence a dramatic increase of cell-photosensitizing efficiency is obtained by binding Hp (and even more HpDME) with liposomes. Electron microscopy investigations on cell damages caused by loading with liposome-bound porphyrins and subsequent illumination show that the plasmatic membrane is one important cell site of porphyrin interaction and photodynamic effect.

Distribution of endogenous and injected porphyrins at the subcellular level in rat hepatocytes and in ascites hepatoma.

Different doses (0.5-20 mg/kg) of hematoporphyrin (HP) have been injected intraperitoneally into normal rats and rats affected by Yoshida ascites hepatoma. About 80% of HP reaching the liver was recovered in the extracellular compartment after liver perfusion, the ratio of extra- to intracellular HP being essentially independent of the administered dose. Similar data were obtained at different times after injection of 20 mg/kg HP. Intracellular HP largely accumulates in the mitochondria and in the membrane components of the nuclear fraction of isolated hepatocytes. Kinetic studies suggest that the cell receptors of highest affinity for HP are present in the external membrane. The latter result obtains for ascites hepatoma cells in an even more evident way, although the latter cells exhibit secondary HP binding sites probably constituted by cytoplasmatic proteins. Moreover, the clearance of intracellular HP from malignant cells occurs at a remarkably lower rate as compared with HP clearance from liver cells.

Characterisation of a specific phycocyanin-hydrolysing protease purified from Spirulina platensis.

A novel protease has been identified, purified and partially characterised from complete medium grown Spirulina platensis, which could be responsible for the selective proteolysis of phycobiliproteins. It is an 80 kDa homodimeric enzyme; its N-terminal sequence is not related to any known protease sequence. It hydrolyses native phycocyanins in both crude extracts and reconstructed systems with purified Allo- or C-phycocyanin. It is inactive on several native proteins, including ribulose-1,5-bisphosphate carboxylase. The two phycocyanins are degraded at different velocities since C-phycocyanin is the better substrate, in agreement with the earlier observations on the progress of the phycobilisome disassembly. Specificity for synthetic substrates and inhibitors strongly suggests its assignment to the serine-protease family. The enzyme, however, is insensitive to the commercially available protein inhibitors of trypsin-like proteases.

Confocal microscopy and biochemical analysis reveal spatial and functional separation between anandamide uptake and hydrolysis in human keratinocytes.

The signaling activity of anandamide (AEA) is terminated by its uptake across the cellular membrane and subsequent intracellular hydrolysis by the fatty acid amide hydrolase (FAAH). To date, the existence of an AEA membrane transporter (AMT) independent of FAAH activity remains questionable, although it has been recently corroborated by pharmacological and genetic data. We performed confocal microscopy and biochemical analysis in human HaCaT keratinocytes, in order to study the cellular distribution of AMT and FAAH. We found that FAAH is intracellularly localized as a punctate staining partially overlapping with the endoplasmic reticulum. Consistently, subcellular fractionation and reconstitution of vesicles from membranes of different compartments demonstrated that FAAH activity was localized mainly in microsomal fractions, whereas AMT activity was almost exclusively in plasma membranes. These results provide the first morphological and biochemical evidence to support the view that transport and hydrolysis are two spatially and functionally distinct processes in AEA degradation.

Nonspecific acid phosphatase from Schizosaccharomyces pombe. Purification and physical chemical properties.

Repressible nonspecific acid phosphatase from Schizosaccharomyces pombe was purified to apparent homogeneity, as ascertained from ultracentrifugal, electrophoretic, and chromatographic data. The native protein has a molecular weight of 383,000 as determined by sucrose density gradient centrifugation and 381,000 as determined by gel filtration. The native protein can be dissociated in the presence of 8 M urea-1% sodium dodecyl sulfate into sub-units possessing an approximate molecular weight of 104,000. Neutral sugars account for about 66% of the total molecular weight and contribute to the high solubility and some of the other physical properties of this enzyme. Purified enzyme preparations have a Km for 4-nitrophenyl phosphate of 0.17 mM and a broad substrate specificity, but do not show diesterase activity. Phosphate and sulfate are competitive inhibitors. The enzyme is inactivated at neutral and alkaline pH and at relatively low temperatures. Mannose and galactose was found as the main components of the carbohydrate moiety; glucosamine was present in lower amounts. The amino acid analysis revealed a high content of aspartate, threonine, and serine; no sulfhydryl group could be detected. Pi is released in stoichiometric amount (1 mol per enzyme monomer) on protein digestion.

Variation of glutamate decarboxylase activity and gamma-amino butyric acid content of wheat embryos during ripening of seeds.

GAD activity and gamma-ABA content of wheat embryos at 7 ripening stages were verified with the aim of studying the metabolic activity of embryo during dehydration and quiescence of caryopsis. Data showed that in the early stage of ripening GAD activity is very low, increases rapidly at dough-stage, remaining constant up to waxy-stage, and decreases in the last fully-ripe embryos. gamma-ABA content appears to be roughly parallel to the variations of GAD activity.

Purification and general properties of glutamate decarboxylase from Clostridium perfringens.

1. A seven-step procedure for preparing highly purified glutamate decarboxylase from Clostridium perfringens is described. 2. The homogeneity of the pure enzyme was established by sucrose-density-gradient centrifugation and starch-gel electrophoresis. 3. The isoelectric point of the pure enzyme is about pH4.5 and the molecular weight is 290000. 4. The pH optimum for activity is 4.7. The pure enzyme is specific for l-glutamate; beta-hydroxyglutamate is decarboxylated at a lower rate. 5. Evidence is presented that each mol of enzyme contains 2mol of firmly bound pyridoxal 5-phosphate. 6. Resolution does not occur at acid pH; by dilution with neutral or alkaline buffers the enzyme is inactivated and the coenzyme is released. 7. Reconstitution of active enzyme was obtained by protecting the apoenzyme with thiol compounds.

Succinic semialdehyde dehydrogenase of wheat grain.

Succinic semialdehyde dehydrogenase (EC 1.2.1.16) was purified 74-fold from wheat grain (Triticum durum Desf.). The enzyme appears quite specific for succinic semialdehyde (SSA). Both NAD and NADP support the oxidation of the substrate, but the former is 7-fold more active than the latter. The optimum pH for activity is around 9; the enzyme is stable in the pH range 6-9 and retains its whole activity up to 40°C. The enzyme activity is strongly dependent on the presence of mercaptoethanol, other thiol compounds being much less effective. Kinetic data support the formation of a ternary complex between enzyme, substrate and coenzyme. The K m for SSA and for NAD are 7.4x10(-6) M and 2x10(-4) M, respectively. The molecular weight of the enzyme protein was estimated by gel-filtration to be about 130,000.

Controlled targeting of different subcellular sites by porphyrins in tumour-bearing mice.

Unilamellar liposomes of dipalmitoyl-phosphatidylcholine can incorporate various porphyrins in either the phospholipid bilayer or the internal aqueous compartment depending on the water-/lipo-solubility of the drug. Intraperitoneal injection of the liposome-bound porphyrins to mice bearing a MS-2 fibrosarcoma results in remarkably more efficient tumour targeting than that obtained by administration of the same porphyrins dissolved in homogeneous aqueous solution. Moreover, also water-insoluble porphyrins can be transported to the tumour via liposomes. Fractionation of liver and neoplastic cells indicates that the subcellular distribution of liposome-delivered porphyrins is also dependent on their solubility properties: thus, relatively polar porphyrins, such as tetra(4-sulfonatophenyl)porphine and uroporphyrin, are mainly recovered from the soluble fraction, whereas hydrophobic porphyrins, such as haematoporphyrin or porphyrin esters, preferentially partition in the cytoplasmic membrane. As a consequence, different subcellular sites can be targeted by porphyrins and possibly photodamaged through a suitable choice of the drug-carrier system.

Regulation of breakdown and synthesis of L-glutamate decarboxylase in Clostridium perfringens.

L-Glutamate decarboxylase (GAD) activity of Clostridium perfringens (ATCC 8009) cells grown in various culture conditions was investigated. Remarkable variations of GAD level occur during the growth cycle in thioglycollate broth. These changes are affected by the pH of the culture medium. Addition of alkali to the culture media results in decrease of cell GAD activity, whereas increase of enzyme level occurs only in cells growing in unbuffered media. The results indicate that the mechanism regulating the GAD levels is sensitive to the changes of pH (or buffering substances) rather than to the steady pH values. Neither repression by glucose nor induction by L-glutamate was observed. Moreover, high concentrations of the free amino acid substrate in the culture media considerably decrease cell GAD activity, owing to the buffering effect of the amino acid. The molecular mechanism supporting the variations of GAD activity during the growth cycle of the cells were investigated and tentatively related to the structural and functional properties of the pure enzyme. It is shown that the drop of GAD activity during the lag phase is due to protein breakdown. Evidence is presented suggesting a control of protein degradation by its quaternary structure. Data are also reported supporting de novo synthesis of GAD during the late logarithmic phase of cell growth. Finally, the possible role of GAD as part of the pH regulation system of C. perfringens cells is discussed in relation both to physiologic conditions of the bacterial cell and to the molecular mechanisms regulating the GAD activity in vivo.

Interaction among heavy metals and methanol affecting superoxide dismutase activity in Saccharomyces cerevisiae.

1. Three strains of Saccharomyces cerevisiae have been exposed to methanol both in the presence and absence of heavy metal ions. The growth curves and the superoxide dismutase activity were determined. 2. The presence of alcohol, copper or cadmium alone did not give strong cytotoxic effects, while methanol plus cadmium yielded a growth inhibition in two strains. 3. SOD levels were stimulated by copper, while methanol did not affect SOD in this non-methylotrophic yeast, indicating the need of alcohol assimilation to stimulate SOD. Cadmium had no inducing effects on SOD levels.

Pyridoxal phosphate-sensitized photoinactivation of glutamate decarboxylase from Clostridium perfringens.

1. l-Glutamate decarboxylase (EC 4.1.1.15) from Clostridium perfringens was inactivated by exposure to visible light at pH6.2. 2. Inactivation does not occur at pH4.6 or in the absence of bound pyridoxal phosphate. 3. On prolonged photo-oxidation six histidine residues per molecule of enzyme were destroyed. 4. The loss of six cysteine residues per molecule occurred both in irradiated samples and in controls oxygenated in the dark. 5. This dark-oxidation of cysteine residues is apparently required before the photo-oxidation process. 6. The absorbance, fluorescence and circular-dichroism properties of the enzyme as well as its elution volume during Sephadex gel-filtration were unaffected by prolonged irradiation. 7. However, an apparently homogeneous product of photo-oxidation could be separated from the control enzyme by ion-exchange chromatography. 8. The K(m) for l-glutamate was unchanged in an irradiated sample retaining 22% of control activity. 9. These data and the catalytic role of imidazole residues at the active sites of amino acid decarboxylases are discussed.

Effect of the insulin iodination on the reactivity of the inter-chain disulphide bonds towards sodium sulphite.

Insulin iodination interferes with the ability of the interchain S.S bonds to react with sulphite at pH7. In insulin samples containing more than 5 iodine atoms/monomer unit, only one S.S bond/molecule reacts. The effect must be related to the substitution of the iodine into the tyrosyl groups, which probably causes a conformational rearrangement resulting in a steric hindrance of one of the interchain S.S bonds. The effect is removed by increasing the pH or by adding urea (8m) to the reaction mixture. The unreactivity of the S.S bond and the biological inactivation occur at the same ;critical' iodination level, suggesting that a same primary alteration of the molecule is responsible for both the effects.