Preparation and general properties of a soluble adenosine triphosphatase from mitochondria

1. The purification of an adenosine triphosphatase present in aqueous extracts of acetone-dried ox-heart mitochondria is described. 2. No evidence was found for the presence of more than one protein having adenosine-triphosphatase activity in these extracts. 3. The enzyme is less stable at 0 degrees than at 25 degrees but is stabilized by glycerol. 4. The activity is dependent on the presence of Mg(2+) or certain other bivalent metal cations. 5. The adenosine-triphosphatase activity of the Mg(2+)-activated enzyme is enhanced by 2,4-dinitrophenol. 6. The kinetics of Mg(2+) activation indicate that the ATP-Mg(2+) complex is the important substrate: free ATP and Mg(2+) are inhibitory. 7. This preparation of mitochondrial adenosine triphosphatase has many properties in common with the adenosine triphosphatase coupling factor from mitochondria (Racker, 1961).     

ATP- and polyphosphate-mediated stimulation of pp60c-src kinase activity in extracts from vascular smooth muscle

Recently, we reported that pp60c-src kinase activity was present in adult bovine coronary arterial smooth muscle and showed that the activity of the enzyme in in vitro immunoprecipitation assays was stimulated 20-60-fold by ATP (Di Salvo, J., Gifford, D., and Kokkinakis, A. (1988) Biochem. Biophys. Res. Commun. 153, 388-394). In the present study, ATP-mediated stimulation of activity was also demonstrated in extracts from aortic vascular smooth muscle. In contrast, no stimulation was apparent in extracts from brain. Stimulation of activity in vascular preparations was also induced with beta,gamma-imidoadenosine 5'-triphosphate (AMP.PNP), a nonmetabolizable analog of ATP, and with several other polyphosphates including ADP and sodium pyrophosphate. No stimulation occurred in response to monophosphates such as AMP or KH2PO4. As expected, the specific activity of pp60c-src in brain extracts did not change when the amount of extracted protein included in immunoprecipitation mixtures was increased. Unexpectedly, however, the specific activity of the vascular enzyme decreased markedly as the amount of extracted protein subjected to immunoprecipitation was increased. Following stimulation of pp60c-src in vascular extracts with ATP, the enzyme behaved in a fashion similar to pp60c-src extracted from brain. That is, the enhanced specific activity of the stimulated vascular enzyme did not decrease with increasing amounts of extracted protein. Moreover, mixing experiments in which vascular smooth muscle extracts were added to brain extracts showed that the muscle extracts contained a factor which inhibited pp60c-src kinase activity. This inhibition was blocked when the mixed extracts were immunoprecipitated in the presence of ATP, or when inhibitory extract was treated with trypsin. Taken together, these data suggest that pp60c-src kinase activity in vascular tissue may be subject to a novel regulatory mechanism involving an inhibitory protein factor which can be nullified by polyphosphates.     

Alpha-amylase inhibitors from mycelium of an oyster mushroom

Abstract

The α-Amylase and α-glucosidase are two main enzymes involved in carbohydrate metabolism. This study was aimed at detecting alpha-amylase inhibitory activity from edible mushroom mycelia. Oyster mushroom was collected from a natural source, from Indian Institute of Technology (Banaras Hindu University) campus and was maintained in vitro in mycelial form. Chloroform, acetone, methanol, and water were used separately for extraction of an active constituent from mycelial cells grown, for 7?days, in potato dextrose broth. The extracts were tested for alpha-amylase inhibitory activity. Chloroform, acetone, and methanol extracts were found to have alpha-amylase inhibitory activity, with IC₅₀ values of 1.71, 224, and 383?μg/mL, respectively. Aqueous extract had no enzyme inhibitory activity. The acetone extract inhibited α-amylase non-competitively whereas chloroform extract showed competitive inhibition. Acetone extraction yielded highest total phenolic content (TPC) of 0.524?mM of gallic acid equivalent, whereas chloroform extraction resulted in lowest TPC of 0.006?mM. The HPLC and absorbance maxima of acetone and chloroform extracts suggest that the bioactive component responsible for enzyme inhibition could be glycoproteins in chloroform extract and catechins (flavonoids) in acetone extract. Thus, the mushroom mycelia under study may be exploited for production and purification of a lead compound for the development of the α-amylase inhibitory drug.     

Poly(adenosine diphosphate ribose) polymerase activity in neuronal and glial nuclei from bovine cerebrum

Two different preparations isolated from beef cerebrum have been used to compare the polyadenosine diphosphate ribose (polyADPR) polymerase activities in neuronal and glial nuclei: (1) nuclear suspensions (with or without DNase I treatment), and (2) 1 M NaCl nuclear extracts (soluble enzyme). The DNAse I treatment of nuclei and the solubilization of polyADPR polymerase by 1 M NaCl enhances the polyADPR polymerase activity. The polyADPR polymerase activity is similar in neuronal and glial nuclear suspensions, while the neuronal soluble enzyme activity is significantly higher than that of the glial soluble enzyme. Evidence is presented that the difference in soluble enzyme activities is not due to the effects of DNA or degrading enzymes. Some activating factor(s) seem to be present in neuronal soluble extracts, while both inhibiting and activating factor(s) seem to be present in glial soluble extracts.     

Peroxidase and IAA-oxidase in crude and partially purified enzyme extracts of growing and differentiating root cells

Crude enzyme extracts from the zones of division, elongation and differentiation of cells of primary maize (Zea mays) root show peroxidase activity but lack IAA-oxidase activity. After partial purification of the extracts by gel filtration on Sephadex G-25, the specific peroxidase activity increases almost twice and a high IAA-oxidase activity appears. The partial purification of the enzyme extracts does not change the electrophoretic pattern of the peroxidase isoenzymes, but significantly improves the separation and the visualization of isoenzymes with IAA-oxidase activity. The data obtained were interpreted in connection with the different modifying effect of the low molecular compounds (mainly phenolics) on the activity and the isoenzyme patterns of the peroxidase and the IAA-oxidase.     

Prostaglandin-H-synthase (PGHS)-1 and -2 microtiter assays for the testing of herbal drugs and in vitro inhibition of PGHS-isoenzyms by polyunsaturated fatty acids from Platycodi radix

In order to test inhibition of prostaglandin-H-synthase-1 and -2 (PGHS-1 and -2) by plant extracts, we have established two enzyme based in vitro assays with enzyme immunoassay (EIA) evaluation. The assays have been evaluated with known synthetic inhibitors and with plant extracts. In a screening of traditionally used Chinese herbs for anti-inflammatory activity, a series of n-hexane and dichloromethane extracts showed significant inhibitory effect in comparison with the known specific PGHS-2 inhibitors NS-398 (IC(50) = 2.6 microM) and nimesulide (IC(50) = 36 microM). The lipophilic extracts of the Chinese drug Jiengeng, the dried roots of Platycodon grandiflorum (Jacq.) A. DC. (Campanulaceae), showed good inhibitory activity against both PGHS isoenzymes. The directly prepared DCM-extract exhibited better activity against PGHS-2 (IC(50) = 4.0 microg/ml) than against PGHS-1 (IC(50) = 17.6 microg/ml). We identified fatty acids as main active constituents and quantified them. Linoleic acid showed the highest content (ca. 20% of the dried extract) and a high and preferential PGHS-2 inhibitory activity (IC(50) (PGHS-1) = 20 microM; IC(50) (PGHS-2) = 2 microM). The comparison of the concentration of linoleic acid and the inhibitory activity of the direct DCM-extract showed, that linoleic acid is mainly responsible for the in vitro activity of the extract on PGHS-2.     

The two alcohol dehydrogenases of Zymomonas mobilis. Purification by differential dye ligand chromatography, molecular characterisation and physiological roles

The two alcohol dehydrogenases found in Zymomonas mobilis have each been purified using dye-ligand chromatography and affinity elution with nucleotides. The isoenzyme with lower electrophoretic mobility (ZADH-1) is a zinc enzyme with properties essentially similar to preparations described elsewhere. The faster isoenzyme (ZADH-2) accounted for some 90% of the ethanol-oxidizing activity in freshly prepared extracts and corresponded to the iron-activated enzyme previously described. This enzyme was inactivated by zinc; activity could only be retained during purification by including either ferrous ions or cobaltous ions in the buffers. ZADH-2 has relatively low acetaldehyde reductase activity; consequently ZADH-1 is responsible for about half of the physiological activity (acetaldehyde reduction) in Zymomonas cells. Kinetic studies showed that ZADH-2 is activated by ethanol in both reaction directions; a hypothesis for the mechanism of activation is presented. Metal ion analyses of ZADH-2 prepared in the presence of iron or cobalt indicated one atom of the relevant metal per subunit, with no significant zinc content. N-terminal sequence analyses showed that the ZADH-1 has some homology with the Bacillus stearothermophilus enzyme, whereas ZADH-2 resembles the yeast enzyme more closely.     

Nitroalkane oxidation by streptomycetes.

Crude cell-free extracts of nine strains of Streptomyces tested for nitroalkane-oxidizing activity showed production of nitrous acid from 2-nitropropane, 1-nitropropane, nitroethane, nitromethane, and 3-nitropropionic acid. These substrates were utilized in most strains but to a decreasing extent in the order given, and different strains varied in their relative efficiency of oxidation. p-Nitrobenzoic acid, p-aminobenzoic acid, enteromycin, and omega-nitro-l-arginine were not attacked. d-Amino acid oxidase, glucose oxidase, glutathione S-transferase, and xanthine oxidase, enzymes potentially responsible for the observed oxidations in crude cellfree extracts, were present at concentrations too low to play any significant role. A nitroalkane-oxidizing enzyme from streptozotocin-producing Streptomyces achromogenes subsp. streptozoticus was partially purified and characterized. It catalyzes the oxidative denitrification of 2-nitropropane as follows: 2CH(3)CH(NO(2))CH(3) + O(2) --> 2CH(3)COCH(3) + 2HNO(2). At the optimum pH of 7.5 of the enzyme, 2-nitropropane was as good a substrate as its sodium salt; t-nitrobutane was not a substrate. Whereas Tiron, oxine, and nitroxyl radical acted as potent inhibitors of this enzyme, superoxide dismutase was essentially without effect. Sodium peroxide abolished a lag phase in the progress curve of the enzyme and afforded stimulation, whereas sodium superoxide did not affect the reaction. Reducing agents, such as glutathione, reduced nicotinamide adenine dinucleotide, and nicotinamide adenine dinucleotide phosphate, reduced form, as well as thiol compounds, were strongly inhibitory, but cyanide had no effect. The S. achromogenes enzyme at the present stage of purification is similar in many respects to the enzyme 2-nitropropane dioxygenase from Hansenula mrakii. The possible involvement of the nitroalkane-oxidizing enzyme in the biosynthesis of antibiotics that contain a nitrogen-nitrogen bond is discussed.     

Purification and characterization of protein tyrosine phosphatase PTP-MEG2

PTP-MEG2 is an intracellular protein tyrosine phosphatase with a putative lipid-binding domain at the N-terminus. The present study reports expression, purification, and characterization of the full-length form of the enzyme plus a truncated form containing the catalytic domain alone. Full-length PTP-MEG2 was expressed with an adenovirus system and purified from cytosolic extracts of human 293 cells infected with the recombinant adenovirus. The purification scheme included chromatographic separation of cytosolic extracts on fast flow Q-Sepharose, heparin-agarose, l-histidyldiazobenzylphosphonic acid agarose, and hydroxylapatite. The enrichment of PTP-MEG2 from the cytosol was about 120-fold. The truncated form of PTP-MEG2 was expressed in E. coli cells as a non-fusion protein and purified by using a chromatographic procedure similar to that used for the full-length enzyme. The purified full-length and truncated enzymes showed single polypeptide bands on SDS-polyacrylamide gel electrophoresis under reducing conditions and behaved as monomers on gel exclusion chromatography. With para-nitrophenylphosphate and phosphotyrosine as substrates, both forms of the enzyme exhibited classical Michaelis-Menten kinetics. Their responses to pH, ionic strength, metal ions, and protein phosphatase inhibitors are similar to those observed with other characterized tyrosine phosphatases. Compared with full-length PTP-MEG2, the truncated DeltaPTP-MEG2 displayed significantly higher V(max) and lower K(m) values, suggesting that the N-terminal putative lipid-binding domain may have an inhibitory role. The full-length and truncated forms of PTP-MEG2 were also expressed as GST fusion proteins in E. coli cells and purified to near homogeneity through affinity columns. However, the specific phosphatase activities of the GST fusion proteins were 10-25-fold below those obtained with the correspondent non-fusion proteins.     

Purification and characterization of a 40 S ribosomal protein S6 kinase from vanadate-stimulated Swiss 3T3 cells

Recently we have identified a mitogen-activated S6 kinase from Swiss 3T3 cells (Jen?, P., Ballou, L. M., Novak-Hofer, I., and Thomas, G. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 406-410). Here we describe the detailed purification of this enzyme from high-speed supernatants (400,000 x g) of vanadate-treated cell extracts. The enzyme is purified through six sequential steps including cation- and anion-exchange, sizing, and affinity chromatography. At each step, the enzyme behaves as one entity and, on the final step of purification, is revealed on silver-stained sodium dodecyl sulfate-polyacrylamide gels as a single protein of Mr 70,000. As reported earlier, the overall purification factor is 3,000-fold, and the specific activity of the homogeneously purified enzyme is 0.6 mumol/min/mg of protein. However, recovery of total activity is only 0.2%. This large loss of activity appears to be due to freeze-thawing the enzyme between each step of purification. The purified kinase does not phosphorylate casein, histones 2A and 3S, or phosvitin. It has a Km for ATP of 28 microM and a broad optimum for Mg2+ between 5 and 20 mM. Mn2+ does not affect the basal level of kinase activity, and at concentrations as low as 1 mM, it completely suppresses the effect of 20 mM Mg2+ on kinase activity. The relationship of this enzyme to two other purified S6 kinases is discussed.     

Purification and characterization of a phospholipase A2 associated with rabbit lung microsomes: some evidence for its mitochondrial origin

Phospholipase A2 (EC 3.1.1.4) activity appeared to be unevenly distributed among the subcellular fractions of rabbit lung homogenates. The mitochondrial/lysosomal fraction, which possessed the highest specific activity, was the second most abundant source of enzyme, following the 1000 x g pellet. Crude microsomes, which were the poorest source of enzyme, had a specific activity intermediate between that of crude mitochondria and of cytosol. Despite these observations, in view of the putative role of microsomal phospholipase A2 in remodelling phosphatidylcholines for pulmonary surfactant biosynthesis, the purification of phospholipase A2 from microsomal membranes was investigated. The activity was solubilized from rabbit lung microsomes with 1 M KCl and resolved into two distinct peaks by ion-exchange chromatography. The larger peak (95% of the recovered activity) was subjected to a combination of hydroxyapatite and gel-filtration chromatography, resulting in a purification factor in excess of 70,000 relative to the microsomal membranes. There was no indication for the removal of endogenous inhibitor(s) during the purification. Application of the same purification protocol to a 1 M KCl extract of lung mitochondria resulted in phospholipase A2 profiles in each of the four columns employed that had exactly the same elution characteristics as those generated by the microsomal extracts. The purified enzyme is specific for the sn-2 ester bond of phosphatidylcholine, requires Ca2+ for activity and has an alkaline pH optimum. It is heat-labile and susceptible to treatment by p-bromophenacyl bromide and by 2-mercaptoethanol but remains unaffected by NaF, diisopropylfluorophosphate and thiol reagents.     

Purification of an endonuclease from adenovirus-infected KB cells.

This report describes the purification of an endonuclease from extracts of adenovirus-type-2-infected KB cells. Endonuclease activity can also be detected in extracts of uninfected KB cells and the enzyme activities from extracts of uninfected and adenovirus-infected cells are very similar, if not identical. The enzyme has its maximal activity at pH 4.0. The enzyme found in uninfected and adenovirus-infectedcells is, however, strikingly different from an endonuclease isolated from calf serum. Hence, the endonuclease described is probably not a contaminant derived from the medium in which the KB cells were propagated. The endonuclease in crude extracts from uninfected or adenovirus-infected KB cells can be activated or its activity enhanced by treatment of the extracts with proteolytic enzymes, like pronase or trypsin. Evidence has been presented suggesting that this activation is due to proteolytic cleavage of an inhibitor present in crude extracts of uninfected and adenovirus-type-2-infected KB cells. A second endonuclease has been found in extracts of infected and uninfected cells with optimal activity at pH 7.2 and this endonuclease can be separated from the one with a pH optimum at 4.0.     

Ent-Kaurene Biosynthesis in Extracts of Helianthus annuus L. Seedlings

Kaurene synthetase B activity (conversion of copalyl pyrophosphate to ent-kaurene) is readily detectable in crude cell-free extracts of 3- to 4-day old dark-grown sunflower (Helianthus annuus cv. Mammoth) seedlings, whereas little or no kaurene synthetase AB activity (conversion of geranylgeranyl pyrophosphate to ent-kaurene) can be found in these extracts under comparable assay conditions. A low amount of AB activity is evident only if an extensively dialyzed extract is used in low concentrations as the enzyme source. One factor which may contribute to the low apparent levels of AB activity is the presence of inhibitory factors in the crude sunflower extract since these extracts can be shown to act as a potent inhibitor of Marah macrocarpus endosperm kaurene synthetase AB activity. Heat treatment (100°C) or dialysis of the sunflower extract reduces the amount of its inhibitory activity. Also, it was observed that low concentrations of extensively dialyzed sunflower extracts act to stimulate M. macrocarpus AB activity. There is no evidence for the presence of an inhibitory factor for M. macrocarpus kaurene synthetase B activity in sunflower extracts. However, there does appear to be present in the crude preparation of sunflower extract a dialyzable factor(s) that impedes its own B activity. There is little information to date on the nature of these inhibitory and stimulatory factors for kaurene synthetase activity or their possible roles in physiological regulation. The possible presence of such factors should be considered, however, when attempting to evaluate kaurene synthetase activities in extracts of vegetative plants.     

Oxidation of hydroxylamine by cytochrome P-460 of the obligate methylotroph Methylococcus capsulatus Bath.

An enzyme capable of the oxidation of hydroxylamine to nitrite was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The absorption spectra in cell extracts, electron paramagnetic resonance spectra, molecular weight, covalent attachment of heme group to polypeptide, and enzymatic activities suggest that the enzyme is similar to cytochrome P-460, a novel iron-containing protein previously observed only in Nitrosomonas europaea. The native and subunit molecular masses of the M. capsulatus Bath protein were 38,900 and 16,390 Da, respectively; the isoelectric point was 6.98. The enzyme has approximately one iron and one copper atom per subunit. The electron paramagnetic resonance spectrum of the protein showed evidence for a high-spin ferric heme. In contrast to the enzyme from N. europaea, a 13-nm blue shift in the soret band of the ferrocytochrome (463 nm in cell extracts to 450 nm in the final sample) occurred during purification. The amino acid composition and N-terminal amino acid sequence of the enzyme from M. capsulatus Bath was similar but not identical to those of cytochrome P-460 of N. europaea. In cell extracts, the identity of the biological electron acceptor is as yet unestablished. Cytochrome c-555 is able to accept electrons from cytochrome P-460, although the purified enzyme required phenazine methosulfate for maximum hydroxylamine oxidation activity (specific activity, 366 mol of O2 per s per mol of enzyme). Hydroxylamine oxidation rates were stimulated approximately 2-fold by 1 mM cyanide and 1.5-fold by 0.1 mM 8-hydroxyquinoline.     

Nitrous oxide reductase from denitrifying Pseudomonas perfectomarina. Purification and properties of a novel multicopper enzyme

Nitrous oxide reductase from the denitrifying bacterium Pseudomonas perfectomarina has been isolated and purified to homogeneity. The enzyme contained about eight copper atoms/120 kDa and was composed of two presumably identical subunits. The isoelectric point was 5.1. Several spectroscopically distinct forms of the enzyme were identified. A 'pink' form of the enzyme was obtained when the purification was done aerobically. The specific activity of this species was around 30 nkat/mg protein as measured by the nitrous-oxide-dependent oxidation of photochemically reduced benzyl viologen. A 'purple' form of the enzyme, whose catalytic activity was 2-5-fold higher, was obtained when the purification was done anaerobically. The activity of both forms of the enzyme was substantially increased by dialyzing the protein against 2-(N-cyclohexylamino)ethanesulfonate buffer at pH approximately equal to 10. A maximal activity of 1000 nkat/mg protein has been obtained for the purple form using this procedure. A 'blue', enzymatically inactive form of the enzyme resulted when either the pink or the purple species was exposed to excess dithionite or ascorbate. Anaerobic, potentiometric titrations of both the purple and the pink form of the enzyme gave a Nernst factor, n540, of 0.95 and a midpoint potential, E'0,540 of +260 mV (vs SHE, 25 degrees C, Tris/HCl buffer, pH 7.5). Electron paramagnetic resonance (EPR) and optical spectra of N2O reductase suggested the presence of an unusual type 1 copper center. Type 2 copper was absent. The hyperfine splitting in the g parallel region consisted of a seven-line pattern. In the presence of excess of reductant, a broad EPR signal with g values at 2.18 and 2.06 was observed. The EPR spectra of the pink and purple forms of the enzyme were similar; however, the spectrum of the purple form was better resolved with g parallel = 2.18 (A parallel = 3.83 mT) and g perpendicular = 2.03 (A perpendicular = 2.8 mT). Most of the copper in N2O reductase was removed by anaerobic dialysis against KCN. Reaction of the apoprotein with Cu(en)2SO4 partially regenerated the optical and EPR spectra of the holoprotein; the resulting protein was enzymatically inactive. Monospecific antibodies against the copper protein strongly inhibited the N2O reductase activity of purified samples and cell-free extracts.     

Regulation of biosynthesis of secondary metabolites

The activity of PEP carboxylase (E.C.4.1.1.31) was demonstrated in cell free extracts ofStreptomyces aureofacines. The enzyme was purified 610 fold. AcetylCoA increased the affinity of the purified enzyme for substrate approximately tenfold and doubled the specific activity of the enzyme preparation. Inorganic phosphate was not essential for the reaction; on the contrary, it had an inhibitory effect. Essential cofactors were divalent cations, the most potent of which was Mn²⁺. The kinetic characters of the purified enzyme were similar to figures for PEP carboxylase from other sources. The substrate saturation function replotted according to the Hill equation showed the extent of intramolecular interactions, reflecting the allosteric nature of the enzyme.     

Binding of a Phosphorylated Inhibitor to Ribulose Bisphosphate Carboxylase/Oxygenase during the Night

The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase was measured at various times during the purification of the enzyme from leaves of Nicotiana tabacum which were collected either 1 hour before the start of the photoperiod (predawn) or in the middle of the photoperiod (midday). The activity of the enzyme in extracts of the predawn leaves (0.8 units/mg enzyme) was consistently about 2-fold lower than that measured in extracts of midday leaves (1.7 units/mg enzyme). The activity of the predawn enzyme was increased to that of the midday enzyme following removal of CO₂ and Mg²⁺ (deactivation), (NH₄)₂SO₄ precipitation, or incubation in SO₄²⁻ (18 millimolar required for one-half maximal increase). Following purification to >95% homogeneity, the predawn enzyme was found to have ~0.5 moles of bound organic phosphate per mole of enzyme active sites, while the midday enzyme had only ~0.08 moles of bound organic phosphate per mole of enzyme active sites. Deactivation of the predawn enzyme or treatment with 0.2 molar SO₄²⁻ resulted in the removal of most of the bound organic phosphate. These findings support the hypothesis that following the night period about 50% of the enzyme is catalytically inactive because of the tight-binding of a small molecular weight, phosphorylated inhibitor at the active site.     

Purification and characterization of glucose 6-phosphate dehydrogenase from sheep erythrocytes and inhibitory effects of some antibiotics on enzyme activity

Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from sheep erythrocytes, using a simple and rapid method. The purification consisted of three steps; preparation of haemolysate, ammonium sulphate fractionation and 2', 5'-ADP Sepharose 4B affinity chromatography. The enzyme was obtained with a yield of 37.1% and had a specific activity of 4.64 U/mg proteins. Optimal pH, stable pH, molecular weight, and KM and Vmax values for NADP+ and glucose 6-phosphate (G6-P) substrates were also determined for the enzyme. The overall purification was about 1,189-fold. A temperature of +4 degrees C was maintained during the purification process. In order to control the purification of the enzyme SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done in 4% and 10% acrylamide concentration for stacking and running gel, respectively. SDS-PAGE showed a single band for enzyme. Enzymatic activity was spectrophotometrically measured according to Beutler's method at 340 nm. In addition, in vitro effects of gentamicin sulphate, penicillin G potassium, amicasin on sheep red blood cell G6PD enzyme activity were investigated. These antibiotics showed inhibitory effects on enzyme activity. I50 values were determined from Activity%-[Drug] graphs and Ki values and the type of inhibition (noncompetitive) were determined by means of Lineweaver-Burk graphs.     

Purification and Characterization of Glucose 6-phosphate Dehydrogenase from Sheep Erythrocytes and Inhibitory Effects of some Antibiotics on Enzyme Activity

Glucose 6-phosphate dehydrogenase (d -glucose 6-phosphate: NADP + oxidoreductase, EC 1.1.1.49; G6PD) was purified from sheep erythrocytes, using a simple and rapid method. The purification consisted of three steps; preparation of haemolysate, ammonium sulphate fractionation and 2′, 5′-ADP Sepharose 4B affinity chromatography. The enzyme was obtained with a yield of 37.1% and had a specific activity of 4.64 U/mg proteins. Optimal pH, stable pH, molecular weight, and K M and V max values for NADP + and glucose 6-phosphate (G6-P) substrates were also determined for the enzyme. The overall purification was about 1,189-fold. A temperature of +4°C was maintained during the purification process. In order to control the purification of the enzyme SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done in 4% and 10% acrylamide concentration for stacking and running gel, respectively. SDS-PAGE showed a single band for enzyme. Enzymatic activity was spectrophotometrically measured according to Beutler's method at 340 nm. In addition, in vitro effects of gentamicin sulphate, penicillin G potassium, amicasin on sheep red blood cell G6PD enzyme activity were investigated. These antibiotics showed inhibitory effects on enzyme activity. I 50 values were determined from Activity %-[Drug] graphs and K i values and the type of inhibition (noncompetitive) were determined by means of Lineweaver-Burk graphs.     

Multiple pharmacological approaches on hydroalcoholic extracts from different parts of Cynoglossum creticum Mill. (Boraginaceae)

Cynoglossum creticum Mill (Boraginaceae) is used traditionally as a remedy to manage several human ailments. In this context, the present study aimed to perform multiple pharmacological investigations on the hydroalcoholic extracts prepared from Cynoglossum roots and aerial parts (leaves and flowers). We evaluated the antioxidant and enzyme inhibitory (against cholinesterases, α-glucosidase, α-amylase, lipase and tyrosinase) activity of the extracts. The protective effect(s) of the extracts on cardiomyocyte C2C12 and intestinal HCT116 cell lines challenged with hydrogen peroxide (H₂O₂) was studied. We found that the aerial parts harbored the highest amount of phenolic compounds. Generally, aerial parts showed significant antioxidant and enzyme inhibitory effects. Leaves exhibited the best lipase inhibitory activity (173.15 mgOE/g extract), followed by flowers and roots. The root and aerial extracts were equally able to blunt intracellular H₂O₂ induced reactive oxygen species production from both C2C12 and HCT116 cell lines. Both cells lines could be treated with scalar concentrations of root and flower extracts in the range 50–300?μg/mL without interferences on cell viability. In conclusion, the present study showed protective effects exerted by Cynoglossum extracts, which could serve as a foundation for the development of pharmaceuticals and nutraceuticals derived from Cynoglossum.