KCl-Dependent Release of Mitochondrial Membrane-Bound Arginase Appears to Be a Novel Variant of Arginase-II

Arginase regulates arginine metabolism, ornithine-urea cycle, and immunological surveillance. Arginase-I is predominant in cytosol, and arginase-II is localised in the mitochondria. A mitochondrial membrane-bound arginase has also been proposed to be adsorbed with outer membrane of mitochondria which gets released by 150 mM potassium chloride (KCl). It is presumed that inclusion of 150 mM KCl in the homogenization medium would not only facilitate release of arginase bound with outer membrane of mitochondria but also affect functional anatomy of mitochondria, mitochondrial enzymes, and proteins. Therefore, it has been intended to characterize KCl-dependent release of mitochondrial membrane-bound arginase from liver of mice. Results provide advancement in the area of arginase biology and suggest that fraction of mitochondrial membrane-bound arginase contains mitochondrial arginase-II and a variant of arginase-II.

Adaptation of the Phosphotungstate Method for the Determination of Vitamin C Contents in Animal and Human Tissues

The usefulness of phosphotungstate reagent for vitamin C determination in tissue homogenates has been confirmed. An optimal homogenization medium was selected: 1.8 м solution of HPO3 in 1.3 м CH3COOH. With this medium the analytical curve (at 700 nm) demonstrated the right linearity, correlation and recovery coefficients were appropriately high (0.999 and 99.8%) and the values of intraserial and interserial variation coefficient were low (< 5% and < 10%, respectively). It makes this method sensitive, easily repeatable, and useful for vitamin C determination in animal and human tissues, including neoplastic ones.

Protection of Ca pump of coronary artery against inactivation by superoxide radical

Superoxide radicals inactivate endoplasmic reticular (ER) Ca2+ pump in membranes isolated from smooth muscle of pig right coronary artery [Am. J. Physiol. 255 (Cell Physiol. 24): C297-C303, 1988]. We report on protective mechanisms against such inactivation. This tissue contained superoxide dismutase (SOD) and catalase. SOD was distributed primarily in cytosolic fraction, was cyanide sensitive, and was also present in mitochondrial fraction, and approximately 25% of this was cyanide insensitive. Catalase was distributed mainly in mitochondrial fraction and did not protect against inactivation of ER Ca2+ pump by superoxide radicals generated using xanthine plus xanthine oxidase. However, cytosolic fraction protected against this inactivation by two mechanisms: 1) DTT carried over from homogenization medium and 2) its intrinsic SOD content. Soluble fraction was concentrated, dialyzed to remove 1,4-dithiothreitol (DTT), lyophilized, and suspended in a small volume of DTT-free buffer. It still protected against superoxide inactivation of Ca2+ pump. On Sephacryl-300 gel chromatography, protecting activity comigrated with SOD. DTT protected against inactivation, but glutathione and cysteine protected only partially. Neither sulfhydryl agents nor SOD could reverse the inactivation process. Ca2+ pump activity was abolished by dithionitrobenzoate and p-chloromercuric benzoate. Superoxide may inactivate ER Ca2+ pump by irreversibly modifying key sulfhydryl group(s) on pump molecule and SOD in coronary artery smooth muscle may partially protect against this inactivation.

Diurnal variation in the fraction of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in the active form in the mammary gland of the lactating rat

‘Expressed’ and ‘total’ activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) were measured in freeze-clamped samples of mammary glands from lactating rats at intervals throughout the 24 h light/dark cycle. ‘Expressed’ activities were measured in microsomal fractions isolated and assayed in the presence of 100 mM-KF. ‘Total’ activities were determined in microsomal preparations from the same homogenates but washed free of KF and incubated with exogenously added sheep liver phosphoprotein phosphatase before assay. Both ‘expressed’ and ‘total’ activities of HMG-CoA reductase underwent a diurnal cycle, which had a major peak 6 h into the light phase and a nadir 15 h later, i.e. 9 h into the dark period. Both activities showed a secondary peak of activity (around 68% of the maximum activity) at the time of changeover from dark to light, with a trough in the value of the ‘expressed’ activity that was close to the nadir value. ‘Expressed’ activity was lower than ‘total’ at all time points, indicating the presence of enzyme molecules inactivated by covalent phosphorylation. Nevertheless the ‘expressed’/‘total’ activity ratio was comparatively constant and varied only between 43% and 75%. Immunotitration of enzyme activity, with antiserum raised in sheep against purified rat liver HMG-CoA reductase, confirmed the presence of both active and inactive forms of the enzyme and indicated that at the peak and nadir the variation in ‘expressed’ HMG-CoA reductase activity resulted from changes in the total number of enzyme molecules rather than from covalent modification. The sample obtained after 3 h of the light phase exhibited an anomalously low ‘total’ HMG-CoA reductase activity, which could be increased when Cl- replaced F- in the homogenization medium. The result suggests that at that time the activity of the enzyme could be regulated by mechanisms other than covalent phosphorylation or degradation.

Characteristics of rat pancreatic zymogen granules prepared by different methods

Zymogen granules isolated from tissue homogenates by differential centrifugation in isotonic sucrose solutions show substantial release of digestive enzyme when suspended in isotonic NaCl and in sucrose solutions at pH values above neutrality. A recent study reported a new method for isolating granules, involving the use of a complex homogenization medium and a Percoll gradient that was claimed to produce "stable" granules, i.e., granules that do not release their content in salt solutions and at pH values at or above neutrality. In the present study, we compare granules prepared in both ways, particularly in terms of their tendency to release amylase in isotonic ionic solutions and as a function of pH. The relative absence of amylase release from granules isolated by the new technique was found to be attributable to simple differences in the details of the experimental procedures that were used and not to actual differences in the characteristics of the two granule preparations. For example, previous studies with granules prepared in sucrose solutions reported substantial salt-induced release at 37 degrees C, whereas the recent study reporting the absence of salt-induced release from granules obtained from a Percoll gradient was done at 24 degrees C. Under the identical experimental conditions as used in the present study, little amylase release was seen at 24 degrees C for granules isolated by either technique, but substantial release was seen for both at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Prunasin Biosynthesis by Cell-Free Extracts from Black Cherry (Prunus serotina Ehrh.) Fruits and Leaves

Immature fruits and leaves of black cherry (Prunus serotina Ehrh.) accumulate the cyanogenic glucoside prunasin (the β-glucoside of (R)-mandelonitrile). Cell-free extracts from these tissues catalysed the stereospecific glucosylation of (R,S)-mandelonitrile to (β)-prunasin at rates of 0.2-2.0 μmol/h/mg protein. Uridine diphosphate glucose (Km = 0.32 mᴍ) acted as glucose donor. The optimum concentration of (R,S)-mandelonitrile was 20 mᴍ. Highest activity was exhibited at pH 7.0-8.0 in Tris-phosphate buffer, and no additional cofactors were required. β- Mercaptoethanol (14.5 mᴍ), provided in the homogenization medium to prevent browning of homogenates, did not stimulate the rate of prunasin production. In addition to (R)-mandelonitrile (100%), significant activity was shown towards mandelamide (21%), benzyl alcohol (15%), mandelic acid (8%) and benzoic acid (153%), but not towards prunasin. Mandelonitrile glucosyltransferase activity was most stable at - 20 °C in the presence of 10% glycerol.

The Para-O-Methylation of Apigenin to Acacetin by Cell-Free Extracts of Robinia pseudoacacia L.

Crude extracts from young Robinia pseudoacacia seedlings, shoots, and callus tissue catalyze the para-O-methylation of apigenin to acacetin using S-adenosyl-ʟ-methionine as methyl donor. Optimum activity was exhibited at pH 9.0, and Mg2+ was not required for maximum activity. EDTA (10 mᴍ) did not affect the reaction rate, but 47% inhibition was observed with SAH (100 μᴍ). β-Mercaptoethanol (5 mᴍ) was required in the homogenization medium for optimum O-methyltransferase activity. Apigenin (Km, 50 μᴍ) was the best substrate, but significant activity was shown towards caffeic acid, 5-hydroxyferulic acid, naringenin, and quercetin. Paracoumaric, ferulic, and sinapic acids were not methylated. The Km for S-adenosyl-ʟ-methionine was 31 μᴍ. Our demonstration of a para-O-methyltransferase activity methylating apigenin, but not para-coumaric acid, strongly supports the conclusion that the B-ring methylation pattern of acacetin is determined at the C15-level in Robinia pseudoacacia.

Tissue and subcellular distribution of the lectin from Datura stramonium (thorn apple)

Plants of Datura stramonium (thorn-apple) were dissected into their component tissues and examined for the presence of the Datura lectin. This lectin was easily detected in seeds and in various parts of the flowers of adult plants. Traces were also found in green (emerged) cotyledons and roots of seedlings. The specific lectin activity in seeds contained within the fruits increased as the seeds matured. Mature seeds were homogenized in sucrose and separated by differential centrifugation into four fractions, three of which were clearly of distinct composition. Most of the lectin activity sedimented with the low-speed (cell-wall/protein-body) pellet, but a similar specific activity was recovered from the other fractions. However, if EDTA was included in the homogenization medium, three or four times more lectin activity was recovered in the soluble fraction. Immunofluorescent staining of formaldehyde-fixed sections showed that the lectin was localized in the cytoplasm, with little associated with cell walls. The possible relevance of these results to the function of the lectin in plant cells is discussed.