Influence of magnesium in the homogenization medium on the state of a divalent cation-stimulated,diethystilbestrol-sensitive adenylnucleotidyl phosphatase activity from pea stem microsomal preparations

The amount of divalent cation-activated, diethylstilbestrol-sensitive adenylnucleotidyl phosphatase activity recovered in the ‘microsomes’ ($\text{13 000–80 000 x g}\text{sediment}$) from pea stem tissue is strongly influenced by the concentration of Mg²⁺ in the homogenization medium. The absence of Mg²⁺ during homogenization results in a marked decrease of the activity found in the microsomal fraction, compensated by its increase in the soluble fraction. Part of the solubilized activity becomes sedimentable at $\text{80 000 × g}$ upon addition of 5–10 mM Mg²⁺ (or Mn²⁺, Ca²⁺, Zn²⁺) to the supernatant. This sediment shows a very high specific activity, and can be re-solubilized by treatment with either EDTA or 0.3 M monovalent salts, or deoxycholate. When the supernatant containing the solubilized activity is incubated together with low-adenylnucleotidyl phosphatase microsomes and with 10 mM MgCl₂ the activity recovered in the sediment is much larger than the sum of the activity of the microsomes plus that of the sediment obtained by incubating the same supernatant with Mg²⁺. Microsomes prepared with Mg²⁺ in the homogenization medium do not show this effect. The supernatant/microsomes saturation curves as well as a change of the temperature coefficient of the activity following combination of the soluble preparation with the microsomal particles suggest an at least partial reconstitution of the original enzyme-membrane structure.     

Cytochrome P-450 and hydroxylating activity of microsomal preparations from whole houseflies

1. A new microsomal preparation, obtained from whole houseflies is described in terms of its cytochrome P-450 content and its hydroxylating activity. 2. Microsomes prepared from whole-fly brei, obtained with the aid of a mortar (a procedure that avoids the destruction of sarcosomes), contain 0.265nmol of cytochrome P-450 and hydroxylate naphthalene at a rate of 28.5nmol/mg of microsomal protein in 30min at 30 degrees C. This corresponds to 104nmol of naphthalene hydroxylated/nmol of cytochrome P-450. This is the highest rate ever reported for housefly and rat liver microsomal preparations. 3. Microsomal fractions prepared by procedures that do not retain the integrity of sarcosomes show the presence in the CO-difference spectrum of a 428nm peak. This cytochrome is associated with sarcosomal microsomes and it may be involved in the inhibition of insect microsomal mixed-function oxidases, although other factors cannot be discarded at present. 4. The inability to show cytochrome P-450 in microsomal fractions isolated from whole houseflies by other procedures may be at least partially due to a masking effect brought about by contamination with the sarcosomal cytochrome.     

3-phosphoglycerate phosphatase activity in chloroplast preparations as a result of contamination by Acid phosphatase

The presence of a nonspecific acid phosphatase which had high activity with 3-phosphoglycerate as substrate has recently been reported in Spinacia oleracea L. chloroplasts (Mulligan, Tolbert 1980 Plant Physiol 66: 1169-1173). The subcellular localization of this activity has been reinvestigated by differential centrifugation of spinach leaf homogenates. The fraction sedimenting at 1,200g comprised mostly intact chloroplasts and contained more than half the chlorophyll but only 5% of the 3-phosphoglycerate phosphatase activity present in the homogenate. The fraction of the homogenate pelleting at 5,000g contained broken chloroplasts and had considerable 3-phosphoglycerate phosphatase activity. Further purification of the 1,200g pellet fraction on a Percoll step gradient yielded greater than 95% intact chloroplasts, yet the phosphatase activity was reduced more than 15-fold on a chlorophyll basis by this purification.

When the intact chloroplast and cytoplasmic fractions of mesophyll protoplasts were separated by silicone oil filtering centrifugation, the chloroplast fraction contained more than 90% of the chlorophyll but had less than 12% of the 3-phosphoglycerate phosphatase activity. By contrast, more than 60% of the 2-phosphoglycolate phosphatase was recovered in this chloroplast fraction supporting previous evidence that this phosphatase is localized in the chloroplast stroma.

It is concluded that 3-phosphoglycerate phosphatase activity is not localized in the chloroplast but that the activity present in chloroplast preparations results from contamination by acid phosphatase, which either binds to the thylakoid membranes during preparation or is present as some other contaminant in the preparation. Inasmuch as the enzyme acts on a broad range of substrates its presence in chloroplast preparations, particularly when the percentage of intact chloroplasts is low, could produce artifacts in metabolic studies such as measurement of phosphorylation.

     

Apyrase from pea stems: Isolation, purification, characterization and identification of a NTPase from the cytoskeleton fraction of pea stem tissue

The cytoskeleton pellet from the first internode of dark-grown pea stems was disintegrated in a high salt buffer, ultracentrifuged to remove ribosomes and the post-ribosomal supernatant was applied to a heparin affinity column. Significant ATPase activity was present in the cytoskeleton fraction and this was eluted from the column at 0.6–0.7 M KOAc, in the same fractions as a 49-kDa protein (which we called B3). B3 was desalted and further purified by cation exchange column chromatography. Purified B3 catalyzed hydrolysis of ATP, CTP, GTP, TTP, UTP and ADP and thus appears to be an apyrase (ATP diphosphohydrolase, EC 3.6.1.5). Partial amino acid sequences of three major fragments were obtained by digestion of B3 by Staphylococcus aureus V8 protease (EC 3.4.21.19), and all these sequences were consistent with the previously reported amino acid sequences for pea nucleoside triphosphatase (NTPase, EC 3.6.1.15) (PIR S48859), which is thought to be an apyrase.     

Effect of angiotensin II infusion in rats on Na,K-ATPase activity in renal cortical microsomal preparations

Direct dose-dependent effects of angiotensin II on renal tubular sodium reabsorption have been demonstrated. Alterations in tubular sodium reabsorption may occur via modulation of renal Na,K-ATPase activity. Thus, these experiments were undertaken to ascertain whether angiotensin II could influence renal cortical Na,K-ATPase activity. Angiotensin II, 495 ng/microliters/h, or vehicle (controls) was infused for 24 h via miniosmotic pumps 48 h after rats were adrenalectomized and implanted with osmotic pumps containing 12.5 micrograms/microliters corticosterone (Treatment I) or both corticosterone and 0.2 microgram/microliter aldosterone (Treatment II), and in rats receiving 3% NaCl in their food (sodium loaded, Treatment III). Rats receiving Treatments I and III received saline to drink. Renal cortical microsomal membranes were prepared, and the effects of angiotensin II infusion on the K1/2 and Vmax for Na, K, and ATP determined. Angiotensin II infusions were associated with (i) a decrease (P less than 0.001) in the K1/2 for Na activation of Na,K-ATPase from 14 +/- 3 to 6 +/- 1 (n = 4 experiments), 16 +/- 1 to 12 +/- 1 (n = 5), and 12 +/- 3 to 7 +/- 1 (n = 5) mM (means +/- SE) for treatments I, II, and III, respectively; (ii) no changes in the K1/2 for K activation or the Km for ATP; (iii) no changes in the Vmax for Na, K, or ATP; and (iv) no change in Mg-ATPase activity. We conclude that angiotensin II infusion is associated with a decrease in the K1/2 of renal cortical Na,K-ATPase activity for sodium. This action of angiotensin II on the enzyme activity may contribute to the regulation of tubular sodium transport.     

Purification and characterization of a divalent cation-independent, spermine-stimulated protein phosphatase from bovine kidney mitochondria

A divalent cation-independent and spermine-stimulated phosphatase (protein phosphatase SP) that is active toward the phosphorylated pyruvate dehydrogenase complex has been purified about 15,000-fold to near homogeneity from extracts of bovine kidney mitochondria. Half-maximal stimulation, 1.5- to 3-fold at pH 7.0-7.3, occurred at 0.5 mM spermine. Protein phosphatase SP exhibited an apparent Mr = 140,000-170,000 as estimated by gel-filtration chromatography on Sephacryl S-300. Two major subunits, with apparent Mr = 60,000 and 34,000, were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel-permeation chromatography of protein phosphatase SP on Sephacryl S-200 in the presence of 6 M urea and 1.4 M NaCl increased its activity 3- to 6-fold and was accompanied by conversion to the catalytic subunit with an apparent Mr = approximately 34,000. Protein phosphatase SP was inactive with p-nitrophenyl phosphate and was not inhibited by protein phosphatase inhibitor 1, inhibitor 2, or the protein inhibitor of branched-chain alpha-keto acid dehydrogenase phosphatase. Protein phosphatase SP was inhibited by sheep antibody to the catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle. It appears that protein phosphatase SP is related to protein phosphatase 2A.     

Influence of sporulation medium and divalent ions on the heat resistance of Alicyclobacillus acidoterrestris spores

The influence of divalent cations on the heat resistance of spores of the thermoacidophilic spoilage bacterium Alicyclobacillus acidoterrestris was studied. The heat resistance of A. acidoterrestris spores was not affected by the presence of the different divalent cations (Ca²⁺, Mg²⁺, Ba²⁺, Mn²⁺ and Sr²⁺) in the sporulation medium, and by the demineralization or remineralization. And the Ca and Mn contents in A. acidoterrestris spores were scarcely changed by these treatments. However, the heat resistance of Bacillus subtilis spores was affected with the changes of Ca content in the spores. The Ca contents in A. acidoterrestris spores of the different forms were greater than the DPA content. In contrast, the DPA content in the B. subtilis spores was greater than the Ca content. These findings suggest that the presence of constant amount of Ca-DPA and a stronger binding characteristic of divalent ions, especially Ca and Mn, is reflected in the specific heat resistance of A. acidoterrestris spores.     

Modulatory effect of divalent metal cations on the phosphotyrosine activity of the frog liver acid phosphatase.

Frog liver acid phosphatase hydrolyzes phosphotyrosine at acidic pH optimum. Mn2+, Ca2+ and Mg2+ (but not Zn2+) ions modulate this activity by shifting its pH optimum to physiological pH. This effect is not observed when p-nitrophenylphosphate is used as a substrate. Phosphoserine and phosphothreonine are not hydrolyzed under the same conditions.     

Participation of a phosphatase in the Ca regulation in actomyosin preparations from smooth vascular muscle

The superprecipitation of actomyosin from the A. carotis of cattle is suppressed by a phosphatase containing protein fraction. This inhibition is compensated by ammonium molybdate, an inhibitor of phosphatase activity. The results are related to a phosphorylation-dephosphorylation cycle to realize the Ca-sensitivity of superprecipitation of the actomyosin.     

Effects of high-pressure homogenization on the survival of Alicyclobacillus acidoterrestris in a laboratory medium

AIMS: This study was aimed to investigate the effectiveness of high-pressure homogenization (HPH) against Alicyclobacillus acidoterrestris. METHODS AND RESULTS: The susceptibility of three different strains of A. acidoterrestris (DSMZ 2498, Gamma4 and c8) to HPH (500-1700 bar) was studied. The experiments were performed in a laboratory medium (malt extract broth) on cells and spores. HPH caused a significant reduction of the initial cell number (1-2 log CFU ml(-1) at the highest pressures) in Gamma4 and DSMZ 2498 strains, whereas the effect on the spores was less significant. CONCLUSIONS: This study showed that the susceptibility of A. acidoterrestris to HPH was strain-dependent: DSMZ 2498 seemed the most susceptible strain, whereas c8 was the most resistant one. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of our study will provide useful information on the sensitivity of an emerging spoilage micro-organism, such as A. acidoterrrestris, to HPH.     

Aromatase activity in microsomal preparations of human genital skin fibroblasts: influence of glucocorticoids

Skin is an important site of estrogen production in men. Although the aromatase complex in these cells appears to be similar to that of other human cells, the regulation of aromatase by glucocorticoids in cultured human skin fibroblasts is unique. We examined aromatase activity in microsomal-enriched fractions of cultured human skin fibroblasts in order to characterize better the factors that regulate the aromatase in these cells. The optimum pH for aromatase activity in microsomal preparations ranged between 7.0 and 7.5. When androstenedione was the substrate, the mean Vmax was 0.58 pmol/mg protein/h (range: 0.09-1.26 pmol/mg protein/h) and the mean Km was 27 nM (range: 9-50 nM). When aromatase activity was determined as a function of NADPH concentration, the mean Vmax was 0.39 pmol/mg protein/h (range 0.11-0.82 pmol/mg protein/h) and the mean Km was 180 microM (range: 86-300 microM). For skin fibroblasts exposed to DEX, aromatase activity in isolated microsomes and intact cells was stimulated demonstrating a typical time course with peak levels at 14h and a decline toward baseline with prolonged (48-60 h) exposure. Cytosol from DEX-stimulated cells did not stimulate the aromatase activity in microsomal-enriched preparations from untreated cells. In addition, cytosol from cells incubated with DEX for a prolonged period (60 h) did not inhibit the higher aromatase activity of microsomes from cells incubated with DEX for only 14 h. We previously demonstrated that skin fibroblasts incubated with DEX and CHX produced a superinduction phenomenon for aromatase activity. This superinduction of enzyme activity also occurred in the microsomal-enriched fraction and was unaffected by the cytosol of these cells. These studies exclude the possibility that the unique effects of DEX on the aromatase in human skin fibroblasts are due to the production of either inhibitory or stimulatory soluble factors within cytosol.