Regulation of synthesis and secretion of acid and alkaline phosphatases in Neurospora crassa

Summary We show that N. crassa represses the production of acid phosphatase at pH higher than 8.0, irrespective of the carbon source used, whereas production was stimulated by sucrose at slightly acidic pH. The same profile of acid phosphatase production was observed in the pho-2A, pho-3A, nuc-1A, nuc-2A and preg ^c mutant strains. We also show that acid phosphatase synthesized by the preg ^c mutant strain grown on high phosphate medium has pronounced differences when compared to the enzyme synthesized by the wild-type strain grown on low phosphate medium in terms of heat stability, steady-state kinetic properties and DEAE-cellulose chromatography. In addition, the synthesis and/or secretion of only phosphate-repressible alkaline phosphatase is affected by mutations in acu-1, and acu-5 and acu-7 genes. These results, which indicate distinct pathways for the synthesis and secretion of acid and alkaline phosphatases in N. crassa, contradict the dosage titration model proposed by Metzenberg et al. (1974) whereby the synthesis of these enzymes should occur through a single hierarchical regulatory circuit as a response to phosphate starvation.     

On the correlation between alkaline phosphatase and phosphate transport in rat renal brush border membrane vesicles

Alkaline phosphatase activity in renal cortex homogenates and in isolated brush border membrane vesicles was compared with the rates of sodium-dependent transport of inorganic phosphate (P_i) by isolated brush border membranes. Brush border membrane vesicles were isolated from renal cortical homogenates of rats adapted during a period of 5–7 weeks to diets with different dietary contents of P_i (low P_i diet=0.15 g%, high P_i diet=2.0 g%).Alkaline phosphatase activity was not increased in the low P_i diet group as compared to the standard P_i diet group but was reduced in the high P_i diet group. Sodium-dependent transport of P_i was increased 2–3-fold in the low P_i diet group as compared to the standard P_i diet group, whereas transport activity was only unsignificantly decreased in the high P_i diet group.Studying kinetik parameters in the two extreme dietary groups it has been found that these differences are due to alteredV _max of the transport activity as well as of alkaline phosphatase activity. TheK _m for both activities remained unaltered.Alkaline phosphatase activity and transport of P_i in brush border membrane vesicles were also compared in the presence of EDTA or Zn²⁺ at concentrations which inhibit alkaline phosphatase activity. Transport of P_i was not affected by the inhibitors even when alkaline phosphatase was inhibited by more than 70% (0.5 mmol/l Zn²⁺) or completely (0.5 mmol/l EDTA).The experiments suggest that no correlation between alkaline phosphatase activity and transport of P_i exists in isolated brush border membrane vesicles.     

ENZYME HISTOCHEMICAL STUDY ON HUMAN THYMUS AND ITS AGE CHANGE

Human thymuses, ranging in age from newborn to 62 years old, were studied enzyme histochemically. The thymic epithelial cells covering cortical surface and bordering vascular areas in the medulla were positive for 5'–nucleotidase, but not for other enzymes. The thymic epithelial cells composing Hassall's corpuscles were positive for acid phosphatase, esterases, –glucuronidase, and alkaline phosphatase, regardless of age, but totally negative for 5'–nucleotidase and ATPase. All enzymes examined except for glucuronidase were demonstrated income of the thymie epithelial cells scattered in the medulla, although the pattern of distribution and the degree of positivity were different by enzymes. These findings suggest that the thymic epithelial cells are composed of functionally heterogenous subpopula–tions. Acid phosphatase was demonstrated in thymocytes in both cortex and medulla, but 5'–nucleotidase and ATPase were observed in some thymocytes in the medulla of young thymus. ACTA PATHOL. JPN. 33: 275–285, 1983.     

Acid phosphatase deficient mutants of Schizosaccharomyces pombe are defective in tyrosine uptake

Summary The uptake of tyrosine and arginine into wild type and acid phosphatase deficient mutants (pho 1) of Schizosaccharomyces pombe was investigated. All 11 pho 1-alleles tested exhibited a reduced tyrosine uptake and impaired uptake cosegregated with the lack of acid phosphatase activity. Kinetic analyses using wild type cells grown in high phosphate medium (acid phosphatase repressed) and low phosphate medium (acid phosphatase derepressed) showed staturation kinetics for tyrosine with a K_M of about 2 × 10^–4 M for both media and a V of about 5 nmol min^–1mg^–1 and 2 nmol min^–1mg^–1 for derepressed and repressed cells respectively. The pho 1–118 strain completely lacked this saturable uptake system for tyrosine. Preliminary evidence suggests that tyrosine uptake may be via a general amino acid permease system and we conclude that mutations in the structural gene of acid phosphatase which abolish enzyme activity lead to a loss of this uptake system. In contrast to tyrosine, arginine uptake seems not to be significantly affected either by different acid phosphatase levels in wild type cells or by the pho 1–118 mutation.     

Mutations affecting cyclic phosphodiesterases and adenylate cyclase in Neurospora

Summary Two regulatory mutants in orthophosphateregulated cyclic phosphodiesterase (CPDase), cpd-3 and cpd-4, were isolated and mapped proximal to arg-1 on L.G. IC and distal to urg-12 on L.G. IIR, respectively. cpd-3 showed short aerial hyphae with dense formation of conidia. The morphology was very similar to that of cr-1, cpd-3 and cr-1 had reduced levels of cyclic 3,5-AMP, adenylate cyclase and cPDases (CPDase I, II and III in low phosphate condition) but had elevated levels of cyclic 3,5-GMP. Although cr-1 showed an enhanced level and enhanced activation of heat activated cyclic phosphodiesterase (ha-PDE), this enzyme was not activated in cpd-3. cpd-4, nuc-1 and nuc-2 produced neither CPDase I, II, III, alkaline phosphatase nor ribonuclease N1 in low phosphate media. These mutants did not produce aerial hypha or conidium when grown in low phosphate liquid medium. Activation of ha-PDE occurred in cpd-4, but not in nuc-1 and nuc-2.     

The Effect of haemolymph extraction on distribution of lysosomal enzymes in Lymnaea stagnalis haemocytes: A cytochemical study

An enzyme cytochemical, light microscopy study was undertaken to evaluate the effect of haemolymph extraction on distribution of acid phosphatase, alkaline phosphatase, alpha-naphthyl acetate esterase, and peroxidase in haemocytes of Lymnaea stagnalis. The study revealed that discrete acid phosphatase, alkaline phosphatase, alpha-naphthyl acetate esterase, and peroxidase-staining subpopulations of haemocytes do exist in L. stagnalis, and that there are differences in distribution. A high percentage, about 96%, of haemocytes showed positive reaction for peroxidase, the percentage of haemocytes showing activity for alpha-naphthyl acetate esterase, acid phosphatase, and alkaline phosphatase, was about 54, 42, and 34, respectively. Haemolymph extraction by foot retraction, significantly affected enzyme distribution. Whereas distribution of acid phosphatase increased significantly from day 1 to 5 after extraction of haemolymph, that of all other enzymes showed significant decrease from day 1 to 7 depending upon the enzyme. The distribution of all enzymes stabilized by day 8 after extraction of haemolymph. Probable reasons for the observed fluctuations in the distribution of enzymes subsequent to haemolymph extraction are discussed.     

Biosynthesis of homoarginine and ornithine as precursors of the phytoeffector phaseolotoxin by the amidinotransfer from arginine to lysine catalyzed by an amidinotransferase in Pseudomonas syringae pv. phaseolicola

In the phytopathogenic Pseudomonas syringae pv. phaseolicola an amidinotransferase was detected and characterized which catalyzes the amidinotransfer from arginine to lysine producing the phaseolotoxin precursors homoarginine and ornithine. The enzyme was purified 419-fold by affinity chromatography on homoarginine-Sepharose. It had an apparent mol. wt. of 200 000 and exhibited MICHAELIS-MENTEN kinetics with K_m 7.7 mM for arginine and K_m 4.2 mM for lysine. Ornithine competitively inhibited the enzyme with a K_i of 0.8 mM. During cultivation of the pseudomonad the highest enzyme activity corresponds with the highest production rate of phaseolotoxin. At low toxin production, mutants exhibit a low enzyme activity and vice versa. Only those strains of different pathovarieties which are able to produce phaseolotoxin contain the enzyme. At 30 °C cultivation temperature, the enzyme activity was 50% of that at 18 °C, and toxin production decreased with increasing temperature. When microbes were grown on ribose, their enzyme activity was 60% lower than grown on glucose or glycerol. Toxin production was decreased to about 10% when growing on ribose. Amidinotransferase is considered to be one of the key enzymes of phaseolotoxin biosynthesis.     

Anion-stimulated ATPase activity of brush border from rat small intestine.

Differential centrifugation of rat small intestinal homogenates produced a crude brush border (BB) fraction that was enriched 15-fold for the marker enzymes, alkaline phosphatase and sucrase; contamination with mitochondrial enzymes, monoamine oxidase and succinate dehydrogenase, was minimal. ATP hydrolysis by this BB fraction was stimulated by addition of several anions to the incubation medium: HCO3 and Cl were equally effective in this regard, with NO3, NO2, SO4, and acetate being less stimulatory. SCN and CNO inhibited ATPase activity, whereas the divalent anion SO3 was stimulatory at low concentrations (less than 25 mM) but inhibitory at 100 mM. Maximum anion stimulation was observed at a Mg concentration of 0.5 mM, and pH optimum was 8.5. Kinetic analysis showed that HCO3 increased the Vmax without altering the Km for ATP; the Ka for this effect of HCO3 was 35 mM. This enzyme activity was completely inhibited by 20 mM L-phenylalanine, 10 mM L-cysteine, and 3 mM EDTA, compounds that also inhibited intestinal alkaline phosphatase. These results demonstrate the presence of anion-stimulated ATPase activity in rat small intestinal brush border and suggest that this activity may be related to intestinal alkaline phosphatase. The role of this enzyme in intestinal transport is not known, but could relate to the regulation of intestinal absorption and secretion.     

Characterization of phosphate binding by alkaline phosphatase in rat kidney brush border membrane

Phosphate binding by rat renal brush border membranes occurs on a single protein, as visualized by SDS polyacrylamide gel electrophoresis. The same protein can also be specifically labelled by gamma-32P ATP at 0 degree C or in the absence of magnesium. The phosphate binding protein co-migrates with monomers of two alkaline phosphatase activity bands previously localized on acrylamide gel. Measurement of binding by TCA precipitation, ion-exchange chromatography and dialysis gave an average of 31.1 +/- 5.7 pmol phosphate bound/mg protein. Alkaline phosphatase would then represent 0.23% of total brush border membrane protein. Maximal binding activity is obtained at pH 6.5, but when membranes are phosphorylated at pH 6.5 and the pH increased to 9.4, 50% of the bound radioactivity is released. The binding of phosphate to this protein presents two different apparent Km: one at 40 microM for low and one at 390 microM for high substrate concentrations. The membrane bound phosphate is readily exchangeable with phosphate in the medium. Phosphate binding and phosphate release are complete within 5 s. Alkaline phosphatase substrates and EDTA are potent inhibitors of phosphate binding and produce over 90% inhibition. Characteristics of phosphate binding for kidney membrane bound alkaline phosphatase seem very similar to the soluble form of the enzyme from various sources.     

Effect of biotin deficiency on some properties of Staphylococcus aureus isolates from humans and other animals.

Staphylococcus aureus isolates from humans and other animals were grown in biotin assay medium containing 12 mug of biotin per liter and compared to isolates from the same sources grown concurrently in medium containing adequate biotin. The two cultures were tested for production of coagulase, phosphatase, and fibrinolysin enzymes and for responses to various antimicrobial agents and bacteriophages. Organisms grown in biotin-deficient medium produced less phosphatase; coagulase and fibrinolytic activity was reduced, and they were more susceptible to antimicrobial agents than were normal organisms, but phage susceptibility was not greatly affected.     

Enzymes of carbohydrate metabolism in root-nodule bacteria during growth on acetate

The key enzymes of the Embden -Meyerhof -Parnas (EMP), Entner -Doudoroff (ED) and pentose phosphate (PP) pathways as well as those of the tricarboxylic acid (TCA) cycle and gluconeogenesis were assayed in the cells of Rhizobium and Bradyrhizobium grown on acetate. Except for glyceraldehyde-3-phosphate dehydrogenase, the activities of all the enzymes of the EMP, ED and PP pathways were found to be low in acetate grown cells in the present study as compared to those of glucose grown cells reported by us earlier (Mandal and Chakrabartty 1993) indicating a probable repression of the enzymes. The enzymes of the tricarboxylic acid cycle were detected in high levels in the acetate grown cells of the root-nodule bacteria. The operation of the glyoxylate pathway for acetate metabolism in the root-nodule bacteria were reported previously by us (Mandal and Chakrabartty 1992). The evidences taken together point to the simultaneous operation of both the TCA cycle and the glyoxylate cycle for acetate metabolism in these bacteria.     

Anion-stimulated phosphohydrolase activity of intestinal alkaline phosphatase

Phosphohydrolase activity of a highly enriched commercial preparation of calf intestinal alkaline phosphatase was stimulated in the presence of HCO3. SO4, Cl, SCN, and acetate did not stimulate hydrolysis, whereas SO3 exhibited a bimodal effect, stimulating at low (25mM) concentration but inhibiting at high (100 mM) concentration. The pH optimum of this stimulation by HCO3 or SO3 was 8.5--9.0 and was maximal at a Mg concentration of 0.5 mM. HCO3 increased the Vmax of the reaction without changing the Km for ATP. ATP, GTP, UTP, and xanthosine triphosphate were equally effective as substrates, whereas AMP and p-nitrophenyl phosphate were much less effective. Alkaline phosphatase activity was inhibited by L-cysteine and L-phenylalanine, compounds that also inhibited the HCO3-ATPase activity of the preparation. Passage of the commercial preparation through an anion-exchange column yielded a fraction with enriched alkaline phosphatase and HCO3-ATPase activities; this fraction proved to be a single protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, on isoelectric focusing, and by immunologic techniques. These studies strongly suggest that alkaline phosphatase and anion-stimulated phosphohydrolase activities are properties of the same protein in small intestine. It is possible that alkaline phosphatase may function as a HCO3-ATPase involved in intestinal absorption and secretion.     

The use of graft copolymers as enzyme supports, 3. The Immobilization of proteins and enzymes on poly(fluorostyrene) and poly(nitrofluorostyrene) — nylon graft copolymers

The radiation induced, graft copolymerization of m- and p-fluorostyrene onto nylon was successfully achieved. Grafting was followed by nitration of the fluorostyrene groups in the copolymer to provide substrates which were active to the coupling of various enzymes and proteins. Bovine serum albumin, ovalbumin, pepsin, β-galactosidase, and alkaline phosphatase proteins all coupled very efficiently but the activities of the coupled enzymes were relatively low.     

Phosphatase activity of staphylococci is constitutive in some species and repressed by phosphates in others.

The phosphatase activities of colonies of staphylococcal strains belonging to the 10 species most frequently isolated from specimens of human origin were evaluated on media with undetermined inorganic phosphate contents and on media supplemented with known amounts of phosphates. All strains of all species tested were phosphatase positive on plates that were not supplemented with inorganic phosphates when the pH of the medium was high. In media supplemented with 0.3% phosphates at low and high pH, all strains of Staphylococcus aureus, almost all strains of S. epidermidis and S. xylosus, and none of the other species tested were phosphatase positive. Thirty S. simulans strains were grown in broths at pH 7 with and without phosphates, and the phosphatase activities of the cells were assayed at pH 5.5 and 8 in the absence of phosphates. At pH 8, all strains gave a strong positive reaction when grown in the absence of phosphates and a negative reaction when grown in the presence of these salts at 0.3%. It was concluded that all the species of staphylococci tested possess phosphatase activity and that the staphylococcal phosphatases are constitutive in some species and repressed in others.     

Uptake of Serotonin in Blood Platelets in vitro. I: The Effects of Chloride

Lingjærde , O. Jr . Uptake of serotonin in blood platelets in vitro. I: The effects of chloride. Acta physiol. scand. 1971. 81. 75–83. The effects of chloride on the “active” uptake of ¹⁴C-serotonin in human blood platelets in vitro have been studied, using a phosphate buffered artificial medium. The uptake of serotonin requires the presence of chloride in the medium. The effect of chloride concentration on the rate of serotonin uptake is in agreement with Michaelis-Menten type kinetics. The apparent Michaelis constant for chloride is about 35 mM at pH 7.2, and it is independent of the concentration of serotonin. Moreover, the apparent Michaelis constant for serotonin is independent of the concentration of chloride. Preincubation with low concentrations of chloride is accompanied by a decrease in the rate of uptake, whereas the converse is true when high concentrations of chloride are used. It is suggested that inward transport of serotonin is dependent on chloride in such a way that serotonin and chloride have to be attached to the same carrier. It is also suggested that the intracellular concentration of chloride is of importance for the rate of re-trans-location of the carrier from the inner to the outer surface of the membrane.     

Calvaria Derived Osteogenic Cells: Phenotypic Expression in Culture

The osteoblast phenotype is characterized by its ability to (a) synthesize a well defined mineralized collage nous matrix, (b) regulate the remodeling process by synthesizing local hormones (PGE₂) and specific molecules (osteocalcin) and enzymes (alkaline phosphatase and collagenase), (c) respond to a variety of hormones (PTH, PGs, vitamin-D metabolites, steroids and growth factors), (d) respond to mechanical stimulation. Most of osteoblast culture systems meet many of the above qualifications though most fail to show the PTH effect on DNA synthesis, (c), and mechanical stimulation, (d). Here we show that by using trypsin digestion and serum-containing low calcium medium (0.25 mM), all the above listed osteoblast phenotypic characteristics are demonstrated including their responsiveness to mechanical stimulation and the PTH effect on DNA synthesis.     

Response of lung enzyme activities in rabbits following short-term exposure ton-hexane: Correlation between morphological and biochemical changes

The activity of lactatedehydrogenase, -glucuronidase, glucose-6-phosphate dehydrogenase, acid and alkaline phosphatase was studied in lung homogenate from New Zealand rabbits exposed to 3000 p.p.m. ofn-hexane 8 h per day for 8 days or filtered air.In hydrocarbon-treated animals all enzymes examined, except alkaline phosphatase, were markedly increased.The biochemical changes correlated well with the morphological changes and the results of cytological evaluation of bronchopulmonary lavage. It is suggested that high values in lung lysosomal enzymes from treated rabbits reflect the acute inflammation whilst the increase in lung glueose-6-phosphate dehydrogenase may depend upon reparative process subsequent ton-hexane-induced lung damage.     

Isolation,identification and germination of spores of Thermoactinomyces from Qatari soils (Arabian Gulf)

40 isolates of Thermoactinomyces were isolated from Qatari soils and identified as T. vulgaris (26 isolates), T. thalpophilus (8), T. putidus (4) and T. sacchari (2). The total count of this genus increased concomitantly with the increase of organic matter in soil samples. The effect of temperature, pH and composition of medium on the germination of spores of T. vulgaris were examined by measuring the reduction of turbidity of spore suspensions. Optimal germination took place at 55 °C, at a pH range of 7.5 to 8.5 and in phosphate buffer (10 mM) plus L-alanine, L-leucine and L-phenylalanine (1 mM).     

Effect of adriamycin on the RPC C2A cloned cell line from rat incisor pulp

Background and Methods: RPC C2A cells, cloned from Wistar rat incisor pulp, were grown in culture 2–7 days after exposure to adriamycin at a concentration of 0.1 mg/L for 2 hours. Morphological changes, labelled proline incorporation and alkaline phosphatase activity were studied in response to this treatment. Results: At the light microscopic level, colonies of enlarged cells appeared 2 days after adriamycin treatment. The size of these colonies increased during the course of this study. The control samples showed no apparent changes. At the electron microscopic level, the small cells in the adriamycin-treated cultures showed fewer vesicles than the controls, but more prominent rough endoplasmic reticulum. However, the enlarged cells contained an abundance of vesicles, and few profiles of rough endoplasmic reticulum. Radiolabelled proline incorporation in the control group was significantly higher than the experimental after 2 days in culture, but showed no significant difference after 7 days. Histological staining for alkaline phosphatase showed that there was a slightly higher intensity in the control samples than in the experimental cultures at this time. Conclusions: This study has shown that the RPC C2A cells will respond to adriamycin treatment in vitro, where the acute effects were quite different from the protracted effects with respect to labelled proline incorporation and alkaline phosphatase activity. © 1995 Wiley-Liss, Inc.     

The localization of the Na+−K+-ATPase in the cells of rat kidney cortex

Summary Plasma membrane fractions of rat kidney cortex were subdivided by centrifugation on a continuous and a discontinuous sucrose gradient and by carrier free continuous electrophoresis. In the different fractions the activity of alkaline phosphatase and aminopeptidase, enzymes which are present in the brushborder membrane, as well as Mg⁺⁺-ATPase, Na⁺–K⁺-ATPase, 5-nucleotidase, acid phosphatase and glucose-6-phosphatase were determined.The distribution of alkaline phosphatase, aminopeptidase and 5-nucleotidase is identical, indicating the localization of these enzymes in the brushborder membrane. Na⁺–K⁺-ATPase does not show an identical distribution with any of the enzymes studied.Using carrier free continuous electrophoresis fractions can be obtained which are enriched in alkaline phosphatase by a factor of 15 when compared to the cortex homogenate, whereas the specific activity of Na⁺–K⁺-ATPase is reduced to one third of the starting material. On the other hand fractions can be isolated in which the specific activity of Na⁺–K⁺-ATPase is 16 times higher than in the homogenate. No enrichment of alkaline phosphatase occurs in these fractions.It is therefore concluded that the Na⁺–K⁺-ATPase is not present in the brushborder membrane nor in the lysosomes or endoplasmatic reticulum. The most probable localization of the Na⁺–K⁺-ATPase are the basal infoldings of the plasma membranes of the cells.A preliminary report has been published by Kinneet al. [28, 29].Major part of this work was done by J. E. Schmitz for his degree of M. D.