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Purification of acid phosphatase I from germinating seeds of Vigna sinensis.

Acid phosphatase I (AP-I) is the major isoform of Vigna acid phosphatase. It is constitutively expressed in seed cotyledons during germination. AP-I was separated from other isoforms and purified to homogeneity by three simple purification steps; (NH4)2SO4 precipitation, and phosphocellulose and DEAE-cellulose column chromatography. The activity of AP-I was not affected by 1 mM Mg2+, Mn2+, Ca2+, Co2+ or Pb2+, but severely inhibited by 1 mM Cu2+, Fe3+, Hg2+, Mo6+ or Zn2+. AP-I has both phosphatase and pyrophosphatase activities, and is highly stable even at 50 degrees.

Acid Phosphatase↗

Intracellular localization of acid phosphatase; a comparative study of biochemical and histochemical methods.

The acid phosphatase activity of rat liver homogenates and of nuclear and cytoplasmic fractions isolated therefrom (by differential centrifugation) was determined biochemically in a series of experiments. For each liver, Gomori's histochemical test for acid phosphatase was run in parallel. No correlation was found between the biochemical and the histochemical results. According to the former, the enzyme appears to be almost entirely (95 per cent) located in the cytoplasm, while according to the latter, the acid phosphatase is predominantly concentrated in the nuclei and in some peribiliary polymorphic structures identified as myelin figures. It was found that the precipitaion pattern obtained in the histochemical test does not reveal, as generally assumed, differences in enzyme concentration among the various cell structures, but actually reveals differences in their lead phosphatase affinity. The usefulnes of the histochemical test for intracellular localization studies was found to be further limited by considerable fixation damage and formation of myelin figure artifacts. The biochemical approach is to be preferred because of better preservation of the material and direct and more reliable methods for the demonstraton of enzyme activity.

Acid Phosphatase↗

An examination of the age-related patterns of decay of acid phosphatase (ACP1) in human red cells from individuals of different phenotypes.

A study has been made of the decay of acid phosphatase (ACP1) in the human red cell using red cell fractions of different mean ages prepared by density gradient centrifugation. Red cells from acid phosphatase type A and type B individuals were used in the study. Acid phosphatase activity of the red cell fractions was determined by two different assay methods. The results obtained were comparable and have been combined. Acid phosphatase type A and type B showed a biphasic decay pattern with a rapid early loss of activity, followed by a more gradual rate of decline. Type A appeared to decay more rapidly than type B in both decay phases. It is proposed that differences in stability between type A and type B in vivo may explain the observed differences in activity between the enzyme types. There was no evidence for the generation of secondary isozymes by acid phosphatase type A or type B during red cell aging.

Acid Phosphatase↗

Structure of Francisella tularensis AcpA: prototype of a unique superfamily of acid phosphatases and phospholipases C.

AcpA is a respiratory burst-inhibiting acid phosphatase from the Centers for Disease Control and Prevention Category A bioterrorism agent Francisella tularensis and prototype of a superfamily of acid phosphatases and phospholipases C. We report the 1.75-A resolution crystal structure of AcpA complexed with the inhibitor orthovanadate, which is the first structure of any F. tularensis protein and the first for any member of this superfamily. The core domain is a twisted 8-stranded beta-sheet flanked by three alpha-helices on either side, with the active site located above the carboxyl-terminal edge of the beta-sheet. This architecture is unique among acid phosphatases and resembles that of alkaline phosphatase. Unexpectedly, the active site features a serine nucleophile and metal ion with octahedral coordination. Structure-based sequence analysis of the AcpA superfamily predicts that the hydroxyl nucleophile and metal center are also present in AcpA-like phospholipases C. These results imply a phospholipase C catalytic mechanism that is radically different from that of zinc metallophospholipases.

Acid Phosphatase↗

Staphylococcal acid phosphatase: extensive purification and characterization of the loosely bound enzyme.

Acid phosphatase of Staphylococcus aureus PS55 was eluted from the surface of these cells with 1.0 m KCl at pH 8.5 by gentle agitation at 25 C and was purified 44-fold (51% recovery) by two cycles of dialysis and gel filtration. The eluted enzyme which had a 280/260 (nm) absorbancy ratio of 0.71 required at least 0.5 m salt solution for solubilization; however, most of the purified product which had a 280/260 (nm) absorbancy ratio of 1.72 was soluble in dilute buffer solution [0.01 m tris(hydroxymethyl)aminomethane chloride, pH 8.5]. Purified acid phosphatase appeared homogeneous according to the criteria of gel filtration, starch-block electrophoresis, and analytical ultracentrifugation. In a starch block, migration was toward the cathode at pH 8.0. Maximal activity occurred at pH 5.2 to 5.3 and salt concentration had little effect on phosphatase activity up to 1.0 m KCl or NaCl. Progressive loss of enzymatic acitivity occurred at higher salt concentrations. Molecular weight of purified acid phosphatase was estimated to be 58,000.

Acid Phosphatase↗

Biochemical and histochemical response to a complete epithelial denudation of the rabbit cornea. Alkaline and acid phosphatase.

Activities of alkaline and acid phosphates were investigated in rabbit corneas after a complete epithelial denudation in vivo (limbus to limbus). Dynamics of enzymatic changes during corneal healing were followed quantitatively in homogenates of regenerated epithelium and stroma (denuded cornea) and in cryostate frozen sections on days 1, 4, 7, 14, and 28. Biochemical and histochemical findings at these times showed a different response in each enzyme. Acid phosphatase displayed a gradual increase of activity in epithelium as well as in stroma; on day 28 after injury its content was normal. In contrast, alkaline phosphatase showed delayed activity during the repair process, and even a month after de-epithelization was still subnormal, particularly in superficial layers of epithelium.

Acid Phosphatase↗

Purification and characterization of an acid phosphatase from Mycoplasma fermentans.

An acid phosphatase associated with the cell membranes of Mycoplasma fermentans was released from the membranes with Triton X-100, then purified by ion-exchange chromatography on DEAE-Sephacel and CM-Sepharose, followed by affinity chromatography on Con A-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed a single band with a molecular mass of 31.2 kilodaltons. The enzyme activity toward p-nitrophenyl phosphate was enhanced remarkably by Cu2+, Co2+ and Mg2+, but the activity was not inhibited by EDTA. The enzyme dephosphorylated O-phospho-L-tyrosine as well as p-nitrophenyl phosphate, but not O-phospho-L-threonine, O-phospho-L-serine, glucose-1-phosphate, phosphoryl choline and adenosine triphosphate. The level of the O-phospho-L-tyrosine phosphatase activity was the highest in Mycoplasma faucium and the second highest in Mycoplasma fermentans of all tested human mycoplasmas.

Acid Phosphatase↗

Identification and characterization of thiamin repressible acid phosphatase in yeast.

We have identified a genetic locus, pho4, in Schizosaccharomyces pombe which encodes a minor expressed cell surface acid phosphatase that is repressed by low concentrations (0.5 microM) of thiamin. The enzyme was purified from a strain that overproduces the enzyme. It is an Asn-linked glycoprotein. Removal of the carbohydrates by endoglycosidase H does not abolish enzymatic activity. The molecular mass of deglycosylated and unglycosylated enzyme that accumulates in membranes when cells are grown in the presence of tunicamycin is 56 kDa as determined by sodium dodecyl sulfate-gel electrophoresis. Thiamin regulation, at least in part, operates by reducing the level of pho4-mRNA. Pho4 is not genetically linked to the phosphate repressible acid phosphatase gene pho1. Phosphate and thiamin repressible acid phosphatase differ in their substrate specificity. Their protein moieties are immunologically related. Pho4 and pho1 are the only genes in S. pombe that express cell surface acid phosphatases being enzymatically active with nitrophenyl phosphate as substrate. S. pombe is not unique in having a thiamin repressible acid phosphatase. In Saccharomyces cerevisiae this enzyme is encoded by PHO3.

Acid Phosphatase↗

Tissue damage in sickle cell disease as assessed by serum acid phosphatase.

The extent of tissue damage caused by vaso-occlusion in sickle cell disease in those organs rich in acid phosphatase was assessed by measuring serum acid phosphatase in 33 patients with homozygous sickle cell disease Hb-SS (sicklers) and comparing the result with that of 31 persons with normal haemoglobin-AA (non-sicklers) matched for age and sex. The result showed a decrease in the level of total, labile and tartrate-resistant serum acid phosphatase in sicklers compared to non-sicklers, though the decrease is not statistically significant (p greater than 0.1). Though serum acid phosphatase is unlikely to be a useful index for the assessment of organ damage, the result is in consonance with reported decreases in other body secretions such as serum testosterone or aldosterone due to organ damage by vaso-occlusion of the micro-circulation by sickled red cells in sickle cell disease.

Acid Phosphatase↗

Changes in acid phosphatase activity during sporulation of Eimeria tenella oocysts.

Extracts of sporulated oocysts of Eimeria tenella have acid phosphatase activity which can be separated into Peak I (basic form) and Peak II (acidic form) on a DEAE-Sepharose Column. 2. Sporulation markedly effects the behavior of Peak I acid phosphatase (basic form) through the DEAE-Sepharose Column. 3. The Km value of Peak I decreases during sporulation. 4. The data obtained suggest that during sporulation the basic form of acid phosphatase (Peak I) undergoes marked degradation and transformation indicating that acid phosphatases may play an important role in the metabolism of E. tenella.

Acid Phosphatase↗

Kinetic properties of derepressible acid phosphatase from the yeast from of Yarrowia lipolytica.

(1) An acid phosphatase from depressed cells of the yeast form of Yarrowia lipolytica has been characterized kinetically by studies on specificity, inhibition, rate equation forms and modelling of the enzyme mechanisms. (2) The study on specificity revealed that the acid phosphatase is a rather unspecific phosphohydrolase that has similar activity on several different phosphate esters. A very weak transphosphorylating activity was also detected. (3) Among the reversible inhibitors, phosphate and vanadate were outstanding, whereas EDTA behaved as an activator. (4) v vs. [S] studies with o-carboxyphenyl phosphate as substrate show that the acid phosphatase of Y. lipolytica exhibits non-Michaelian behaviour, a minimum degree of 2:2 being detected for the rate equation in [S]. (5) The inhibition by phosphate and vanadate seems to have the same pattern of partial inhibition with a certain non-competitive nature, 1:1 being the minimum degree of the rate equation detected in (I).

Acid Phosphatase↗

Transcriptional control and patterning of the pho-1 gene, an essential acid phosphatase expressed in the C. elegans intestine.

We have previously described an acid phosphatase enzyme, PHO-1, present at the lumenal surface of all but the anterior six cells of the Caenorhabditis elegans intestine. In the present paper, we identify the pho-1 structural gene, which encodes a histidine acid phosphatase showing highest similarity to human prostatic acid phosphatase. The pho-1 5'-flanking DNA is capable of directing reporter gene expression that is both gut specific, correctly timed and correctly "patterned", that is, not expressed in the gut anterior. Furthermore, this anterior-posterior patterning of pho-1 expression responds to the C. elegans Wnt pathway as if pho-1 is repressed (directly or indirectly) by high levels of the HMG effector protein POP-1. Transgenic analysis of the pho-1 promoter shows that gut expression is critically dependent on a single WGATAR site. The gut-specific GATA factor ELT-2 binds to this site in vitro and removal of ELT-2 from the embryo destroys expression of the pho-1 reporter. Thus, all our results indicate that pho-1 is a direct downstream target of ELT-2. Finally, the pho-1 loss-of-function mutation shows an interesting and unexpected phenotype for a somatically-expressed hydrolytic enzyme: loss of pho-1 causes arrest of the majority of embryos but this lethality is a maternal effect. We suggest that pho-1 is required by the maternal intestine to assimilate some nutrient or cleavage product that is subsequently provided to the next generation of embryos.

Acid Phosphatase↗

Phosphotyrosine protein phosphatases and diabetic pregnancy: an association between low molecular weight acid phosphatase and degree of glycemic control.

Low molecular weight acid phosphatase encoded by the highly polymorphic locus ACP1 is a member of the protein-tyrosin phosphatase family (PTPases) which plays an essential role in the control of receptor signalling through phosphotyrosine pathways. Recent experiments have shown that purified rat liver ACP, corresponding to human ACP1, is able to hydrolyze a phosphotyrosine-containing synthetic peptide corresponding to the 1146-1158 sequence of the human insulin receptor, and shows a high affinity for it. This prompted us to analyze the degree of glycemic control in relation to ACP1 genetic variability in a sample of 214 diabetic pregnant women including IDDM, NIDDM and gestational diabetes. The ACP1 genotype was also determined in 482 non-diabetic pregnant women. In diabetic women glycemic levels in the last trimester of pregnancy appear to be significantly associated with the ACP1 genotype, and correlate positively with ACP1 enzymatic activity. The data suggest that quantitative variations of ACP1 may influence the clinical manifestations of diabetic disorders, and call for further studies on the role of this enzyme in the modulation of insulin-receptor phosphotyrosine pathways.

Acid Phosphatase↗

Regulation of nonspecific acid phosphatase in Salmonella: phoN and phoP genes.

Mutations in Salmonella typhimurium strains lacking nonspecific acid phosphatase mapped in two unlinked loci. One of these, phoP, was cotransducible by phage P22 with purB, whereas the second, phoN, was cotransducible by phage P1 with purA. Mutants with temperature-sensitive nonspecific acid phosphatase activity (measured in whole cells) were also isolated. A phoN mutant with thermolabile whole-cell activity was isolated directly from wild-type LT-2. Several other mutants with temperature-sensitive enzyme activity were also isolated as revertants of phoN mutants. These data suggest that phoN might be a structural locus for nonspecific acid phosphatase. The observation that a mutation resulting in high level of nonspecific acid phosphatase mapped in phoP suggests a possible regulatory role for this locus.

Acid Phosphatase↗

Effect of the thermal variation on the molecular weight estimation of the acid phosphatase from Physarum polycephalum.

A Binding Protein (BP) of nearly 95,000 daltons interacts with the acid phosphatase at the permissive temperature, elevating its molecular weight from 48,000 +/- 2,000 to 136,000 +/- 7,000 in CL and CHR strains of Physarum polycephalum plasmodia. The same molecule has not been observed in the extracts of M3C VIII and K strains. 28-31 degrees C is the critical temperature range for the breaking of enzyme-BP association in CL strains. This critical temperature is higher than 39 degrees C for the extract of CHR strains. CHR BP recognizes and binds CL acid phosphatase as strongly as CHR acid phosphatase. Km determination at 32 degrees C and thermoinactivation patterns at 66 degrees C indicate that the BP has no apparent regulatory role for the acid phosphatase activities. The presence or the absence of the BP in some of the strains may be used as a marker in the genetical studies of this organism.

Acid Phosphatase↗

[Analyse of zinc and acid phosphatase in seminal plasma and sperm parameters of infertile male].

OBJECTIVE: This study was to investigate the relationship among zinc, acid phosphatase levels in seminal plasma and sperm parameters in infertile male. METHODS: The activities of zinc and acid phosphatase in seminal plasma were detected and leucocytes was stained and counted in 169 infertile men and 21 normal fertility as control. RESULTS: Acid phosphatase levels in infertile groups were significantly lower than those in the normal control when grouped both in terms of sperm vitality and density( P < 0.01), but no statistical differences were observed among infertile groups (P > 0.05). Further, zinc levels in necrospermia groups were lower than those in the control (P > 0.05). The leucocyte numbers in infertile groups were larger than those in the control when grouped according to sperm density (P < 0.001), and the same results were obtained when grouped in terms of sperm vitality except in the necrospermia group (P > 0.05). Acid phosphatase and zinc levels were not correlated with leucocyte counting (r = 0.088, P = 0.162; r = 0.119, P = 0.057). CONCLUSION: The descent of acid phosphatase and ascent of leucocyte number in seminal plasma can result in the fall of sperm vitality and density, and can be used to be diagnostic indexes of male infertility. Seminal zinc levels in infertile groups were lower than those in the control, but there was no statistical significance.

Acid Phosphatase↗

The value of acid phosphatase measurements in predicting extraprostatic cancer growth before radical prostatectomy.

Acid phosphatase levels were determined using both an enzymatic method (32 cases) and radioimmunoassay (35 cases) in 35 patients with clinically localised prostatic cancer. All patients underwent total prostatectomy and pelvic lymphadenectomy. In cases of intracapsular prostatic cancer the level of prostatic acid phosphatase (PAP) measured by radioimmunoassay was 1.4 +/- 0.8 micrograms/l. In patients with either local extraprostatic disease or pelvic lymph node metastases the mean level of PAP was 3.5 +/- 2.8 micrograms/l. The difference was statistically significant. The specificity, sensitivity and accuracy of an elevated PAP (greater than 3.0 micrograms/l) in revealing extraprostatic extension of clinically localised prostatic cancer were 100, 37 and 66% respectively. When the enzymatic method was used, the level of acid phosphatase was elevated (greater than 13 u/l) in only 1 case. The specificity, sensitivity and accuracy of the enzymatic method were 100, 6 and 47% respectively. Elevation of PAP predicts, with a high degree of probability, either local extension outside the prostate or lymph node metastases. A normal PAP does not exclude extraprostatic extension of prostatic cancer.

Acid Phosphatase↗

Human lysosomal acid phosphatase: cloning, expression and chromosomal assignment.

A 2112-bp cDNA clone (lambda CT29) encoding the entire sequence of the human lysosomal acid phosphatase (EC 3.1.3.2) was isolated from a lambda gt11 human placenta cDNA library. The cDNA hybridized with a 2.3-kb mRNA from human liver and HL-60 promyelocytes. The gene for lysosomal acid phosphatase was localized to human chromosome 11. The cDNA includes a 12-bp 5' non-coding region, an open reading frame of 1269 bp and an 831-bp 3' non-coding region with a putative polyadenylation signal 25 bp upstream of a 3' poly(A) tract. The deduced amino acid sequence reveals a putative signal sequence of 30 amino acids followed by a sequence of 393 amino acids that contains eight potential glycosylation sites and a hydrophobic region, which could function as a transmembrane domain. A 60% homology between the known 23 N-terminal amino acid residues of human prostatic acid phosphatase and the N-terminal sequence of lysosomal acid phosphatase suggests an evolutionary link between these two phosphatases. Insertion of the cDNA into the expression vector pSVL yielded a construct that encoded enzymatically active acid phosphatase in transfected monkey COS cells.

Acid Phosphatase↗