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European Journal of Biochemistry Jul 1986We have purified secreted acid phosphatase of Schizosaccharomyces pombe. The enzyme is N-glycosylated, the associated carbohydrate accounts for 90% of the total...
We have purified secreted acid phosphatase of Schizosaccharomyces pombe. The enzyme is N-glycosylated, the associated carbohydrate accounts for 90% of the total molecular mass and the protein moiety has a molecular mass of 54 kDa. The deglycosylated enzyme still exhibits enzymatic activity. Using antibodies recognizing the protein moiety of the enzyme we have identified two intracellular precursors of acid phosphatase: an unglycosylated membrane-bound 54-kDa form that accumulates in the presence of tunicamycin and a partially glycosylated 72-kDa form that accumulates mostly in membranes of cells grown in rich medium. We further showed that the conversion of the 54-kDa and 72-kDa forms to partially glycosylated and fully glycosylated acid phosphatase is a regulated process. Growth conditions determine how much of translated 54-kDa acid phosphatase is glycosylated to the 72-kDa form and how much remains unglycosylated in membranes. When cells are grown in a rich medium, 5% of the total acid phosphatase protein remains as unglycosylated enzyme and 8% as partially glycosylated 72-kDa form. In cells grown in the minimal medium, however, all of the 54-kDa and 72-kDa forms of acid phosphatase are rapidly processed to fully glycosylated enzyme. The 72-kDa form and the unglycosylated form of acid phosphatase are not secreted or transported to the plasma membrane.
Topics: Acid Phosphatase; Autoradiography; Carbohydrate Metabolism; Carbohydrates; Cell Membrane; Culture Media; Saccharomycetales; Schizosaccharomyces
PubMed: 3732265
DOI: 10.1111/j.1432-1033.1986.tb09730.x -
The Biochemical Journal Jul 1989Tartrate-resistant acid phosphatase is one of the major enzymes produced and secreted by osteoclasts. To obtain sufficient enzyme for biochemical characterization, we...
Tartrate-resistant acid phosphatase is one of the major enzymes produced and secreted by osteoclasts. To obtain sufficient enzyme for biochemical characterization, we have purified this enzyme from human osteoclastomas by sequential chromatography on SP-Sephadex, CM-Sephadex, hydroxylapatite, Sephadex G-150 and concanavalin A-Sepharose. The purification over the original tumour extract was about 2000-fold, with a yield of 10%. The enzyme appeared to be homogeneous when assessed by SDS/polyacrylamide-gel electrophoresis. Both gel filtration and SDS/polyacrylamide-gel electrophoresis indicated an Mr of about 30,000. The reduced and alkylated enzyme consists of two subunits with Mrs of 15,000 and 17,500. The N-terminal amino acid sequence of both subunits indicates that there is a high degree of identity between the osteoclastoma enzyme and similar enzymes purified from spleen and uterus. Using 4-methylumbelliferyl phosphate as substrate, the specific activity of the purified enzyme was 387 units.mg-1, and the Km was 284 microns. The pH optimum was 5.7. Unlike similar enzymes purified from human and bovine bone, osteoclastoma acid phosphatase is not activated by reducing agents (2-mercaptoethanol or ascorbic acid). The enzyme contains 4.8 mol of Fe2+/3+, 0.3 mol of Mn2+ and 1.7 mol of Mg2+ per mol of enzyme. Although the enzyme loses 50% of its activity in the presence of EDTA, it is not inhibited by the iron chelator 1,10-phenanthroline. However, the enzyme is activated to a small extent by Mn2+ and Mg2+. Using a variety of substrates and inhibitors, we demonstrate that there are differences between the osteoclastoma acid phosphatase and the enzyme purified from other sources.
Topics: Acid Phosphatase; Amino Acid Sequence; Bone Neoplasms; Giant Cell Tumors; Humans; Molecular Sequence Data; Substrate Specificity; Tartrates
PubMed: 2775236
DOI: 10.1042/bj2610601 -
The Journal of Investigative Dermatology Dec 1979Isoelectric focusing and concanavalin A-Sepharose chromatography were used to study the multiple forms and glycoprotein natures of so-called lysosomal hydrolases from...
Isoelectric focusing and concanavalin A-Sepharose chromatography were used to study the multiple forms and glycoprotein natures of so-called lysosomal hydrolases from psoriatic scales. Acid phosphatase appeared as 5 different forms with pI values of 6.5, 6.1, 5.8, 5.6 and 5.45. Seven isoenzymes of alpha-fucosidase were identified with pI values of 6.4, 6.2, 5.9, 5.75, 5.65, 5.4 and 5.2. Acid alpha-mannosidase activity appeared as one peak with pI value of 6.75 and a weak activity of neutral alpha-mannosidase was present with pI value of 6.7. Incubation of the extract with neuraminidase increased their pI values of acid phosphatase, alpha-fucosidase and alpha-mannosidase to the more basic forms. This finding suggests that epidermal acid phosphatase, alpha-fucosidase and alpha-mannosidase have some N-acetylneuraminic acid residues. In addition concanavalin A-Sepharose column chromatography was also performed to confirm the glycoprotein nature of acid phosphatase. This enzyme was bound to the column and not released from the column even with the treatment of 0.5 M NaCl, but the enzyme was eluted from the column with the treatment of alpha-methyl-D-glucoside.
Topics: Acid Phosphatase; Humans; In Vitro Techniques; Isoelectric Focusing; Mannosidases; Psoriasis; Skin; alpha-L-Fucosidase
PubMed: 512407
DOI: 10.1111/1523-1747.ep12541593 -
Journal of Bacteriology Feb 1973Saccharomyces cerevisiae strain H-42 seems to have two kinds of acid phosphatase: one which is constitutive and one which is repressible by inorganic phosphate. The...
Saccharomyces cerevisiae strain H-42 seems to have two kinds of acid phosphatase: one which is constitutive and one which is repressible by inorganic phosphate. The constitutive enzyme was significantly unstable to heat inactivation, and its K(m) of 9.1 x 10(-4)m for p-nitrophenylphosphate was higher than that of the repressible enzyme (2.4 x 10(-4)m). The constitutive and the repressible acid phosphatases are specified by the phoC gene and by the phoB, phoD, or phoE gene, respectively. Results of tetrad analysis suggested that the phoC and phoE genes are linked to the lys2 locus on chromosome II. Since both repressible acid and alkaline phosphatases were affected simultaneously in the phoR, phoD, and phoS mutants, it was concluded that these enzymes were under the same regulatory mechanism or that they shared a common polypeptide. The phoR mutant produced acid phosphatase constitutively, and the phoR mutant allele was recessive to its wild-type counterpart. The phoS mutant showed a phenotype similar to that of a mutant defective in one of the phoB, phoD, or phoE genes. However, the results of genetic analysis of the phoS mutant clearly indicated that the phoS gene is not a structural gene for either of the repressible acid and alkaline phosphatases, but is a kind of regulatory gene. According to the proposed model, the phoS gene controls the expression of the phoR gene, and inorganic phosphate would act primarily as an inducer for the formation of the phoR product which represses phosphatase synthesis.
Topics: Acid Phosphatase; Cell-Free System; Crosses, Genetic; Enzyme Repression; Genes, Regulator; Genotype; Hot Temperature; Kinetics; Mutation; Nitrophenols; Phenotype; Phosphates; Radiation Effects; Saccharomyces cerevisiae; Spectrophotometry; Ultraviolet Rays
PubMed: 4570606
DOI: 10.1128/jb.113.2.727-738.1973 -
PloS One 2019Prostatic acid phosphatase (PAP), which is secreted by prostate, increases in some diseases such as prostate cancer. PAP is also present in the central nervous system....
Prostatic acid phosphatase (PAP), which is secreted by prostate, increases in some diseases such as prostate cancer. PAP is also present in the central nervous system. In this study we reveal that α-synuclein (Snca) gene is co-deleted/mutated in PAP null mouse. It is indicated that mice deficient in transmembrane PAP display neurological alterations. By using immunohistochemistry, cerebellar cortical neurons and zone and stripes pattern were studied in Pap-/- ;Snca-/- mouse cerebellum. We show that the Pap-/- ;Snca-/- cerebellar cortex development appears to be normal. Compartmentation genes expression such as zebrin II, HSP25, and P75NTR show the zone and stripe phenotype characteristic of the normal cerebellum. These data indicate that although aggregation of PAP and SNCA causes severe neurodegenerative diseases, PAP -/- with absence of the Snca does not appear to interrupt the cerebellar architecture development and zone and stripe pattern formation. These findings question the physiological and pathological role of SNCA and PAP during cerebellar development or suggest existence of the possible compensatory mechanisms in the absence of these genes.
Topics: Acid Phosphatase; Animals; Cerebellar Cortex; Cerebellum; Gene Expression; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Motor Activity; Nerve Tissue Proteins; Transcriptome; alpha-Synuclein
PubMed: 31509576
DOI: 10.1371/journal.pone.0222234 -
Journal of Enzyme Inhibition and... Dec 2023Acid phosphatases (EC 3.1.3.2) are the enzymes that catalyse transphosphorylation reactions and promotes the hydrolysis of numerous orthophosphate esters in acidic... (Review)
Review
Acid phosphatases (EC 3.1.3.2) are the enzymes that catalyse transphosphorylation reactions and promotes the hydrolysis of numerous orthophosphate esters in acidic media, as a crucial element for the metabolism of phosphate in tissues. Inorganic phosphate (Pi) utilisation and scavenging, as well as the turnover of Pi-rich sources found in plant vacuoles, are major processes in which intracellular and secretory acid phosphatases function. Therefore, a thorough understanding of these enzymes' structural characteristics, specificity, and physiochemical properties is required to comprehend the function of acid phosphatases in plant energy metabolism. Furthermore, acid phosphatases are gaining increasing importance in industrial biotechnology due to their involvement in transphosphorylation processes and their ability to reduce phosphate levels in food products. Hence, this review aims to provide a comprehensive overview of the purification methods employed for isolating acid phosphatases from diverse plant sources, as well as their structural and functional properties. Additionally, the review explores the potential applications of these enzymes in various fields.
Topics: Acid Phosphatase; Hydrolysis; Plants; Phosphates
PubMed: 37985663
DOI: 10.1080/14756366.2023.2282379 -
Journal of Experimental Botany Jul 2020Whilst constitutive overexpression of particular acid phosphatases (APases) can increase utilization of extracellular organic phosphate, negative effects are frequently...
Whilst constitutive overexpression of particular acid phosphatases (APases) can increase utilization of extracellular organic phosphate, negative effects are frequently observed in these transgenic plants under conditions of inorganic phosphate (Pi) sufficiency. In this study, we identified rice purple acid phosphatase 10c (OsPAP10c) as being a novel and major APase that exhibits activities associated both with the root surface and with secretion. Two constructs were used to generate the OsPAP10c-overexpression plants by driving its coding sequence with either a ubiquitin promoter (UP) or the OsPAP10c-native promoter (NP). Compared with the UP transgenic plants, lower expression levels and APase activities were observed in the NP plants. However, the UP and NP plants both showed a similar ability to degrade extracellular ATP and both promoted root growth. The growth performance and yield of the NP transgenic plants were better than the wild-type and UP plants in both hydroponic and field experiments irrespective of the level of Pi supply. Overexpression of APase by its native promoter therefore provides a potential way to improve crop production that might avoid increased APase activity in untargeted tissues and its inhibition of the growth of transgenic plants.
Topics: Acid Phosphatase; Gene Expression Regulation, Plant; Organophosphates; Oryza; Phosphates; Phosphorus; Plant Proteins; Plant Roots; Plants, Genetically Modified
PubMed: 32270183
DOI: 10.1093/jxb/eraa179 -
Journal of Bone and Mineral Research :... Oct 2003TRACP is a lysosomal enzyme found in diverse tissues, where it is expressed in dendritic cells as well as osteoclasts and macrophages. To investigate the function of... (Review)
Review
TRACP is a lysosomal enzyme found in diverse tissues, where it is expressed in dendritic cells as well as osteoclasts and macrophages. To investigate the function of TRACP in vivo, we have generated mice in which the gene-encoding TRACP has been selectively disrupted by targeted homologous recombination in murine embryonic stem cells. Homozygous TRACP "knockout" mice have progressive foreshortening and deformity of the long bones and axial skeleton suggesting a role for TRACP in endochondral ossification. There is increased mineralization reflecting a mild osteopetrosis caused by reduced osteoclast modeling activity. These knockout mice also display an impairment of macrophage function with abnormal immunomodulatory cytokine responses. Superoxide formation and nitrite production were enhanced in stimulated macrophages lacking TRACP as was the secretion of the proinflammatory cytokines TNF-alpha, interleukin (IL)-1beta, and IL-12. TRACP knockout mice showed delayed clearance of the microbial pathogen Staphylococcus aureus after sublethal intraperitoneal inoculation. The macrophages lacking TRACP showed an increase in tartrate-sensitive lysosomal acid phosphatase activity (LAP). The TRACP knockout mice were bred with mice lacking LAP. Mice lacking both TRACP and LAP had even shorter bones than the TRACP single knockouts. Osteopontin, identical to the T-cell cytokine eta-1, accumulated adjacent to actively resorbing osteoclasts suggesting that both phosphatases are important for processing this protein. We propose that TRACP may be an important regulator of osteopontin/eta-1 activity common to both the immune system and skeleton.
Topics: Acid Phosphatase; Animals; Bone and Bones; Dendritic Cells; Homozygote; Interleukin-1; Interleukin-12; Isoenzymes; Lysosomes; Macrophages; Mice; Mice, Knockout; Osteopetrosis; Phenotype; Staphylococcus aureus; Superoxides; Tartrate-Resistant Acid Phosphatase; Time Factors; Tumor Necrosis Factor-alpha
PubMed: 14584904
DOI: 10.1359/jbmr.2003.18.10.1905 -
American Journal of Physiology. Cell... Jun 2014We have previously shown that in addition to the well-known secreted isoform of prostatic acid phosphatase (sPAP), a transmembrane isoform exists (TMPAP) that interacts...
We have previously shown that in addition to the well-known secreted isoform of prostatic acid phosphatase (sPAP), a transmembrane isoform exists (TMPAP) that interacts with snapin (a SNARE-associated protein) and regulates the endo-/exocytic pathways. We have also shown that PAP has 5'-ectonucleotidase and thiamine monophosphatase activity and elicits antinociceptive effects in mouse models of chronic inflammatory and neuropathic pain. Therefore, to determine the physiological role of PAP in a typical exocrine organ, we studied the submandibular salivary gland (SMG) of PAP(-/-) and wild-type C57BL/6J mice by microarray analyses, microRNA sequencing, activity tests, immunohistochemistry, and biochemical and physiological analyses of saliva. We show that PAP is the main acid phosphatase in the wild-type male mouse saliva, accounting for 50% of the total acid phosphatase activity, and that it is expressed only in the granular convoluted tubules of the SMGs, where it is the only 5'-ectonucleotidase. The lack of PAP in male PAP(-/-) mice was associated with a significant increase in the salivation volume under secretagogue stimulation, overexpression of genes related to cell proliferation (Mki67, Aurkb, Birc5) and immune response (Irf7, Cxcl9, Ccl3, Fpr2), and upregulation of miR-146a in SMGs. An increased and sustained acinar cell proliferation was detected without signs of glandular hyperplasia. Our results indicate that in PAP(-/-) mice, SMG homeostasis is maintained by an innate immune response. Additionally, we suggest that in male mice, PAP via its 5'-ectonucleotidase activity and production of adenosine can elicit analgesic effects when animals lick their wounds.
Topics: Acid Phosphatase; Animals; Enzyme Activation; Female; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Protein Tyrosine Phosphatases; Saliva; Salivation
PubMed: 24717577
DOI: 10.1152/ajpcell.00062.2014 -
Acta Crystallographica. Section F,... Jun 2015The major acid phosphatase from Legionella pneumophila (LpMAP) belongs to the histidine acid phosphatase superfamily. It contains the characteristic histidine acid...
The major acid phosphatase from Legionella pneumophila (LpMAP) belongs to the histidine acid phosphatase superfamily. It contains the characteristic histidine acid phosphatase (HAP) sequence motif RHGXRXP responsible for the hydrolysis of a phosphoryl group from phosphate monoesters under acidic conditions. Here, the crystallization and preliminary X-ray analysis of crystals of LpMAP in the apo form and in complex with L-(+)-tartrate are described. By using the hanging-drop vapour-diffusion method, apo LpMAP and LpMAP-tartrate were crystallized in space group P2(1), with unit-cell parameters a = 91.50, b = 56.48, c = 146.35 Å, β = 110.01°, and in space group P1, with unit-cell parameters a = 55.51, b = 73.51, c = 98.78 Å, α = 78.82, β = 77.65, γ = 67.73°, respectively. Diffraction data were collected at 100 K and the phases were determined using the molecular-replacement method.
Topics: Acid Phosphatase; Amino Acid Motifs; Bacterial Proteins; Cloning, Molecular; Crystallization; Crystallography, X-Ray; Escherichia coli; Gene Expression; Legionella pneumophila; Models, Molecular; Molecular Sequence Data; Recombinant Proteins; X-Ray Diffraction
PubMed: 26057812
DOI: 10.1107/S2053230X15008213