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PubMed1981

The relation of the soluble thiamine triphosphatase activity of various rat tissues to nonspecific phosphatases.

Polyacrylamide gel electrophoresis was used to investigate the relation of the soluble thiamine triphosphatase activity of various rat tissues to other phosphatases. This technique separated the thiamine triphosphatase of rat brain, heart, kidney, liver, lung, muscle and spleen from alkaline phosphatase (EC 3.1.3.1), acid phosphatase (EC 3.1.3.2) and other nonspecific phosphatase activities. In contrast, the hydrolytic activity for thiamine triphosphate in rat intestine moved identically with alkaline phosphatase in gel electrophoresis. Thiamine triphosphatase from rat liver and brain was also separated from alkaline phosphatase and acid phosphatase by gel chromatography on Sephadex G-100. This gave an apparent molecular weight of about 30,000 and a Stokes radius of 2.5 nanometers for brain and liver thiamine triphosphatase. The intestinal thiamine triphosphatase activity of the rat was eluted from the Sephadex G-100 column as two separate peaks (with apparent molecular weights of over 200,000 and 123,000) which exactly corresponded to the peaks of alkaline phosphatase. The isoelectric point (pI) of the brain thiamine triphosphatase was 4.6 (4 degrees C). The partially purified thiamine triphosphatase from brain and liver was highly specific for thiamine triphosphate. The results suggest that, apart from the intestine, the rat tissues studied contain a specific enzyme, thiamine triphosphatase (EC 3.6.1.28). The specific enzyme is responsible for most of the thiamine triphosphatase activity in these tissues. Rat intestine contains a high thiamine triphosphatase activity but all of it appears to be due to alkaline phosphatase.

Acid Phosphatase↗
PubMedDate not supplied

Determination of serum acid and alkaline phosphatase using 4-methylumbelliferyl phosphate.

The present report compares the performance of several colorimetric substrates presently employed in clinical laboratories for the determination of serum acid and alkaline phosphatase with that of the fluorogenic phosphatase reagent 4-methylumbelliferyl phosphate. Acid and alkaline phosphatase assays were performed on reference hospital populations in Nigeria and the United States. The results of both acid and alkaline phosphatase determinations indicate that the coefficient of variation is smaller for the fluorometric assay than for the colorimetric methods that employ either phenyl phosphate or p-nitrophenyl phosphate as the substrate. Furthermore, serum acid phosphatase determinations using 4-methylumbelliferyl phosphate identified increased serum enzyme levels in 6/6 cases of prostatic carcinoma and 9/9 cases of Gaucher's disease. The occurrence of elevated serum alkaline phosphatase levels in 24/24 cases involving liver and bone disease was also confirmed by the fluorometric alkaline phosphatase assay. In addition, sera from eight patients with the connective tissue disease, osteogenesis imperfecta, were found to contain normal levels of acid and alkaline phosphatase by the fluorometric assays. From these results it appears that the sensitive and rapid fluorometric assay procedures can be readily employed in the clinical pathology laboratory for the determination of serum acid and alkaline phosphatase levels.

Acid Phosphatase↗
PubMed2000

Catalytic activation of mitogen-activated protein (MAP) kinase phosphatase-1 by binding to p38 MAP kinase: critical role of the p38 C-terminal domain in its negative regulation.

Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) is the archetypal member of the dual-specificity protein phosphatase family, the expression of which can be rapidly induced by a variety of growth factors and cellular stress. Since MKP-1 protein localizes in the nucleus, it has been suggested to play an important role in the feedback control of MAP kinase-regulated gene transcription. Recently it has been demonstrated that the interaction of several cytosolic MAP kinase phosphatases with MAP kinases can trigger the catalytic activation of the phosphatases. It is unclear whether such a regulatory mechanism can apply to nuclear MAP kinase phosphatases and serve as an additional apparatus for the feedback control of MAP kinase-mediated gene expression. Here we have shown that MKP-1 associates directly with p38 MAP kinase both in vivo and in vitro, and that this interaction enhances the catalytic activity of MKP-1. The point mutation Asp-316-->Asn in the C-terminus of p38, analogous to the ERK2 (extracellular-signal-regulated kinase 2) sevenmaker mutation, dramatically decreases its binding to MKP-1 and substantially compromises its stimulatory effect on the catalytic activity of this phosphatase. Consistent with its defective interaction with MKP-1, this p38 mutant also displays greater resistance to dephosphorylation by the phosphatase. Our studies provide the first example of catalytic activation of a nuclear MAP kinase phosphatase through direct binding to a MAP kinase, suggesting that such a regulatory mechanism may play an important role in the feedback control of MAP kinase signalling in the nuclear compartment.

Asparagine↗
PubMed1999

Heterogeneity in alkaline phosphatase isozyme expression in human testicular germ cell tumours: An enzyme-/immunohistochemical and molecular analysis.

In humans, alkaline phosphatases are encoded by one tissue-non-specific alkaline phosphatase (TNAP) gene and three tissue-specific alkaline phosphatase genes, intestinal, placental (PLAP), and germ cell-specific alkaline phosphatase (GCAP). Although the presence of alkaline phosphatases in testicular germ cell tumours (TGCTs) of adolescents and adults has been utilized for both detection and patient monitoring, it is not known in detail which isozymes are expressed. Since alkaline phosphatase is detected in carcinoma in situ (CIS), the common precursor of all TGCTs, it might provide a marker for the early diagnosis of TGCTs. Testicular cancers of germ cell and non-germ cell origin along with testicular parenchyma with and without CIS have been analysed for the expression of the different alkaline phosphatase isozymes. Antibodies to TNAP and PLAP/GCAP showed positivity in CIS, seminoma, and embryonal carcinoma. The heterogeneous staining pattern detected in frozen tissue sections was similar to the pattern found in formalin-fixed, paraffin-embedded material, indicating a biological phenomenon and not a handling artefact. Since PLAP and GCAP cannot be distinguished using immunohistochemistry, the expression of these isozymes was studied at the molecular level using a reverse transcriptase-polymerase chain reaction (RT-PCR) approach, in combination with a primer extension assay. The results show that CIS and seminoma predominantly express GCAP, while in embryonal carcinoma the expression of GCAP versus PLAP varies. Due to the presence of alkaline phosphatase transcripts in normal testicular parenchyma, an RT-PCR-based analysis of alkaline phosphatase is not informative for the early detection of TGCTs in biopsy samples.

Alkaline Phosphatase↗
PubMed1985

Gaucher disease: comparative study of acid phosphatase and glucocerebrosidase in normal and type-1 Gaucher tissues.

Acid phosphatase activity was determined in serum, cultured fibroblasts, and peripheral blood lymphocytes of six splenectomized adult patients with non-neuropathic Gaucher disease in two Canadian families. Elevated levels of serum acid phosphatase activity (520-711% of normal) were found in four patients who also developed orthopedic complications associated with Gaucher disease, including intermittent bone pain, arthritis, collapse of femoral head, and pathological fractures. Serum acid phosphatase activity in two patients who do not have bone involvement were found to be within the normal range. Contrary to the serum enzyme, acid phosphatase activity in lymphocytes and cultured fibroblasts of all of the patients was within the normal range. Deficient glucocerebrosidase (7.5-15.5% of normal) and acid beta-glucosidase (13.8-27.8% of normal) activities were noted in all probands. Similarly, normal levels of fibroblast and lymphocyte acid phosphatase activity were found in Gaucher heterozygotes whose glucocerebrosidase activity was about 50% of normal. Acid polyacrylamide gel electrophoresis and acid phosphatase activity staining of the patients' sera showed that the elevated acid phosphatase is isozyme type 5 osteoclastic origin. The apparent Michaelis constant, Km, of fibroblast glucocerebrosidase for the natural substrate was 0.6 +/- 0.1 mM for controls and 0.6 +/- 0.2 mM for the patients. These data suggest that the assay of serum acid phosphatase activity for the presumptive diagnosis of Gaucher disease is not completely reliable and that the elevated level of serum acid phosphatase in Gaucher disease is most likely a secondary phenomenon which may be indicative of bone involvement in some patients with this disorder. It also demonstrates the clinical heterogeneity of type 1 Gaucher disease, even among full sibs of the same heterozygous parents.

Acid Phosphatase↗
PubMed1993

Reduction of low-molecular-weight acid phosphatase activity in Alzheimer brains.

Recent studies in Alzheimer brains have shown aberrant protein phosphorylation, suggesting an alteration in protein kinases and/or phosphoprotein phosphatases. In the present study, the activity of acid phosphatase was investigated in samples prepared from postmortem normal human and Alzheimer brains. p-Nitrophenyl phosphate, a nonprotein phosphoester, was used as a substrate for acid phosphatase. The separation profile on Sephadex G-100 gel filtration chromatography revealed that two major forms of high-molecular-weight and low-molecular-weight acid phosphatase were present in the crude extracts of both rat and human brains. Another class of zinc ion (Zn2+)-dependent acid p-nitrophenyl phosphatase was also detected in rat and human brains. In Alzheimer brains, the low-molecular-weight acid phosphatase activity was significantly decreased compared to that in control brains; however, the high-molecular-weight and Zn(2+)-dependent acid phosphatase activity in control and Alzheimer brains was not different. These results suggest that reduced activity of the low-molecular-weight acid phosphatase, which possesses phosphotyrosine protein phosphatase activity, might be linked to aberrant protein tyrosine phosphorylation found in Alzheimer brains.

Acid Phosphatase↗
PubMed1985

Immunofluorescent localization of an androgen-dependent isoenzyme of prostatic acid phosphatase in rat ventral prostate.

Isoenzymes of rat ventral prostate (RVP) acid phosphatase were isolated and partially purified by ultracentrifugation, Sephadex G-100 column chromatography, and isoelectric focusing. Antisera were raised to the isoenzymes of prostatic acid phosphatase by immunization of New Zealand white rabbits. Rabbit antisera reacting specifically to homologous but not heterologous isoenzymes of acid phosphatase were then reacted with a variety of tissues using indirect immunofluorescence. The tissues included prostate, spleen, bone marrow, liver, kidney, salivary gland complex, small intestine, and adrenal glands. An antiserum against a RVP acid phosphatase isoenzyme with a pI of 4.5 (A-PAP) localized acid phosphatase only in the supranuclear region of rat ventral prostate epithelial cells, and did not react with acid phosphatase in any of the other organs tested. A-PAP did not localize acid phosphatase in the ventral prostate from rats 14 days after castration. A-PAP did localize acid phosphatase in the ventral prostate from castrated animals that were treated with testosterone. These results indicate the A-PAP localized an androgen-dependent isoenzyme of acid phosphatase in RVP epithelial cells that may be secretory in nature. This antiserum should prove to be an ideal marker for studies involving hormonal regulation of prostatic epithelial function in vivo and in vitro.

Acid Phosphatase↗
PubMed1995

An in-gel assay for protein tyrosine phosphatase activity: detection of widespread distribution in cells and tissues.

A method is described for the detection of protein tyrosine phosphatase activity in sodium dodecyl sulfate-polyacrylamide gels. A radiolabeled substrate, 32P-labeled poly(glutamic acid-tyrosine) (random copolymer) is incorporated into gels prior to polymerization. Following electrophoresis, the sodium dodecyl sulfate is removed; the proteins are fully denatured by soaking gels in 6 M guanidine hydrochloride and then renatured by incubation in buffers containing 0.04% Tween 40 and high concentrations of reducing agents. Protein tyrosine phosphatase activity is detected in autoradiographs of dried gels as regions from which the 32P has been selectively removed. Electrophoresis of known cytoplasmic protein tyrosine phosphatases indicates activity as the predicted molecular weights. As little as 10 pg of some cytoplasmic phosphatases is detectable. However, transmembrane tyrosine phosphatases, such as CD45, are detected only at very high protein loadings in this assay. Electrophoresis of whole cell lysates indicates multiple bands of tyrosine phosphatase activity, some of which comigrate with known cytoplasmic protein tyrosine phosphatases. The activity is inhibited by sodium orthovanadate or the omission of reducing agents during the renaturation process. The assay has been used to analyze embryonic and adult tissues, as well as whole cell lysates. A similar profile of bands of tyrosine phosphatase activity is seen with many different cells and tissues. However, some that are highly differentiated, such as adult skeletal muscle, erythrocytes, or sperm, reveal either a reduced level of tyrosine phosphatase activity or a simplified profile of bands.

Adult↗
PubMed1999

Platelet-activating factor induction of secreted phosphatase activity in Trypanosoma cruzi.

The effects of platelet-activating factor (PAF) on the ecto-phosphatase activity of Trypanosoma cruzi were investigated. Living parasites hydrolyzed p-nitrophenyl phosphate (p-NPP) at a rate of 5.71 +/- 0.37 nmol P(i) mg(-1) min(-1). This ecto-phosphatase activity increased to 8.70 +/- 1.12 nmol P(i) mg(-1) min(-1) when the cells were grown in the presence of 10(-9) M PAF. This effect was probably due to stimulation of the release of the ecto-phosphatase and/or the secretion of an intracellular phosphatase to the extracellular medium, as suggested by cytochemical analysis. Modulation of the ecto-phosphatase activity was also observed when PAF was added during the time course of the reaction. WEB 2086, a competitive PAF antagonist, was able to revert PAF effects when both were used at the same concentration. When PAF was added to a membrane enriched fraction preparation of T. cruzi, no alteration on the phosphatase activity was observed. This result suggests an involvement of intracellular signaling, as PAF was only effective on intact cells. Sphingosine and phorbol-12-myristate-13-acetate (PMA) were then used to investigate a possible involvement of protein kinase C (PKC) with PAF-induced phosphatase secretion. Sphingosine by itself stimulated the secretion of a phosphatase but did not significantly interfere with PAF effects on this enzyme. On the other hand, PMA was able to abrogate PAF-induced release of this phosphatase. These data are highly suggestive of a putative involvement of signal transduction mediated by a ligand of mammalian origin (PAF), through PKC and a specific receptor located on the cell surface of the human parasite Trypanosoma cruzi.

Animals↗
PubMed2001

18 kDa protein tyrosine phosphatase in the ocular lens.

Protein tyrosyl phosphorylation and dephosphorylation play essential roles in regulating cellular events such as proliferation and differentiation, and their involvement in the lens development and transparency is also suggested. The level of tyrosine phosphorylation in a given protein is regulated by the opposing actions of protein-tyrosine kinases (Tyr kinases) and protein-tyrosine phosphatases (TyrPases). Recent studies have revealed that some Tyr kinases, such as platelet-derived growth factor receptor and fibroblast growth factor receptor, are present in the lens, however, little is known about TyrPases in the lens. In this study, we found a 18 kDa protein tyrosine phosphatase (18 kDa TyrPase) predominantly present in the ocular lens of various animals. We purified the phosphatase from the lens of chick embryo and characterized its activity.Phosphatase activity was determined in chick embryo, mouse, rabbit and bovine lenses using p -nitrophenyl phosphate (p NPP) as substrate. All lenses examined dephosphorylated p NPP under acidic conditions, and a large portion of the activity resided in a low molecular weight protein, ca. 18 kDa, following high-resolution gel permeation column chromatography. The brain and liver showed high dephosphorylation activities, but most of their activity was present in high molecular weight fractions, unlike that in the lens. The 18 kDa phosphatase was purified from the lens of 17 day old chick embryos to near-homogeneity with two-step rapid chromatography. This phosphatase showed strict substrate specificity for phosphotyrosine and phosphotyrosyl peptides, suggesting that it was a kind of protein tyrosine phosphatases (TyrPases). Several known inhibitors of TyrPases, such as SH blockers, vanadate and phenylarsine oxide, strongly inhibited the enzyme activity. The molecular weight, substrate specificity, and responses to various inhibitors and activators coincide well with those reported for the low molecular weight protein tyrosine phosphatase (LMW-TyrPase), belonging to the TyrPase superfamily. These results suggest that the 18 kDa phosphatase found in the lens is a LMW-TyrPase. The 18 kDa TyrPase is the predominant phosphatase in the ocular lens. It may be involved in regulation of lens cell proliferation, differentiation and/or lens transparency.

Animals↗
PubMed1990

Effects of sulfhydryl agents, trifluoperazine, phosphatase inhibitors and tryptic proteolysis on calcineurin isolated from bovine cerebral cortex.

Calcineurin was discovered as an inhibitor of calmodulin stimulated cyclic AMP phosphodiesterase and its ability to act as a calmodulin binding protein largely explains its inhibitory action on calmodulin regulated enzymes. Recent studies establish calcineurin as the enzyme protein phosphatase whose activity is regulated by calmodulin and a variety of divalent metals. In this work, we have investigated the effects of several agents including sulfhydryl agents, trifluoperazine (a calmodulin antagonist), PPi, NaF and orthovanadate and of tryptic proteolysis on the calcineurin inhibition of cyclic AMP phosphodiesterase (called inhibitory activity) and on protein phosphatase activity. Inhibitors for sulfhydryl groups (pHMB, NEM) inhibited phosphatase activity without any effect on the inhibitory activity. Dithioerythritol completely reversed the inhibition by pHMB. Limited proteolysis of calcineurin caused an activation of basal phosphatase activity with a complete loss of inhibitory activity. Phosphatase activity of the proteolyzed calcineurin was not stimulated by calmodulin. The presence of calmodulin along with calcineurin during tryptic digestion appeared to preserve the stimulation of phosphatase by Ca2(+)-calmodulin. [3H]-Trifluoperazine (TFP) was found to be incorporated irreversibly into calcineurin in the presence of ultraviolet light. This incorporation was evident into the A and B subunits of calcineurin. TFP-caused a decrease in the phosphatase activity and an increase in its inhibitory activity. [3H]-TFP incorporation into the A subunit was drastically decreased in the proteolyzed calcineurin. This was also true when the [3H]-TFP incorporated calcineurin was subjected to tryptic proteolysis. The incorporation into the B unit was essentially unaffected in the trypsinized calcineurin. Phosphatase activity was inhibited by orthovanadate, NaF, PPi, and EDTA. Inhibitions by these compounds were more pronounced when the phosphatase was determined in the presence of Ca2(+)-calmodulin than in their absence.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

Immunohistochemical localisation of glucose-6-phosphatase in developing human kidney.

The objective of our study was to determine the cellular localisation of glucose-6-phosphatase in developing human kidney using monospecific antiserum and a standard immunohistochemical method (peroxidase-antiperoxidase, PAP) on formalin fixed and paraffin embedded tissue. In embryonic and early fetal development of the metanephric kidney, glucose-6-phosphatase is located primarily in derivatives of the ureteric bud such as the pelvis, calyces and collecting ducts. In mid-fetal life as nephrons evolve and develop they become increasingly immunoreactive to glucose-6-phosphatase, such that in mature metanephric kidney the proximal tubules are highly reactive for glucose-6-phosphatase with other elements of the nephron also immunopositive albeit at lower reactivities. In addition the parietal layer of Bowman's capsule and some cells of the visceral layer are immunopositive. Only with the development of nephrons does the early predominance of glucose-6-phosphatase immunoreactivity to ureteric bud derivatives change: in mature kidney the reactivity in the collecting ducts is a small proportion of the total. In proximal tubular cells the distribution of glucose-6-phosphatase immunoreactivity is relatively uniform throughout development in contrast to collecting ducts where in fetal life this reactivity is displaced to the apices and basal areas by intracellular glycogen deposits. The mesonephric kidney has a similar pattern of glucose-6-phosphatase immunoreactivity to that of metanephric kidney. The availability of monospecific antiserum to glucose-6-phosphatase and immunohistochemical methods now allows an alternative approach to cellular localisation. Many of the difficulties in the fixation of tissue and assay of glucose-6-phosphatase activity inherent in conventional histochemical methods are avoided by such methods.

Female↗
PubMed1996

Activation of the ATP.Mg-dependent type 1 protein phosphatase by the Fe2+/ascorbate system.

The ATP.Mg-dependent type 1 protein phosphatase is inactive as isolated but can be activated in several different ways. In this report, we show that the phosphatase can also be activated by the Fe2+/ascorbate system. Activation of the phosphatase requires both Fe2+ ion and ascorbate and the level of activation is dependent on the concentrations of Fe2+ ion and ascorbate. In the presence of 20 mM ascorbate, the Fe2+ ion concentrations required for half-maximal and maximal activation are about 0.3 and 3 mM, respectively. Several common divalent metal ions, including CO2+, Ni2+, Cu2+, Mg2+, and Ca2+ ions, cannot cooperate with ascorbate to activate the phosphatase, and SH-containing reducing agents such as 2-mercaptoethanol and dithiothreitol cannot cooperate with Fe2+ ion to activate the phosphatase, indicating that activation of the phosphatase by the Fe2+/ascorbate system is a specific process. Moreover, H2O2, a strong oxidizer, could significantly diminish the phosphatase activation by the Fe2+/ascorbate system, suggesting that reduction mechanism other than SH-SS interchange is a prerequisite for the Fe2+/ascorbate-mediated phosphatase activation. Taken together, the present study provides initial evidence for a new mode of type 1 protein phosphatase activation mechanism.

Animals↗
PubMed1984

Characterization of bovine brain calmodulin-dependent protein phosphatase.

Calmodulin-dependent protein phosphatase of bovine brain exhibited a pH optimum of 7 and appeared to require sulfhydryl groups for activity. Phosphatase activity was inhibited by both NaF and ZnCl2, but was stimulated approximately 2-fold by MnCl2. The enzyme exhibited broad substrate specificity, dephosphorylating casein, troponin I, protamine, histone, and phosvitin, and was not phosphorylated by cAMP-dependent protein kinase. With 32P-labeled casein as a substrate, phosphatase was activated 15-fold by calmodulin; the dissociation constant of phosphatase for calmodulin was 30 nM. Activation of the enzyme by calmodulin as a function of Ca2+ was highly cooperative; the Hill coefficient was 4.9. At a saturating concentration of calmodulin, half-maximal activation of phosphatase was obtained at 0.3 microM Ca2+. Calmodulin increased the Vmax from 1.7 to 41 nmol mg protein-1 min-1 with no significant change in its Km. Formation of a Ca2+-dependent complex between calmodulin and the phosphatase was demonstrated by a calmodulin-Sepharose affinity column, gel-filtration chromatography, and sedimentation on a sucrose density gradient. The rate of formation and dissociation of the calmodulin X phosphatase complex was rapid and readily reversible in response to changes in Ca2+ concentration. The calmodulin X phosphatase complex consists of 1 mol of calmodulin and 1 mol of phosphatase.

Animals↗
PubMed1985

Properties of an acid phosphatase from Legionella micdadei which blocks superoxide anion production by human neutrophils.

The high-speed supernatant (100,000 g, 1 h) obtained after centrifuging a suspension of Legionella micdadei that had been freeze-thawed and sonicated contained (i) considerable acid phosphatase activity when assayed using 4-methylumbelliferyl phosphate (MUP) as the substrate, and a factor that blocked superoxide anion production by human neutrophils stimulated with f-Met-Leu-Phe. Chromatography of the extract on a hydroxylapatite column resolved two acids phosphatases (designated ACP1 and ACP2). Subsequent chromatography of ACP2 on a Sephadex G-150 column revealed coincident elution of phosphatase activity and neutrophil blocking activity. When heated at 45 degrees C for various periods of time, the phosphatase activity of the acid phosphatase preparation was lost at the same rate as the ability of the preparation to block superoxide anion production by neutrophils. Furthermore, preincubation of neutrophils and acid phosphatase together in the presence of a heteropolymolybdate complex that inhibits the phosphatase eliminated the effect of the L. micdadei phosphatase on neutrophil superoxide anion production. ACP2 had the following properties: pH optimum, 6.0; Km for MUP, 3.8 mM; isoelectric point, 4.5; substrate specificity, MUP greater than ADP greater than phosphoenolpyruvate greater than phosphothreonine greater than phosphoserine greater than phosphotyrosine; molecular weight (estimated by sucrose density gradient centrifugation and gel filtration chromatography), 71,000-86,000. These results indicate that a cell-associated phosphatase may play a role in the virulence of L. micdadei.

Acid Phosphatase↗
PubMed1989

Polyclonal antibodies to rabbit skeletal muscle protein phosphatases C-I and C-II.

Polyclonal antibodies against rabbit skeletal muscle phosphatases C-I and C-II were raised in goats and in mice. The goat polyclonal antibodies to phosphatases C-I and C-II were examined for their ability to immunoblot the purified enzymes and crude rabbit muscle extracts. In preparations of phosphatases C-I and C-II that were apparently homogeneous, the expected ca. 35- to 38-kDa polypeptides were immunoblotted, but, in addition, immunoblotting of a 67-kDa polypeptide was observed. Both the antisera blotted only the 67-kDa polypeptide in crude rabbit muscle extracts and not the expected 35- to 38-kDa polypeptides. These findings are qualitatively similar to those reported previously (D.L. Brautigan et al. (1985) J. Biol. Chem. 260, 4295-4305) where immunoblotting experiments with a sheep antisera to phosphatase C-I indicated that the ca. 35-kDa polypeptide originates from a 70-kDa precursor. On further investigation, it was found that our antisera were strongly immunoreactive to rabbit serum albumin. The antisera blotted purified rabbit albumin, but not bovine serum albumin. After passage through a rabbit albumin-Sepharose column, the antisera lost immunoreactivity to rabbit albumin, and no longer blotted the ca. 70-kDa band in muscle extracts or in purified enzyme preparations. These findings show that the phosphatase preparations contained traces of albumin which produced a strong antigenic reaction. Production of antisera in BALB/c mice produced similar results; i.e., an antibody to the low-molecular-weight phosphatases was produced that was also a strong antibody to rabbit albumin. This antibody could be removed by affinity adsoption on rabbit albumin-Sepharose columns. In addition, the antibodies to phosphatase C-I displayed no cross-reactivity to phosphatase C-II, while antibodies to C-II showed no cross-reactivity to phosphatase C-I by immunoblotting methods.

Animals↗
PubMed1990

Alterations to N-linked oligosaccharides which affect intracellular transport rates and regulated secretion but not sorting of lysosomal acid phosphatase in Dictyostelium discoideum.

The importance of N-linked oligosaccharides and their associated modifications in the transport, sorting, and secretion of lysosomal acid phosphatase was investigated using three mutant Dictyostelium cell lines. These mutants synthesize altered N-linked oligosaccharides with the following properties: (i) in strain HL244 carbohydrate side chains lack mannose 6-sulfate residues, (ii) in strain M31 the side chains retain the two alpha-1,3-linked glucose residues resulting in less sulfate and methylphosphate modifications, and (iii) in strain HL243 the nonglucosylated branches are missing three of the outer mannose sugars and the oligosaccharides contain fewer sulfate and phosphate modifications. Lysosomal enzymes in both HL243 and HL244 are also missing a shared epitope termed common antigen-1 (CA-1), which consists in part of mannose 6-sulfate moieties. No increases were observed in the secretion of radiolabeled acid phosphatase or acid phosphatase activity during growth in any of the mutant cell lines, suggesting that the enzyme was correctly sorted to lysosomes. In support of this, Percoll gradient fractionations and indirect immunofluorescence microscopy indicated that acid phosphatase was transported to lysosomes in all cell lines. However, radiolabel pulse chase protocols indicated that newly synthesized acid phosphatase was transported out of the endoplasmic reticulum (ER) and into lysosomes at a two- to threefold slower rate in HL243 and at a sixfold slower rate in M31. The rate of transport of acid phosphatase from the ER to the Golgi was reduced only twofold in M31 as determined by digestion of newly synthesized enzyme with endoglycosidose H. This suggests that certain alterations in carbohydrate structure may only slightly affect transport of the enzyme from the ER to the Golgi but these alterations may greatly delay transport from the Golgi or post-Golgi compartments to lysosomes. Finally all three mutants secreted acid phosphatase at significantly lower rates than the wild-type strain when growing cells were placed in a buffered salt solution (conditions which stimulate the secretion of mature lysosomally localized enzymes). In contrast, alpha-mannosidase was secreted with similar kinetics from the mutant and wild-type strains. Together, these results suggest that the mechanism(s) operating to sort acid phosphatase in Dictyostelium can tolerate a wide range of changes in N-linked oligosaccharides including a reduction in phosphate and the absence of CA-1 and sulfate, while in contrast, these same alterations can profoundly influence the rate of transport of acid phosphatase from the ER and post-ER compartments to lysosomes as well as the secr

Acetylglucosaminidase↗
PubMed1991

Modulation of the activity of hepatic glucose-6-phosphatase by methylthioadenosine sulfoxide.

Methylthioadenosine sulfoxide (MTAS), an oxidized derivative of the cell toxic metabolite methylthioadenosine has been used in elucidating the relevance of an interrelationship between the catalytic behavior and the conformational state of hepatic glucose-6-phosphatase and in characterizing the transmembrane orientation of the integral unit in the microsomal membrane. The following results were obtained: (1) Glucose 6-phosphate hydrolysis at 37 degrees C is progressively inhibited when native microsomes are treated with MTAS at 37 degrees C. In contrast, glucose 6-phosphate hydrolysis of the same MTAS-treated microsomes assayed at 0 degrees C is not inhibited. (2) Subsequent modification of the MTAS-treated microsomes with Triton X-114 reveals that glucose-6-phosphatase assayed at 37 degrees C as well as at 0 degrees C is inhibited. (3) Although excess reagent is separated by centrifugation and the MTAS-treated microsomes diluted with buffer before being modified with Triton the temperature-dependent effect of MTAS on microsomal glucose-6-phosphatase is not reversed at all. (4) In native microsomes MTAS is shown to inhibit glucose-6-phosphatase noncompetitively. The subsequent Triton-modification of the MTAS-treated microsomes, however, generates an uncompetitive type of inhibition. (5) Preincubation of native microsomes with MTAS completely prevents the inhibitory effect of 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS) as well as 4,4'-diazidostilbene 2,2'-disulfonate (DASS) on glucose-6-phosphatase. (6) Low molecular weight thiols and tocopherol protect the microsomal glucose-6-phosphatase against MTAS-induced inhibition. (7) Glucose-6-phosphatase solubilized and partially purified from rat liver microsomes is also affected by MTAS in demonstrating the same temperature-dependent behavior as the enzyme of MTAS-treated and Triton-modified microsomes. From these results we conclude that MTAS modulates the enzyme catalytic properties of hepatic glucose-6-phosphatase by covalent modification of reactive groups of the integral protein accessible from the cytoplasmic surface of the microsomal membrane. The temperature-dependent kinetic behavior of MTAS-modulated glucose-6-phosphatase is interpreted by the existence of distinct catalytically active enzyme conformation forms. Detergent-induced modification of the adjacent hydrophobic microenvironment additionally generates alterations of the conformational state leading to changes of the kinetic characteristics of the integral enzyme.

Acid Anhydride Hydrolases↗