Bone-Targeted Alkaline Phosphatase Treatment of Mandibular Bone and Teeth in Lethal Hypophosphatasia via an scAAV8 Vector

The regulation of rat liver S-adenosylmethionine synthetase (AdoMet synthetase), a key enzyme in methionine metabolism, by protein kinase C (PKC) phosphorylation has been studied. Both enzyme forms, tetramer and dimer, are phosphorylated by this kinase in the same residue, Thr-342, of the sequence. Phosphorylation of the dimer leads to its dissociation, with production of a fully-active monomer. The kinetics of the monomer have been studied, and a KmMet of 931.9 microM, a KmATP of 708 microM and a Vmax of 66.8 nmol/min/mg have been calculated. Alkaline phosphatase treatment of both enzyme forms (tetramer and dimer) produces a reduction in their activity with no change in the oligomeric state. On the other hand, PKC phosphorylation of the alkaline phosphatase-treated AdoMet synthetase forms leads to the dissociation of the dimer to produce a monomer. Rephosphorylation occurs again in the same residue, Thr-342, of the sequence. The significance of AdoMet synthetase regulation by PKC phosphorylation is further discussed.

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Dephosphorylation of activated protein kinase C contributes to downregulation by bryostatin

AJP Cell Physiology ◽

10.1152/ajpcell.1996.271.1.c304 ◽

1996 ◽

Vol 271 (1) ◽

pp. C304-C311 ◽

Cited By ~ 56

Author(s):

H. W. Lee ◽

L. Smith ◽

G. R. Pettit ◽

J. Bingham Smith

Keyword(s):

Alkaline Phosphatase ◽

Protein Synthesis ◽

Protein Kinase C ◽

Protein Kinase ◽

Bryostatin 1 ◽

Rapid Loss ◽

Renal Epithelial Cells ◽

Kinase C ◽

Phosphatase Treatment ◽

Pkc Alpha

We show that bryostatin 1 (Bryo) rapidly produces an inactive, incompetent 76-kDa form of protein kinase C-alpha (PKC-alpha) in the LLC-MK2 line of renal epithelial cells. Bryo, like phorbol 12-myristate 13-acetate (PMA), acutely activated PKC, as indicated by autophosphorylation and translocation of PKC-alpha, the predominant PMA-sensitive isoform expressed by the cells. Bryo concomitantly increased the 32P labeling of 80-kDa PKC-alpha by autophosphorylation and produced a 76-kDa form of PKC-alpha that lacked detectable 32P. The 76-kDa form was in the particulate rather than the cytosolic fraction, which suggests that it was produced from activated kinase. Alkaline phosphatase treatment of immunoprecipitated PKC-alpha converted the 80-kDa form to 76 kDa, but it had no effect on the mobility of the 76-kDa form, suggesting that it was not phosphorylated. Pulse-chase labeling of PKC-alpha with [35S]Met/Cys indicated that there is a precursor-product relationship between the 80- and 76-kDa forms, respectively. Inhibition of protein synthesis had no effect on the production of 76-kDa PKC-alpha by Bryo. PMA also produced 76-kDa PKC-alpha but was less potent and efficacious than Bryo. Bryo produced a more rapid loss of 80-kDa PKC-alpha protein and total Ca(2+)- and phospholipid-dependent PKC activity than PMA. The 76-kDa form is inactive and incompetent because it lacked detectable 32P under conditions that strongly autophosphorylated the 80-kDa form. We suggest that dephosphorylation predisposes PKC to proteolysis, and greater production of the 76-kDa form explains the more efficient downregulation of the kinase by Bryo vs. PMA.

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Hepatoprotection by carotenoids in isoniazid–rifampicin induced hepatic injury in rats

Biochemistry and Cell Biology ◽

10.1139/o10-023 ◽

2010 ◽

Vol 88 (5) ◽

pp. 819-834 ◽

Cited By ~ 10

Author(s):

S. V. Rana ◽

R. Pal ◽

K. Vaiphei ◽

R. P. Ola ◽

K. Singh

Keyword(s):

Oxidative Stress ◽

Lipid Peroxidation ◽

Alkaline Phosphatase ◽

Superoxide Dismutase ◽

Body Mass ◽

Hepatic Injury ◽

Control Group ◽

Adult Rats ◽

Partial Protection ◽

Phosphatase Treatment

This study evaluates the hepatoprotective effect of carotenoids against isoniazid (INH) and rifampicin (RIF). Thirty-six adult rats were divided into the following 4 groups: (1) control group treated with normal saline; (2) INH + RIF group treated with 50 mg·(kg body mass)–1·day–1 of INH and RIF each; (3) INH + RIF+ carotenoids group treated with 50 mg·(kg body mass)–1·day–1 of INH and RIF each and 10 mg·(kg body mass)–1·day–1 of carotenoids; and (4) carotenoids group treated with 10 mg·(kg body mass)–1·day–1 of carotenoids for 28 days intragastrically. Oxidative stress and antioxidant levels in liver and blood, liver histology and change in transaminases were measured in all the above-mentioned groups. There was an increase in lipid peroxidation with a reduction in thiols, catalase, and superoxide dismutase (SOD) in the liver and blood of rats accompanied by an increase in transaminases, bilirubin, and alkaline phosphatase. Treatment with carotenoids along with INH + RIF partially reversed lipid peroxidation, thiols, catalase, and SOD in the liver and blood of rats. Elevated levels of the enzymes in serum were also reversed partially by this treatment. The degree of necrosis, portal triaditis, and inflammation were also lowered in the carotenoids group. In conclusion, carotenoids supplementation in INH + RIF treated rats showed partial protection.

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Alkaline Phosphatase Treatment of Phosphopeptides: In-Solution Dephosphorylation after MALDI-MS Analysis

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot4611 ◽

2008 ◽

Vol 2008 (6) ◽

pp. pdb.prot4611-pdb.prot4611

Author(s):

H. Steen ◽

A. Stensballe ◽

O. N. Jensen

Keyword(s):

Alkaline Phosphatase ◽

Maldi Ms ◽

Ms Analysis ◽

Phosphatase Treatment

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Exogenous alkaline phosphatase treatment complements endogenous enzyme protection in colonic inflammation and reduces bacterial translocation in rats

Pharmacological Research ◽

10.1016/j.phrs.2012.04.006 ◽

2012 ◽

Vol 66 (2) ◽

pp. 144-153 ◽

Cited By ~ 34

Author(s):

P. Martínez-Moya ◽

M. Ortega-González ◽

R. González ◽

A. Anzola ◽

B. Ocón ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Bacterial Translocation ◽

Colonic Inflammation ◽

Phosphatase Treatment ◽

Enzyme Protection

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Alkaline phosphatase treatment improves renal function in patients with severe sepsis or septic shock

Critical Care ◽

10.1186/cc5174 ◽

2007 ◽

Vol 11 (Suppl 2) ◽

pp. P14 ◽

Cited By ~ 1

Author(s):

S Heemskerk ◽

R Masereeuw ◽

O Moesker ◽

M Bouw ◽

J van der Hoeven ◽

...

Keyword(s):

Septic Shock ◽

Alkaline Phosphatase ◽

Renal Function ◽

Severe Sepsis ◽

Phosphatase Treatment

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Analysis of the metabolic turnover of the individual phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Validation of novel analytical techniques by using 32P-labelled lipids from erythrocytes

Biochemical Journal ◽

10.1042/bj2180785 ◽

1984 ◽

Vol 218 (3) ◽

pp. 785-793 ◽

Cited By ~ 35

Author(s):

P T Hawkins ◽

R H Michell ◽

C J Kirk

Keyword(s):

Alkaline Phosphatase ◽

Analytical Techniques ◽

Erythrocyte Membranes ◽

Intact Cells ◽

First Order Kinetics ◽

Phosphatase Treatment ◽

The Many ◽

The Individual ◽

Phosphatidylinositol 4 ◽

Phosphate Groups

We have developed methods that yield estimates of the 32P content of each of the individual phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, thus extending the information available from studies of the labelling of these lipids in intact cells or membrane preparations. The analyses are undertaken with the deacylated lipids. Assay of the 5-phosphate of phosphatidylinositol 4,5-bisphosphate is achieved by the use, under conditions of first-order kinetics, of a 5-phosphate-specific phosphomonoesterase present in isolated erythrocyte membranes [Downes, Mussat & Michell (1982) Biochem. J. 203, 169-177]. Assay of the 4-phosphate of phosphatidylinositol 4-phosphate and of the total monoester phosphate content (4-phosphate plus 5-phosphate) of phosphatidylinositol 4,5-bisphosphate employs alkaline phosphatase from bovine intestine. The radioactivity of the 1-phosphate is that remaining as organic phosphate after exhaustive alkaline phosphatase treatment. The methodology has been validated by using lipids from human erythrocytes: these contain no 32P in their 1-phosphate. These methods should be of substantial value in studies of the many cells that show rapid hormonal perturbations of phosphatidylinositol 4,5-bisphosphate metabolism.

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Alkaline Phosphatase Treatment of Phosphopeptides: On-Probe Dephosphorylation after MALDI-MS Analysis

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot4612 ◽

2008 ◽

Vol 2008 (6) ◽

pp. pdb.prot4612-pdb.prot4612

Author(s):

H. Steen ◽

A. Stensballe ◽

O. N. Jensen

Keyword(s):

Alkaline Phosphatase ◽

Maldi Ms ◽

Ms Analysis ◽

Phosphatase Treatment

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Fibroblast receptor for lysosomal enzymes mediates pinocytosis of multivalent phosphomannan fragment

The Journal of Cell Biology ◽

10.1083/jcb.84.1.77 ◽

1980 ◽

Vol 84 (1) ◽

pp. 77-86 ◽

Cited By ~ 45

Author(s):

HD Fischer ◽

M Natowicz ◽

WS Sly ◽

RK Bretthauer

Keyword(s):

Molecular Weight ◽

Alkaline Phosphatase ◽

Lysosomal Enzymes ◽

Human Fibroblasts ◽

Recognition System ◽

High Uptake ◽

Acid Hydrolases ◽

Phosphatase Treatment ◽

Hydrolysis Of ◽

Endoglycosidase H

Mild acid hydrolysis of phosphomannan secreted by the yeast hansenula holstii (NRRL Y- 2448) produces two phosphomannyl fragments which differ strikingly in their potency as inhibitors of pinocytosis of human β-glucuronidase by human fibroblasts. The larger molecular weight polyphosphomonoester fragment is 100,000-fold more potent an inhibitor of enzyme uptake than the smaller penta-mannosyl-monophosphate fragment. Binding to attached fibroblasts at 3 degrees C was much greater with the polyphosphomonoester fragment than with the pentamannosyl-monophosphate. The larger molecular weight fragment was also subject to adsorptive pinocytosis and was taken up by fibroblasts at a rate 30- fold greater than the rate of uptake of pentamannosyl-monophosphate. Evidence that the polyphosphomonoester fragment is taken up by the phosphomannosyl-recognition system that mediates uptake of lysosomal enzymes includes: (a) its pinocytosis is inhibited by the same compounds that competitively inhibit enzyme pinocytosis (mannose-6-phosphate and phosphomannan from saccharomyces cerevisiae mutant mnn-1); (b) alkaline phosphatase treatment greatly reduces its susceptibility to pinocytosis; (c) its pinocytosis is competitively inhibited by high-uptake human β-glucuronidase; and (d) this inhibition by high-uptake enzyme is dramatically reduced by prior treatment of the enzyme with alkaline phosphatase or endoglycosidase-H. Endoglycosidase-H treatment human β-glucuronidase dramatically reduced its susceptibility to pinocytosis by fibroblasts. The phosphomannosyl components of high- uptake enzyme released by endoglycosidase-H treatment were much less effective inhibitors of polyphosphomonoester pinocytosis than when present on the phosphomannyl-enzyme. These results suggest that high-uptake acid hydrolases may be polyvalent ligands analogous to the polyphosphomonoester mannan fragment whose pinocytosis depends on interaction of more than one phospho-mannosyl recognition marker with pinocytosis receptors on fibroblasts.

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