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PubMed2005

ABA-hypersensitive germination3 encodes a protein phosphatase 2C (AtPP2CA) that strongly regulates abscisic acid signaling during germination among Arabidopsis protein phosphatase 2Cs.

The phytohormone abscisic acid (ABA) regulates physiologically important developmental processes and stress responses. Previously, we reported on Arabidopsis (Arabidopsis thaliana) L. Heynh. ahg mutants, which are hypersensitive to ABA during germination and early growth. Among them, ABA-hypersensitive germination3 (ahg3) showed the strongest ABA hypersensitivity. In this study, we found that the AHG3 gene is identical to AtPP2CA, which encodes a protein phosphatase 2C (PP2C). Although AtPP2CA has been reported to be involved in the ABA response on the basis of results obtained by reverse-genetics approaches, its physiological relevance in the ABA response has not been clarified yet. We demonstrate in vitro and in vivo that the ahg3-1 missense mutation causes the loss of PP2C activity, providing concrete confirmation that this PP2C functions as a negative regulator in ABA signaling. Furthermore, we compared the effects of disruption mutations of eight structurally related PP2C genes of Arabidopsis, including ABI1, ABI2, HAB1, and HAB2, and found that the disruptant mutant of AHG3/AtPP2CA had the strongest ABA hypersensitivity during germination, but it did not display any significant phenotypes in adult plants. Northern-blot analysis clearly showed that AHG3/AtPP2CA is the most active among those PP2C genes in seeds. These results suggest that AHG3/AtPP2CA plays a major role among PP2Cs in the ABA response in seeds and that the functions of those PP2Cs overlap, but their unique tissue- or development-specific expression confers distinct and indispensable physiological functions in the ABA response.

Abscisic Acid↗
PubMed2000

Characterization of Arabidopsis acid phosphatase promoter and regulation of acid phosphatase expression.

The expression and secretion of acid phosphatase (APase) was investigated in Indian mustard (Brassica juncea L. Czern.) plants using sensitive in vitro and activity gel assays. Phosphorus (P) starvation induced two APases in Indian mustard roots, only one of which was secreted. Northern-blot analysis indicated transcriptional regulation of APase expression. Polymerase chain reaction and Southern-blot analyses revealed two APase homologs in Indian mustard, whereas in Arabidopsis, only one APase homolog was detected. The Arabidopsis APase promoter region was cloned and fused to the beta-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. GUS expression was first evident in leaves of the P-starved Arabidopsis plants. In P-starved roots, the expression of GUS initiated in lateral root meristems followed by generalized expression throughout the root. GUS expression diminished with the addition of P to the medium. Expression of GFP in P-starved roots also initiated in the lateral root meristems and the recombinant GFP with the APase signal peptide was secreted by the roots into the medium. The APase promoter was specifically activated by low P levels. The removal of other essential elements or the addition of salicylic or jasmonic acids, known inducers of gene expression, did not activate the APase promoter. This novel APase promoter may be used as a plant-inducible gene expression system for the production of recombinant proteins and as a tool to study P metabolism in plants.

Acid Phosphatase↗
PubMed1990

The Bacillus subtilis 168 alkaline phosphatase III gene: impact of a phoAIII mutation on total alkaline phosphatase synthesis.

The first alkaline phosphatase (APase) structural gene mutant of Bacillus subtilis 168 was constructed by using a clone identified by hybridization to a synthetic degenerative oligonucleotide. The design of the probe was based on the first 29 amino acids of the sequenced mature APase III protein, which had been isolated from the secreted fraction of vegetative, phosphate-starved cells. DNA sequencing of the clone revealed the first 80 amino acids of the APase III protein, including a typical procaryotic signal sequence of 32 amino acids preceding the start of the mature protein. The 29 amino acids encoded by the predicted open reading frame immediately following the signal sequence are identical to the first 29 amino acids of the sequenced mature protein. This region shows 80% identity to strand A of the beta sheet that is very well conserved in Escherichia coli and mammalian APases. A phoAIII structural mutant was constructed by insertional mutagenesis with a fragment internal to the coding region. The effects of this mutation on APase production in B. subtilis 168 were analyzed under both phosphate starvation and sporulation conditions. The mutation in APase III reduced the total vegetative APase specific activity by approximately 40% and sporulation APase specific activity by approximately 45%. An APase protein was isolated from sporulating cells at stage III and was identified as APase III by protein sequencing of the amino terminus and by its absence in the phoAIII mutant. The APase III gene has been mapped to approximately 50 degrees on the B. subtilis chromosome.

Alkaline Phosphatase↗
PubMed1990

The complete nucleotide sequence of the Escherichia coli gene appA reveals significant homology between pH 2.5 acid phosphatase and glucose-1-phosphatase.

The whole nucleotide sequence of Escherichia coli gene appA, which encodes periplasmic phosphoanhydride phosphohydrolase (optimum pH, 2.5), and its flanking regions was determined. The AppA protein is significantly homologous to the product of the nearby gene agp, acid glucose-1-phosphatase. Because identical amino acids are distributed over the whole lengths of the proteins, it is likely that appA and agp originate from the same ancestor gene.

Acid Phosphatase↗
PubMed1994

Tyrosine phosphorylation of CD45 phosphotyrosine phosphatase by p50csk kinase creates a binding site for p56lck tyrosine kinase and activates the phosphatase.

Src family protein tyrosine kinases (PTKs) play an essential role in antigen receptor-initiated lymphocyte activation. Their activity is largely regulated by a negative regulatory tyrosine which is a substrate for the activating action of the CD45 phosphotyrosine phosphatase (PTPase) or, conversely, the suppressing action of the cytosolic p50csk PTK. Here we report that CD45 was phosphorylated by p50csk on two tyrosine residues, one of them identified as Tyr-1193. This residue was not phosphorylated by T-cell PTKs p56lck and p59fyn. Tyr-1193 was phosphorylated in intact T cells, and phosphorylation increased upon treatment with PTPase inhibitors, indicating that this tyrosine is a target for a constitutively active PTK. Cotransfection of CD45 and csk into COS-1 cells caused tyrosine phosphorylation of CD45 in the intact cells. Tyrosine-phosphorylated CD45 bound p56lck through the SH2 domain of the kinase. Finally, p50csk-mediated phosphorylation of CD45 caused a severalfold increase in its PTPase activity. Our results show that direct tyrosine phosphorylation of CD45 can affect its activity and association with Src family PTKs and that this phosphorylation could be mediated by p50csk. If this is also true in the intact cells, it adds a new dimension to the physiological function of p50csk in T lymphocytes.

Amino Acid Sequence↗
PubMed2005

Serum alkaline phosphatase activity is regulated by a chromosomal region containing the alkaline phosphatase 2 gene (Akp2) in C57BL/6J and DBA/2J mice.

Quantitative trait locus (QTL) analyses were conducted to identify chromosomal regions that contribute to variability in serum alkaline phosphatase (AP) enzyme activity in mice derived from the C57BL/6J (B6) and DBA/2J (D2) inbred strains. Serum AP was measured in 400 B6D2 F2 mice at 5 mo and 400 B6D2 F2 mice at 15 mo of age that were genotyped at 96 microsatellite markers, and in 19 BXD recombinant inbred (RI) strains at 5 mo of age. A QTL on the distal end of chromosome 4 was present in all sex- and age-specific analyses with a peak logarithm of odds (LOD) score of 20.36 at 58.51 cM. The Akp2 gene, which encodes the major serum AP isozyme, falls within this QTL region at 70.2 cM where the LOD score reached 13.2 (LOD significance level set at 4.3). Serum AP activity was directly related to the number of D2 alleles of a single nucleotide polymorphism in the 5'-flanking region of the Akp2 gene, although no strain-related differences in hepatic expression of Akp2 RNA were found. A variety of sequence polymorphisms in this chromosomal region could be responsible for the differences in serum AP activity; the Akp2 gene, however, with several known amino acid substitutions between protein sequences of the B6 and D2 strains, is a leading candidate.

Alkaline Phosphatase↗
PubMed1996

Phosphotyrosine-protein-phosphatases and human reproduction: an association between low molecular weight acid phosphatase (ACP1) and spontaneous abortion.

ACP1 (low molecular weight acid phosphatase) genetic polymorphism has been studied in 173 women with a history of two or more consecutive spontaneous abortions and in 1508 control subjects, including 482 normal pregnant women. The proportion of carriers of ACP1*C allele (*A/ *C, *B/*C) in women with a history of repeated spontaneous abortion is lower than in normal pregnant women and other control groups. Women with repeated spontaneous abortion show a specific decrease of ACP1 S isoform concentration as compared to normal pregnant women. The other component of ACP1 activity, the F isoform, does not show a significant difference between the two groups. The data suggest that women with ACP1 genotypes showing a high concentration of S isoform are relatively 'protected' against spontaneous abortion. Preliminary analysis of a sample of 352 normal puerperae along with their newborn babies supports this hypothesis.

Abortion, Habitual↗
PubMed1998

Phosphotyrosine-protein-phosphatase and diabetic disorders. Further studies on the relationship between low molecular weight acid phosphatase genotype and degree of glycemic control.

We have studied a new sample of 276 NIDDM patients from the population of Penne (Italy). Comparison of the new data with those of 214 diabetic pregnant women from the population of Rome reported in a previous paper has shown that the pattern of association between low molecular weight acid phosphatase genotype and degree of glycemic control is similar in the two classes of diabetic patients. Among nonobese subjects the proportion of ACP1*A (the allele showing the lowest enzymatic activity) is lower in diabetic patients with high glycemic levels (mean value greater than 8.9 mmol/l) than in diabetic patients with a low glycemic level (mean value less than 8.9 mmol/l). Among obese subjects no significant association is observed between glycemic levels and ACP1. Among nonobese subjects the concentration of f isoform of ACP1 is higher in patients showing a high glycemic level than in patients showing a low glycemic level. No significant difference is observed for s isoform.

Acid Phosphatase↗
PubMed1999

Profile of placental alkaline phosphatase expression in human malignancies: effect of tumour cell activation on alkaline phosphatase expression.

Cellular alkaline phosphatases (ALP) are increasingly recognised as important markers for monitoring tumour cell behaviour in human malignancies. Colorimetric, flow-cytometric, and immunocytochemical assays were employed to assess the influence of activation on expression of cellular ALP in human tumour cell lines. The results showed the following: (1) Testis tumour biopsies (16/16) unlike bladder (0/14) and head and neck (0/16) tumours showed positive staining for ALP, particularly the placental type, i.e. PLAP, although this was not always present on all the cells of non-seminoma biopsies. (2) The intensity of ALP expression differed widely in tumour cell lines. Based on biochemical analysis, the profile of ALP fell into two categories: (a) low expressing (MW 70 kD, placental type ALP) like Hep2 and KB lines, and (b) those expressing both low and high molecular (MW 95 kD) bands like testis lines Tera II and Ep2102. In all cases treatment of tumour cell lysates with heat prior to biochemical analysis showed the disappearance of the higher and sharpening of the lower molecular weight ALP band. (3) Exposure of tumour cells to epidermal growth factor (EGF) expressing EGF receptor led to a decreased ALP expression by as much as 54% as assessed by biochemical or flow-cytometric techniques. These data demonstrated that testis tumour tissues and cell lines expressed ALP which were different from others. The data also showed that exposure of tumour cell lines expressing EGFr to EGF resulted in suppression of ALP expression. These observations are consistent with the notion that EGFr and PLAP expression may be taken as a marker of proliferation and differentiation in human malignancies, respectively.

Alkaline Phosphatase↗
PubMed1997

Microheterogeneity of rat serum alkaline phosphatase in fasting state: characterization of two duodenal alkaline phosphatase glycoforms.

Using concanavalin A-Sepharose affinity chromatography (Con A) we found that the serum of normal fasted adult rats contains two alkaline phosphatase (APase) glycoforms, one weakly bound (II) and the other strongly (III) bound to the column. Both serum APase glycoforms had an apparent molecular mass of 163 kD on Sepharose CL-6B and 118 kD on SDS-PAGE under nondenaturing conditions. We consider the molecular forms as dimeric, since monomers of 60.3 and 58.5 kD for the Con A weakly and strongly bound glycoform, respectively, were obtained. However, these two dimeric glycoforms were different in their pH optimum, affinity to p-nitrophenyl phosphate as substrate, the degree of L-phenylalanine inhibition and relative thermostability. Judging by the relative thermostability and by L-phenylalanine inhibition, it seems that both serum APase glycoforms in fasted rats are mainly of duodenal mucosal cell origin. The Con A weakly bound (II) glycoform could be derived from the cytosol, and the Con A strongly bound (III) one from both the cytosolic and membranous fractions of duodenal mucosal cells. However, in addition to the heat-stable component, the Con A strongly bound serum APase glycoform also contains a minor heat-labile and L-phenylalanine-resistant component which could be of nonspecific tissue origin since such a fraction was not discovered by us in rat duodenal mucosal cells.

Alkaline Phosphatase↗
PubMed1994

Skeletal muscle protein tyrosine phosphatase activity and tyrosine phosphatase 1B protein content are associated with insulin action and resistance.

Particulate and cytosolic protein tyrosine phosphatase (PTPase) activity was measured in skeletal muscle from 15 insulin-sensitive subjects and 5 insulin-resistant nondiabetic subjects, as well as 18 subjects with non-insulin-dependent diabetes mellitus (NIDDM). Approximately 90% of total PTPase activity resided in the particulate fraction. In comparison with lean nondiabetic subjects, particulate PTPase activity was reduced 21% (P < 0.05) and 22% (P < 0.005) in obese nondiabetic and NIDDM subjects, respectively. PTPase1B protein levels were likewise decreased by 38% in NIDDM subjects (P < 0.05). During hyperinsulinemic glucose clamps, glucose disposal rates (GDR) increased approximately sixfold in lean control and twofold in NIDDM subjects, while particulate PTPase activity did not change. However, a strong positive correlation (r = 0.64, P < 0.001) existed between particulate PTPase activity and insulin-stimulated GDR. In five obese NIDDM subjects, weight loss of approximately 10% body wt resulted in a significant and corresponding increase in both particulate PTPase activity and insulin-stimulated GDR. These findings indicate that skeletal muscle particulate PTPase activity and PTPase1B protein content reflect in vivo insulin sensitivity and are reduced in insulin resistant states. We conclude that skeletal muscle PTPase activity is involved in the chronic, but not acute regulation of insulin action, and that the decreased enzyme activity may have a role in the insulin resistance of obesity and NIDDM.

Adult↗
PubMed1987

The effect of pH and temperature on the stability and enzymatic activity of prostatic acid phosphatase. Studies on the optimization of a continuous monitored determination of acid phosphatase, II.

The catalytic activity and the stability of prostatic acid phosphatase were studied with respect to pH and temperature: 1. Enzymatic activity in serum decreases with time, and the rate of decrease depends on pH and temperature. Half life times were estimated. 2. To preserve at least 90% of its original activity, serum must be cooled as soon as possible below room temperature and/or the pH must be lowered to 6. 3. Considering the effect of pH on side reactions and kinetic parameters, a pH of 5.2 is recommended for the assay. 4. Between 25 and 37 degrees C, the value for Km app, in the absence of alcohols, is constant within the limits of error. In the presence of alcohols there is a significant increase of Km app at lower temperatures, and higher substrate concentrations are needed to avoid nonsaturation of the enzyme. vmax increases with temperature. Inactivation is observed above 45 degrees C, especially in the presenc of alcohols. 5. The Arrhenius plot shows a strict linear regression between 20 degrees C and 40 degrees C, in the presence or absence of 1,4-butanediol, 1,5-pentanediol or 1,6-hexanediol. 6. Temperature conversion factors for catalytic activity were calculated to be: 1.33 (25 to 30 degrees C), 1.96 (25 to 37 degrees C) and 1.47 (30 to 37 degrees C).

Acid Phosphatase↗
PubMed1996

Role of phosphoprotein phosphatases in the corpus luteum: I identification and characterisation of serine/threonine phosphoprotein phosphatases in isolated rat luteal cells.

Although the role of protein kinases and phosphorylation in steroidogenesis has received much attention, very little is known about the activities of phosphoprotein phosphatases (PP) and dephosphorylation in steroidogenic tissues. The aims of the present study were therefore to identify which of those serine/threonine PPs more commonly involved in intracellular signalling are expressed in rat luteal cells; to quantify, in vitro, the effects of inhibitors on PP activity extracted from purified rat luteal cells; and to measure the effects of PP inhibitors on the phosphorylation of endogenous luteal cell proteins. Polyclonal antibodies raised against the catalytic subunits of PP types 1 and 2A, and a monoclonal antibody raised against the Ca(2+)-binding subunit of PP2B, were used to identify immunoreactive proteins that migrated on SDS-PAGE with approximate molecular masses of 37, 34 and 16 kDa, corresponding well with the reported molecular mass of PP1, PP2A and PP2B respectively. Five selective inhibitors of PP1/PP2A: okadaic acid, calyculin A, cantharidin, tautomycin and microcystin-RR, each caused a dose-dependent decrease in the activity of PPs in luteal cell homogenates, and also enhanced 32P incorporation into numerous luteal cell proteins; most notably, proteins with approximate molecular masses of 20 and 22 kDa. The results of this study suggest that PPs may play an important role in the regulation of rat luteal cell functions.

Animals↗
PubMed1999

Mutational analysis of the coding regions of the genes encoding protein kinase B-alpha and -beta, phosphoinositide-dependent protein kinase-1, phosphatase targeting to glycogen, protein phosphatase inhibitor-1, and glycogenin: lessons from a search for genetic variability of the insulin-stimulated glycogen synthesis pathway of skeletal muscle in NIDDM patients.

The finding of a reduced insulin-stimulated glucose uptake and glycogen synthesis in the skeletal muscle of glucose-tolerant first-degree relatives of patients with NIDDM, as well as in cultured fibroblasts and skeletal muscle cells isolated from NIDDM patients, has been interpreted as evidence for a genetic involvement in the disease. The mode of inheritance of the common forms of NIDDM is as yet unclear, but the prevailing hypothesis supports a polygenic model. In the present study, we tested the hypothesis that the putative inheritable defects of insulin-stimulated muscle glycogen synthesis might be caused by genetic variability in the genes encoding proteins shown by biochemical evidence to be involved in insulin-stimulated glycogen synthesis in skeletal muscle. In 70 insulin-resistant Danish NIDDM patients, mutational analysis by reverse transcription-polymerase chain reaction-single strand conformation polymorphism-heteroduplex analysis was performed on genomic DNA or skeletal muscle-derived cDNAs encoding glycogenin, protein phosphatase inhibitor-1, phophatase targeting to glycogen, protein kinase B-alpha and -beta, and the phosphoinositide-dependent protein kinase-1. Although a number of silent variants were identified in some of the examined genes, we found no evidence for the hypothesis that the defective insulin-stimulated glycogen synthesis in skeletal muscle in NIDDM is caused by structural changes in the genes encoding the known components of the insulin-sensitive glycogen synthesis pathway of skeletal muscle.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2001

Inhibition of GSK-3 selectively reduces glucose-6-phosphatase and phosphatase and phosphoenolypyruvate carboxykinase gene expression.

A major action of insulin is to regulate the transcription rate of specific genes. The expression of these genes is dramatically altered in type 2 diabetes. For example, the expression of two hepatic genes, glucose-6-phosphatase and PEPCK, is normally inhibited by insulin, but in type 2 diabetes, their expression is insensitive to insulin. An agent that mimics the effect of insulin on the expression of these genes would reduce gluconeogenesis and hepatic glucose output, even in the presence of insulin resistance. The repressive actions of insulin on these genes are dependent on phosphatidylinositol (PI) 3-kinase. However, the molecules that lie between this lipid kinase and the two gene promoters are unknown. Glycogen synthase kinase-3 (GSK-3) is inhibited following activation of PI 3-kinase and protein kinase B. In hepatoma cells, we find that selectively reducing GSK-3 activity strongly reduces the expression of both gluconeogenic genes. The effect is at the level of transcription and is observed with induced or basal gene expression. In addition, GSK-3 inhibition does not result in the subsequent activation of protein kinase B or inhibition of the transcription factor FKHR, which are candidate regulatory molecules for these promoters. Thus, GSK-3 activity is required for basal activity of each promoter. Inhibitors of GSK-3 should therefore reduce hepatic glucose output, as well as increase the synthesis of glycogen from L-glucose. These findings indicate that GSK-3 inhibitors may have greater therapeutic potential for lowering blood glucose levels and treating type 2 diabetes than previously realized.

Aminophenols↗
PubMed1975

Disc electrophoresis of turkey serum alkaline phosphatase isoenzymes using polyacrylamide gel. 2. Turkey serum alkaline phosphatase zymograms.

The isoenzymes of alkaline phosphatase (akp) in turkey serum were separated by disc electrophoresis using polyacrylamide gel as the supporting medium Test subjects consisted of 103 turkeys maintained for the "Vibrator" condition at Michigan State University; and 46 female turkey of the Nicholas Broad Breasted White egg laying strain. Thirteen distinct akp bands were isolated. The 13 bands were observed to occur in 13 different patterns, each pattern being classified as a separate zymogram. Specific types of zymograms were detected for "Vibrator" birds vs. Nicholas strain; females vs, males; high intensity egg production vs. low intensity egg production and molting vs. laying.

Alkaline Phosphatase↗
PubMed1999

Residual alkaline phosphatase activity in pasteurized milk heated at various temperatures--measurement with the fluorophos and Scharer rapid phosphatase tests.

Milk containing three levels of milkfat (skim [0.5%], lowfat [2.0%], and whole [3.25%]) were heat treated at five different temperatures (59, 61, 63, 65, and 67 degrees C) using a laboratory scale, batch pasteurization method. Heated milk samples were removed at 5-min intervals, immediately cooled, and then assayed using the quantitative fluorometric method and the qualitative Scharer rapid test. Mean alkaline phosphatase (ALP) activity values as measured with the Fluorophos method decreased in all milk preparations as the time of sampling and temperature of heating increased. Samples representing the three fat levels and heat treated at 63 degrees C for 30 min, the minimum time/temperature allowed by the 1995 pasteurized milk ordinance (PMO), had ALP activity values <100 mU/liter. All values were below the 350 mU/liter standard for fluid milk products established by the Food and Drug Administration and cited in the 1995 PMO. Evaluation of the milks for adequacy of pasteurization with the Scharer rapid method indicated that those same milks were adequately pasteurized.

Alkaline Phosphatase↗
PubMed2000

Expression of neuron specific phosphatase, striatal enriched phosphatase (STEP) in reactive astrocytes after transient forebrain ischemia.

We studied the distribution and change of striatal enriched phosphatase (STEP) in the gerbil hippocampus after transient forebrain ischemia. STEP was expressed in the perikarya and in neuronal processes; it was not detected in non-neuronal cells of control animals. After 5-min forebrain ischemia, STEP immunoreactivity (STEP-IR) was preserved for 2 days; it disappeared 4 and more days after ischemia with completion of delayed neuronal death (DND) in the CA1 subfield. Furthermore, only in the CA1 after ischemia, STEP was expressed in reactive astrocytes for 4 to 28 days, showing different patterns of glial fibrillary acidic protein (GFAP)-positive reactive astrocytes. After non-or less-than lethal ischemia, STEP expression in reactive astrocytes corresponded with the degree of neuronal degeneration. Immunoblot analysis of the CA1 subfield revealed the expression of three isoforms, STEP45, -56 and -61; their expression patterns changed with time after ischemia. These data suggest that neuronal STEP is preserved until cell degeneration after ischemia and that STEP is expressed in reactive astrocytes only after lethal ischemia, with different expression patterns for its isoforms. Of STEP45, -56 and -61, STEP61 was the most strongly expressed in the reactive astrocytes; both STEP45 and -61 were expressed in neurons and the expression of STEP56 was weak. STEP may play an important role not only in neurons but also in reactive astrocytes after ischemia, depending on neuronal degeneration.

Animals↗