Presence Of Phosphatase Research Articles

Temporal dissociation of frequency-dependent acceleration of relaxation and protein phosphorylation by CaMKII

Frequency-dependent acceleration of relaxation (FDAR) is an important intrinsic mechanism that allows for diastolic filling of the ventricle at higher heart rates, yet its molecular mechanism is still not understood. Previous studies showed that FDAR is dependent on functional sarcoplasmic reticulum (SR) and can be abolished by phosphatase or by Ca/CaM kinase (CaMKII) inhibition. Additionally, CaMKII activity/autophosphorylation has been shown to be frequency-dependent. Thus, we tested the hypothesis that CaMKII phosphorylation of SR Ca 2+-handling proteins (Phospholamban (PLB), Ca 2+ release channel (RyR)) mediates FDAR. Here we show that FDAR occurs abruptly in fluo-4 loaded isolated rat ventricular myocytes when frequency is raised from 0.1 to 2 Hz. The effect is essentially complete within four beats (2 s) with the τ of [Ca 2+] i decline decreasing by 42 ± 3%. While there is a detectable increase in PLB Thr-17 and RyR Ser-2814 phosphorylation, the increase is quantitatively small (PLB < 5%, RyR ∼ 8%) and the time-course is clearly delayed with regard to FDAR. The low substrate phosphorylation indicates that pacing of myocytes only mildly activates CaMKII and consistent with this CaMKIIδ autophosphorylation did not increase with pacing alone. However, in the presence of phosphatase 1 inhibition pacing triggered a net-increase in autophosphorylated CaMKII and also greatly enhanced PLB and RyR phosphorylation. We conclude that FDAR does not rely on phosphorylation of PLB or RyR. Even though CaMKII does become activated when myocytes are paced, phosphatases immediately antagonize CaMKII action, limit substrate phosphorylation and also prevent sustained CaMKII autophosphorylation (thereby suppressing global CaMKII effects).

Interactions Between Kinases and Phosphatases in the Rapid Control of Brain Aromatase

Aromatization of testosterone into oestradiol plays a key role in the activation of male sexual behaviour in many vertebrate species. Rapid changes in brain aromatase activity have recently been identified and the resulting changes in local oestrogen bioavailability could modulate fast behavioural responses to oestrogens. In quail hypothalamic homogenates, aromatase activity is down-regulated within minutes by calcium-dependent phosphorylations in the presence of ATP, MgCl2 and CaCl2 (ATP/Mg/Ca). Three kinases (protein kinases A and C and calmodulin kinase; PKA, PKC and CAMK) are potentially implicated in this process. If kinases decrease aromatase activity in a reversible manner, then it would be expected that the enzymatic activity would increase and/or return to baseline levels in the presence of phosphatases. We showed previously that 0.1 mM vanadate (a general inhibitor of protein phosphatases) significantly decreases aromatase activity but specific protein phosphatases that could up-regulate aromatase activity have not been identified to date. The reversibility of aromatase activity inhibition by phosphorylations was investigated in the present study using alkaline and acid phosphatase (Alk and Ac PPase). Unexpectedly, Alk PPase inhibited aromatase activity in a dose-dependent manner in the presence, as well as in the absence, of ATP/Mg/Ca. By contrast, Ac PPase completely blocked the inhibitory effects of ATP/Mg/Ca on aromatase activity, even if it moderately inhibited aromatase activity in the absence of ATP/Mg/Ca. However, the addition of Ac PPase was unable to restore aromatase activity after it had been inhibited by exposure to ATP/Mg/Ca. Taken together, these data suggest that, amongst the 15 potential consensus phosphorylation sites identified on the quail aromatase sequence, some must be constitutively phosphorylated for the enzyme to be active whereas phosphorylation of the others is involved in the rapid inhibition of aromatase activity by the competitive effects of protein kinases and phosphatases. Two out of these 15 putative phosphorylation sites occur in an environment corresponding to the consensus sites for PKC, PKA (and possibly a CAMK) and, in all probability, represent the sites whose phosphorylation rapidly blocks enzyme activity.

Role of the TCF phosphorylation state and the chromatin structure in the negative transcription regulation of the c-fos proto-oncogene in E1A + c-Ha-ras transformed cells

As compared with normal rat embryo fibroblasts (REF), c-fos transcription is suppressed in REF transformed with the E1A and cHa-ras complementing oncogenes. Negative regulation of the fos promoter is due to the serum-responsive element (SRE) constantly bound with the serum response factor (SRF) and with the ternary complex factor (TCF), which form the SRF/SRF/TCF complex. Possible mechanisms of the c-fos suppression were studied, including changes in the TCF content and phosphorylation and replacement of TCF by repressor proteins. In addition, the transcription factors were tested for DNA-binding activity, and c-fos transcription was analyzed by RT-PCR in various cell culture conditions and in the presence of phosphatase and histone deacetylase inhibitors. The c-fos suppression in transformed REF was explained by formation of a transcriptionally inactive chromatin structure in the fos promoter region as a result of histone deacetylation, rather than by insufficient TCF phosphorylation.

Structural analysis of the carbohydrate components of the outer membrane of the lipopolysaccharide-lacking cellulolytic ruminal bacterium Fibrobacter succinogenes S85.

The polysaccharides from the outer membrane of the Gram-negative ruminal bacterium Fibrobacter succinogenes were isolated by phenol/water extraction and separated by size-exclusion chromatography in the presence of deoxycholate detergent into a lower-molecular-mass fraction designated 'glycolipid' and a high-molecular-mass 'capsular polysaccharide' fraction. Both fractions lacked typical lipopolysaccharide components including 2-keto-3-deoxyoctulosonic acid and 3-hydroxy fatty acids. Carbohydrate components of these fractions were represented by two polysaccharides and one oligosaccharide (possibly glycolipid) with the following structures: : : where HEAEP is N-(2-hydroxyethyl)-2-aminoethylphosphonic acid, found for the first time in natural compounds. The polysaccharides contained pentadecanoic acid and anteisopentadecanoic acid, possibly present as the acyl components. All constituent monosaccharides except L-rhamnose had a D-configuration. In addition to having a structural role in the outer membrane, these polysaccharides may provide protection for this lipopolysaccharide-less bacterium in the highly competitive ruminal environment, as phosphonic acids covalently linked to membrane polymers have in the past been attributed the function of stabilizing membranes in the presence of phosphatases and lipases.

Protein kinase and protein phosphatase presence in the stria vascularis.

Na+, K+-ATPase contributes to the high potassium concentration in the endolymph and the resulting endocochlear potential, which are both essential for the function of the sensory part of the inner ear. Na+, K+-ATPase is present in the stria vascularis and it has lately been suggested that its activity is hormonally regulated. The intracellular signalling system for hormonal short-term regulation of Na+, K+-ATPase activity by phosphorylation in renal tubular cells has been well described. In this study, the presence of the intracellular components of this phosphorylation system in the stria vascularis from guinea-pig has been investigated with immunoblotting. The concentrations found were related to those in renal medullary tissue or the corpus striatum. Protein kinase C was present with isoforms alpha, delta and zeta in the stria vascularis. Calcium- and calmodulin-dependent protein kinase II and protein phosphatase-1 isoforms alpha and gamma were found in the stria vascularis. Protein phosphatase-2B, on the other hand, could not be detected. I-1, an inhibitor of protein phosphatase activity, was present, whereas the phosphatase inhibitor dopamine- and cAMP-regulated phosphoprotein (DARPP-32), was not present in the stria vascularis. These results demonstrate that several intracellular components of the phosphorylation/dephosphorylation system are present in the stria vascularis, and suggest that hormonal short-term regulation of Na+, K+-ATPase activity is also possible in the stria vascularis.

A Microtiter Well Assay System to Measure Insulin Activation of Insulin Receptor Kinase in Intact Human Mononuclear Cells: Decreased Insulin Effect in Cells From Patients With NIDDM

A sensitive microtiter well-based assay for the measurement of insulin activation of insulin receptor kinase in intact human circulating mononuclear cells has been developed and characterized. Mononuclear cells from 100-150 ml blood were incubated with various insulin concentrations to activate the receptor kinase. The cells were then solubilized in the presence of phosphatase and kinase inhibitors and the receptors immobilized to microwells coated with anti-insulin receptor antibody (efficiency of receptor immobilization > 85%). Receptor kinase activity and binding activity were then consecutively measured in the same wells. Insulin incubation of the cells increased the kinase activity three- to fourfold with a half-maximal effect at 5 nM and a maximal effect at 87 nM. In mononuclear cells from 16 subjects with NIDDM, the insulin effect on receptor kinase activation was significantly reduced compared with 16 nondiabetic control subjects (0.135 +/- 0.016 vs. 0.195 +/- 0.024 fmol P.fmol binding activity-1 x min-1, respectively; P < 0.05). We conclude that; 1) it is possible to determine insulin activation of receptor kinase in intact cells in this easily accessible human tissue; 2) insulin activation of insulin receptor kinase is impaired in intact mononuclear cells from patients with NIDDM; and 3) the finding that kinase activation in NIDDM is reduced in a tissue that, according to the literature, contains only the A isoform of the insulin receptor, suggests that mechanisms other than a different abundance of the A and B insulin receptor isoforms must exist that contribute to the decreased kinase activity in NIDDM.

Histone H1 phosphorylated by protein kinase C is a selective substrate for the assay of protein phosphatase 2A in the presence of phosphatase 1

A protein phosphatase assay, selective for protein phosphotase 2A, has been developed. Bovine histone H1 phosphorylated by protein kinase C and [γ 32P]ATP, designated H1(C), was tested as the substrate for various preparations of protein phosphatases 1 and 2A. The phosphatase 2A preparations were 10–60 times more active with H1(C) as the substrate when compared to phosphorylase a. The phosphatase 1 enzymes showed very little dephosphorylation of the H1(C) substrate, the activity being less than 5% of the phosphorylase phosphatase activity. This preference and selectivity was demonstrated for purified phosphatase preparations in addition to fresh tissue extracts. The assay provides a rapid, simple assay for the routine analysis of phosphatase 2A in the presence of phosphatase 1, without the use of heat-stable inhibitor proteins.

Tyrosine phosphorylation of insulin receptor beta subunit activates the receptor tyrosine kinase in intact H-35 hepatoma cells.

The phosphorylation characteristics of insulin receptor from control and insulin-treated rat H-35 hepatoma cells 32P-labeled to equilibrium have been documented. The 32P-labeled insulin receptor is isolated by immunoprecipitation with patient-derived insulin receptor antibodies in the presence of phosphatase and protease inhibitors to preserve the native phosphorylation and structural characteristics of the receptor. The unstimulated insulin receptor contains predominantly [32P] phosphoserine and trace amounts of [32P]phosphothreonine in its beta subunit. In response to insulin, the insulin receptor beta subunit exhibits marked tyrosine phosphorylation and a 2-fold increase in total [32P]phosphoserine contents. High pressure liquid chromatography of the tryptic hydrolysates of the 32P-labeled receptor beta subunit from quiescent cells results in the resolution of up to 9 fractions containing [32P]phosphoserine. The insulin-stimulated tyrosine phosphorylation is concentrated in two of these receptor phosphopeptide fractions, whereas the increase in [32P]phosphoserine content is scattered in low abundance over all receptor tryptic fractions. Insulin receptors affinity-purified by lectin- and insulin-agarose chromatographies from insulin-treated, 32P-labeled cells exhibit a 22-fold increase in the Vmax of receptor tyrosine kinase activity toward histone when compared to controls. The elevated kinase activity of the insulin receptor derived from insulin-treated cells is not due to the presence of hormone bound to the receptor because the receptor kinase activity is assayed while immobilized on insulin-agarose. Furthermore, the insulin-activated receptor kinase activity is reversed following dephosphorylation of the receptor beta subunit with alkaline phosphatase in vitro. The correlation between the insulin-stimulated site specific tyrosine phosphorylation on receptor beta subunit and the elevation of receptor tyrosine kinase activity strongly suggests that the insulin receptor kinase is activated by hormone-stimulated autophosphorylation on tyrosine residues in intact cells, as previously demonstrated for the purified receptor.

Enzymic ribonucleoside reduction at the non-phosphorylated level in Acetabularia.

The occurrence of an enzyme that catalyses the conversion of cytidine into deoxycytidine was demonstrated in homogenates of Acetabularia. Cytidine was identified as the substrate by comparing cytidine, cytidine 5'-monophosphate, cytidine 5'-diphosphate and cytidine 5'-triphosphate as potential substrates. Experiments with ATP analogues whose inhibitory effect on kinase reactions is well established, supplied evidence that the nucleoside is reduced without a phosphorylation step before the reduction. Further evidence in this line came from incubations with cytidine in the presence of phosphatase and from trap-type experiments in which the effects of excess non-labelled cytidine 5'-phosphate and deoxycytidine, respectively, on the formation of deoxycytidine phosphates from cytidine were studied.

Activation mechanism of insect fat body phosphorylase by cold Phosphorylase kinase, phosphatase and ATP level

Fat body glycogen phosphorylase of overwintering insects is considerably activated by cold; two enzymes, phosphorylase kinase and phosphatase, may be involved in this activation. In this paper, we demonstrate the mechanism by which phosphorylase is activated by cold. Initially, a latent phosphorylase phosphatase was stimulated by freeze-thawing with 67% saturated ammonium sulphate and mercaptoethanol, enabling partial purification and characterization of this enzyme. Unlike the phosphorylase kinase studied previously, the phosphatase did not function at 0°C. There was no appreciable activation of phosphorylase at 25°C if the phosphatase was present, whereas the phosphorylase was greatly activated at 0°C even in the presence of phosphatase. It is suggested that ATP regulates phosphorylase activity through its effect on kinase and phosphatase. The activation of fat body phosphorylase that naturally occurs in overwintering insects is also discussed.

11] Isopentenyldiphosphate Δ-isomerase

Isopentenyldiphosphate Δ-Isomerase catalyzes the reversible reaction providing dimethylallyl pyrophosphate, the 5-carbon allylic pyrophosphate subunit (isoprene), which initiates the formation of all olyterpenoid molecules by the action of prenyltransferase. Isomerase was first found in bakers' yeast by Lynen et al. Since then isomerase has been studied in pig liver, pumpkin: orange peel, pine seedlings, camphor tree, cotton root, Penicillium cyclopium, avian liver, and Claviceps purpurea. One of the purifications from pig liver was extensive, and the enzyme was reported to be nearly homogeneous. The instability of this enzyme and presence of phosphatase and prenyltransferase found in these and other preparations have severely limited the mechanistic characterization of isomerase. Recently, investigators reported multiple forms of avian isomerase prepared by DEAE cellulose chromatography. However, these may have resulted from proteolysis.

Increase of calmodulin-stimulated striatal particulate phosphorylation response in chronic haloperidol-treated rats

Calcium- and calmodulin-dependent protein kinase and phosphatase activities were studied in rat striatal particulate preparations. The effect of Ca 2+ (0.1–0.5 mM) on phosphorylation was completely abolished in the preparation which had been washed 3 times with a buffer containing ethylene glycol bis-(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA, 1.2 mM). Ca 2+-stimulated phosphorylation was restored in a dose-dependent manner after calmodulin (1 μg) was added to the assays. Ca 2+ and calmodulin promoted the phosphate incorporation into two major striatal protein bands with estimated M r at 52 and 40 kdaltons. The presence of phosphatase in the EGTA-pretreated preparations was negligible. Chronic treatment in rats with haloperidol (1 mg/kg, 20 days) produced a significant decrease in the Ca 2+-independent phosphorylation but an increase in the extent of Ca 2+ and calmodulin-dependent phosphorylation in the striatum. The chronic haloperidol treatment did not alter the striatal [ 125I]calmodulin binding curve. In vitro, haloperidol (even at 10 −4 M) had no effect on calmodulin-dependent phosphorylation. Haloperidol (10 −4 M) did not reduce the number but decreased the rate of [ 125I]calmodulin binding to the striatal particulates. These data suggest that the link between dopamine receptors and calmodulin-dependent enzyme is dissociated in vitro. On the other hand, the potential sensitivity of calmodulin-dependent protein kinase in the chronic haloperidol-treated rats correlated with the supersensitive dopamine receptor responses occurred in these rats. Therefore, calmodulin-dependent protein kinase may biochemically regulate dopamine receptor function in the striatum in vivo.

A method for quantitation of vitamin B2 by using an enzyme (producing vitamin B2-aldehyde) from Schizophyllum commune.

A method for enzymatic determination of riboflavin was developed by enzymatic reduction of 2,6-dichlorophenolindophenol using a vitamin B2-aldehyde-forming enzyme prepared from Schizophyllum commune. The enzymatic method enabled us to determine riboflavin in a mixture containing riboflavin, FMN and FAD without separation of riboflavin. Moreover, the total amount of flavins in the mixture was shown to be determined by the enzyme in the presence of phosphatase.

HISTOCHEMICAL STUDIES OF BONE IN NORMAL AND PATHOLOGICAL CONDITIONS

1. Histochemical studies have been made of the distribution of alkaline phosphatase, glycogen and acid mucopolysaccharides in normal growing bones (mice, rats and men) and also in forty cases of pathological bone processes (neoplastic and dystrophic). 2. The study of normal material confirmed that alkaline phosphatase is plentiful in calcification of cartilage and even more plentiful in bone formation (whether enchondral or direct). 3. It was observed that glycogen increased in the cartilage areas about to be calcified, and that it disappeared in those calcified. It seemed that osteoblasts did not always contain glycogen. 4. In the pathological material (tumours and dystrophic processes) there was great phosphatase activity in the osteogenic areas and also in the cartilage about to be calcified. Whereas glycogen was plentiful in some cases of neoplastic or reactive osteogenesis, it was absent from others. 5. In every area of normal or pathological ossification, the presence of phosphatase seems to be a rule; glycogen is often but not always present. 6. It appears that alkaline phosphatase plays an important role in the formation of the protein matrix of bone, but is not associated with the elaboration of the mucoprotein cartilage matrix. We believe it is premature to draw any definite conclusion on the behaviour and role of the metachromatic substances in the processes of calcification and ossification. The histochemical study of alkaline phosphatase has shown that this is a valuable method in the detection of reactionary or pathological osteogenic processes which in some cases are difficult to demonstrate with the usual histological methods.

麦芽根飼料より分離せる有毒物質産生糸状菌の研究 (第2報)

1) The range of optimal temperatures for the growth of the hyphomycetes which produce toxic substance is 8-40°, the best range being 22-25°.2) The allowable pH for growth is in a range of 3.5-8.5, the best being pH 4.5-6.5.3) Essential nutritional sources are sugars, nitrogen, phosphates, and magnesium salts, the growth being dependent on the ratio of sugar to nitrogen content.4) The fungus grows in aerobic conditions, absorbing oxygen from the air in contact and producing carbon dioxide.5) The sporodium of this fungus dies in 1 minute in 0.1% AgNO3 solution, in 24 hours in 0.001% AgNO3 solution, and in 5 minutes in 70% ethanol, but is comparatively resistant to other chemicals. The sporodium was also made inert by the ultraviolet rays (2537 Å) of 1350 radiation energy and by moist steam of 58° in 10 minutes.6) The enzyme of this fungus possessed weak amylase, lipase, and protease actions, and a markedly strong disaccharide decomposing action. The presence of phosphatase, reductose, and catalase was marked.

Absence of Acid Phosphatase from Chloroplasts of Spinach and Iris

The presence of phosphatases in leaves is well known. The acid phosphatase of leaves appears to lack specificity with respect to hydrolyzable substrates (1, 6, 7, 8). Glycerophosphate has always been found to be an effective substrate (1, 5, 6, 7, 8). However, there is some evidence for the plurality of the acid phosphatase of leaves in that specific effects of activators and inhibitors have been found (1). The objective of the present study was to determine whether or not chloroplasts contain acid phosphatase. This investigation was prompted by Yin's (8) inability to discern whether most of the acid phosphatase of the leaves of Iris and Lamium was concentrated in the chloroplasts themselves or in the surrounding cytoplasm at the interface with the chloroplasts. Freehand sections of leaves of Iris and market spinach, and fractionated macerates of spinach leaves were tested for acid phosphatase by means of Gomori's microchemical method (3). All samples were washed with 0.2% versene and with several changes of distilled water, then incubated for periods of up to three hours at 37? C in the medium containing the substrate a-glycerophosphate at pH 5.1. Almost no phosphatase activity was found in the freehand sections of Iris leaves. Less than 1% of the chlorenchyma cells showed activity, and this activity did not appear to be associated with the chloroplasts. In sections of spinach leaves the phosphatase was active in irregular patches of chlorenchyma. The precipitated lead sulphide was seen to occur in the extra-chloroplastic cytoplasm and at nuclei. There was very high activity on the crystals (druses) which occurred regularly in enlarged cells in the mesophyll. As was observed by Glick and Fischer (2), some cell walls, especially those of tracheide and epidermal cells, gave a positive reaction. However, this reaction was not the result of enzymatic activity since it also appeared in sections that had been boiled for three minutes. Spinach leaves were macerated with a Waring Blendor in 0.2% versene. After filtration through muslin, the mixture was centrifuged at 6000 x g. for 15 minutes. The green sediment (fraction I), containing the chloroplasts and various unidentified particles, was resuspended in distilled water. The supernatant (fraction II) was light yellow in color. Both fractions were saturated with ammonium sulphate and the resulting precipitates were

ACID PHOSPHATASE IN THE SENILE BRAIN

GOMORI1and Takamatsu2in 1939 showed that the presence of phosphatase can be demonstrated in tissue sections. Their work referred to alkaline phosphatase; however, later, Gomori3described a method by which acid phosphatase, too, can be made visible in sections. Wolf, Kabat and Newman4examined the nerve tissue for acid phosphatase. They found that it contains large amounts. With proper technic, normal nerve cells, as well as nerve fibers, may give a strong positive reaction. Hard and Lassek5utilized the method for examination of growing axons and of degenerating nerve tissue. They found that tracts which become myelinated early exhibit enzyme activity early and that those which become myelinated late, such as the pyramidal tract, show phosphatase late. If a tract degenerates, the entire degenerating field gives a strong positive reaction for acid phosphatase. The authors discuss the possibility that the enzyme may be liberated

The Pancreas and Alkaline Phosphatase

IN various accounts of histochemical studies on the presence of alkaline phosphatase (substrate: sodium-β-glycerophosphate, pH. 9) in homologous organs, correspondences (for example, in kidney and intestine) have usually been stressed rather than differences. It seems, however, appropriate to direct attention to the latter in order to place the value of experiments on animals in its proper perspective. If the pancreas is selected for discussion, it is because of the confusion which seems to exist in the literature concerning the phosphatase content of this organ. It is often stated in a general way that the enzyme occurs or has been shown to be present in pancreatic tissue1,2. The fact is that Grosser and Husler3, who investigated the pancreas of cattle, sheep, cat and man biochemically, expressly emphasize the complete absence of the enzyme. Again, Takamatsu4, and Kabat and Furth5 failed to produce histochemical evidence for the presence of phosphatase in the pancreas of man. Gomori6 reported that only in the pancreas of the dog are the smallest ducts strongly outlined in black (phosphatase-positive). I found7, in histochemical tests, the epithelial cells, which are related to the duct system of the external secretion of the pancreas, entirely negative in the guinea pig and rat, but strongly positive in the dog (from the centro-acinar cells to the epithelium of large ducts; Fig. 1), thus confirming and extending Gomori's observation.

CONSTITUENTS OF ELEMENTARY BODIES OF VACCINIA

Purified elementary bodies of vaccinia have been tested with a variety of substrates and found to possess phosphatase, catalase, and lipase activity. Tests for malate, succinate, pyruvate, and lactate dehydrogenases were negative. Interpretation of these results is complicated by the observation that elementary bodies of vaccinia adsorb relatively large quantities of certain enzymes from dilute solutions. These enzymes are not eluted by procedures of washing and centrifuging similar to those carried out in the preparation of the virus. For this reason, the presence of phosphatase, catalase, and lipase in the purified virus may well be accounted for on the basis of adsorption from the host tissues which are known to be rich in these particular enzymes. That some degree of specificity in this adsorption is to be recognized is shown by the failure of the virus to adsorb urease, an enzyme of vegetable origin. Until some method can be devised which will distinguish between the enzymes of the host cell and those which may be integral parts of the virus it would seem that the problem of the enzyme constituents of vaccine virus is incapable of definite solution.