Determination of inorganic phosphate in the presence of nonionic detergents: A modified isobutanol-benzene extraction method

Inorganic phosphate can be determined either radiometrically or spectrophotometrically after extraction of its complex with molybdate into an organic phase. Triton X-100 interferes with this extraction. Determination of the inorganic phosphate can be carried out in the presence of up to 0.8% ($\text{w}\text{v}$) Triton X-100 by modification of the method: After addition of the silicotungstate, the sample is centrifuged, the yellow oily phase removed, and a sufficient amount of silicotungstate added again. Lubrol WX interfered even more drastically than Triton X-100, but the modified method was effective in the presence of 0.04% ($\text{w}\text{v}$) Lubrol WX. The method was useful in biochemical assays such as those of ATPase and cyclic nucleotide-dependent phosphodiesterase.     

Phosphoprotein phosphatase inhibits flagellar movement of Triton models of sea urchin spermatozoa

Phosphoprotein phosphatase prepared from bovine cardiac muscle was used to study the roles of axonemal phosphoproteins in the flagellar motility of sea urchin spermatozoa. When isolated axonemes were incubated with cyclic AMP-dependent protein kinase, gamma-[32P]ATP and cyclic AMP, more than 15 polypeptides were phosphorylated. Most were dephosphorylated by treatment with phosphoprotein phosphatase. When Triton models of sea urchin spermatozoa were treated with phosphoprotein phosphatase followed by an addition of ATP, the flagellar motility of the models was drastically reduced in comparison with that of the untreated models. The motility of the phosphatase-treated Triton models was partially restored by an addition of cyclic AMP and cyclic AMP-dependent protein kinase. These data give strong support to the idea that the motility of eukaryotic flagella is controlled by a protein phosphorylation-dephosphorylation system.     

Dephosphorylation of 40S ribosomal subunits by phosphoprotein phosphatase activity from rabbit reticulocytes.

Phosphoprotein phosphatase activities which remove phosphoryl groups from ribosomal protein have been partially purified from rabbit reticulocytes by chromatography on DEAE-cellulose. Two major peaks of phosphoprotein phosphatase activity were observed when 40S ribosomal subunits, phosphorylated $\text{in vitro}$ with cyclic AMP-regulated protein kinases and (γ-³²P)ATP, were used as substrate. The phosphatase activity eluting at 0.14 M KCl was characterized further using ribosomal subunits phosphorylated $\text{in situ}$ by incubation of intact reticulocytes with radioactive inorganic phosphate. Phosphate covalently bound to 40S ribosomal subunits and 80S ribosomes was removed by the phosphatase activity. The enzyme was not active with phosphorylated proteins associated with 60S ribosomal subunits.     

Changes in hepatic fatty acid synthesis following glucagon injections in vivo

When retinol was incubated in the presence of ATP, UDP-galactose, magnesium ions and Triton X-100, with a whole homogenate of rat thyroid as an enzyme source the formation of a retinol containing phosphate compound was observed besides retinol galactoside described before. This phosphate compound is soluble in chloroform-methanol (3:2, $\text{v}\text{v}$) and can be separated by chromatography on a column prepared with butanol-washed cellulose powder. The isolated compound was retinol pyrophosphate since it contained retinol and phosphate in a molar ratio of 1:2 shown by double isotopic labelling techniques and was found to be free of galactose.     

Phosphoprotein phosphatase in bovine tracheal smooth muscle. Multiple fractions and multiple substrates.

Phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) from bovine tracheal smooth muscle extracts was isolated and its activity determined using two [32P]phosphorylated proteins as substrates, i.e. phosphorylated histone (H-P) and a phosphorylated muscle specific substrate protein (MS-P) for the tracheal smooth muscle protein kinase. The enzyme was purified by the use of DEAE-cellulose followed by a two stage chromatography on a histone-Sepharose affinity column. Elution from the affinity column resolved the phosphoprotein phosphatase into four activity fractions. While fractions expressed phosphatase activity against both tested substrates the relative amounts of either activity varied. The ratio of activity towards H-P to activity towards MS-P changed from 11.5 to 0.12. The characterization of four phosphoprotein phosphatase fractions was based on the differences found in the following parameters: substrate specificity; sensitivity to NaF; influences of nucleotides (ATP, 5'-AMP, cyclic AMP, cyclic GMP) and the requirement of Mn2+ for maximal activity. Mg2+, Ba2+ or Ca2+ could not substitute for Mn2+.     

Compartmentation of creatine kinases during perinatal development of mammalian heart

Maturation of the cardiac cell is characterized by increasing diversity of isozymic expression of creatine kinases. Expression of the M-CK isozyme always precedes that of mitochondrial isozyme (mi-CK), however the expression of an isoform does not inform about its localization or cellular function. The functional role of isozymes binding to sites of energy utilization and production characteristic of the adult myocardium can be evidenced by the functional coupling of M-CK to myofibrillar ATPase and mito-CK to translocase in Triton X-100 and saponin skinned fibers. Functional activity of M-CK and mito-CK were investigated during perinatal development. Both functional activities appear during late fetal life in species mature at birth like guinea pig, and in the first postnatal weeks in immature species like rat or rabbit. Thus, the functional activity of bound CK isozymes is not associated with birthper se but with the general process of cell maturation. Localization of CK in the cytosol appears optimal for the transfer of glycolytic production of ATP to sites of utilization in an immature heart. During cell maturation, the increasing contribution of oxidative phosphorylation to ATP production, the apparition and binding of mi-CK to mitochondria, the binding of M-CK to myofibrils, turn the cell in a compartmentalized system of energy production. This provides the cellular basis for energy transfer by the PCr-Cr-CK system between sites of ATP production and utilization. Compartmentation of both Ca handling and energy turnover leads to a highly structured cell organization and could be essential for the efficiency of heart function.     

Phosphoprotein phosphatase in the central nervous system of Manduca sexta.

The existence and some enzymological properties of phosphoprotein phosphatase (EC 3.1.3.16) have been established in the larval central nervous system of the tobacco hornworm, Manduca sexta (Lepidoptera: Sphingidae). A simple, sensitive and reproducible assay employing 32-P-labeled protamine as a phosphoprotein substrate was employed to measure phosphatase activity in both soluble and particulate fractions of the insect nerve cord. The specific activity of soluble phosphatase in the Manduca sexta central nervous system is of the same order of magnitude as that in mammalian brain. Nerve cord phosphoprotamine phosphatase activity may be stimulated by a variety of monovalent salts, the optimal concentration of NaCl or KCl being 0.2 molar. Activity does not appear to be dependent on bivalent metals and is stimulated by EDTA. A reduced sulfhydryl group is obligatory for maximum activity. Phosphatase could be greatly inhibited by sodium fluoride, ATP and GTP. Cyclic AMP and cyclic GMP are without effect on enzyme activity. Although most of the phosphatase activity in the insect nerve cord appears to be of cytosolic origin, much latent activity can be unmasked by incubating membranous fractions with Triton X-100. In contrast to soluble phosphatase, the detergent-solubilized activity is moderately stimulated by Mn-2+.?     

The hormonal stimulation of ureogenesis in isolated hepatocytes through increases in mitochondrial ATP production

Isolated hepatocytes incubated with 2 mm ornithine-10 mm glutamine as substrates and challenged with either glucagon, epinephrine, or phenylephrine exhibited stimulated rates of urea production, and mitochondria isolated from these cells displayed an increased rate of energy-dependent citrulline formation. There was no change in the total carbamyl phosphate synthetase I activity, nor mitochondrial content of the positive effector N-acetyl glutamate after acute hormonal treatment. The time of onset of ureogenesis and its sensitivity to glucagon were compared with stimulation of glucose production from lactate-pyruvate. No apparent differences in time of onset or sensitivity of the responses were observed indicating both pathways may be stimulated by a common mechanism. Mitochondria prepared from cells treated with catecholamines exhibited increased rates of State 3 respiration and increased uncoupler-dependent ATPase activity, in addition to the increased rates of citrulline formation. There was also an elevated intramitochondrial content of ATP and an increased $\text{ATP}\text{ADP}$ ratio. The catecholamine-induced stimulation of ureogenesis was mediated by an α-adrenergic cyclic AMP independent mechanism. The addition of the α-adrenergic antagonist, dihydroergotamine, blocked both the epinephrine-induced stimulation of ureogenesis and also the stimulated functions in the isolated mitochondria. dl-Propranolol, a β-antagonist, inhibited the rise in cyclic AMP due to epinephrine, but had no effect on any of the other reactions measured. The effects of catecholamines on citrulline formation and urea production are correlated with the increased capacity of the mitochondria to generate ATP. It is suggested that both glucagon and catecholamines, acting via independent mechanisms, stimulate electron transport and the activity of the ATP-forming enzyme complex. The consequent elevated intramitochondrial ATP levels and $\text{ATP}\text{ADP}$ ratio enhance the rate of citrulline formation and hence ureogenesis.     

Protein-bound histidine,as well as protein-bound serine,residues are sites of phosphorylation in the synaptic plasma membrane

When synaptic plasma membrane fragments are incubated with ATP in the presence of Mg²⁺, phosphate is transferred, not only to protein-bound serine, but also to protein-bound histidine. The phosphorylation of protein-bound serine is stimulated by cyclic AMP and has a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP of about 0.12 mM, both in the presence and absence of cyclic AMP. By contrast, the phosphorylation of protein-bound histidine is unaffected by cyclic AMP and does not follow Michaelis-Menton kinetics since a non-linear double reciprocal plot is given when activity is measured at various ATP concentrations.     

Myofibrillar and membrane-bound enzymes in skeletal muscle from myodystrophic mice.

1. Experiments were carried out to examine the biochemical changes, such as contractile protein biochemistry and membrane bound enzyme alterations associated with skeletal muscles of myd/myd. 2. Our studies demonstrate that there was a progressive decline in myofibrillar ATPase activity, and this decrease is greatest in 30 weeks old animals of myd/myd as compared to controls. 3. The proteolytic activity of myofibrils isolated from myd/myd was significantly higher than controls. 4. There was no significant difference in Ca2+ ATPase activity of myosin and actin-activated myosin ATPase activity of myd/myd and their controls. 5. Mg2+ ATPase and Na(+)+K(+)-ATPase of myodystrophic SL showed significant increase compared to controls. 6. Isoproterenol stimulated adenylate cyclase activity was significantly lower in the SL of dystrophic mice compared to controls. 7. GTP+isoproterenol stimulate adenylate cyclase was significantly higher in control SL and SR when compared to SL and SR isolated from myd/myd. 8. Guanylate cyclase activity was greater in myodystrophic mice both in the absence and presence of Triton X-100. cGMP and cAMP phosphodiesterase activities were greater in dystrophic mice as compared to controls. 9. These observations suggest that there are significant changes in myofibrillar ATPase, myofibrillar protease and membrane bound enzymes of myd/myd compared to control.     

Phosphorylation of cardiac sarcolemma by endogenous and exogenous protein kinases.

Sarcolemmal membranes isolated from guinea pig heart ventricles contained endogenous protein kinase activity and protein substrates for this enzyme. Phosphorylation of sarcolemma was modestly stimulated by cyclic AMP with the half-maximal stimulation at 0.5 μm cyclic AMP. The phosphorylation of sarcolemma due to endogenous kinase was dependent on Mg²⁺. The apparent affinity for Mg²⁺ was found to be 1.4 and 0.53 mm in the absence and presence of 1 μm cyclic AMP, respectively. The apparent affinity for ATP was 55 μm. Sarcolemmal membranes were also phosphorylated by exogenous (purified) cyclic AMP-dependent protein kinase(s). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of phosphorylated membranes, followed by slicing and determination of the radioactivity in the gel slices, showed that endogenous protein kinase activity promoted the phosphorylation of specific protein peaks, arbitrarily designated a–g in order of increasing relative mobility (relative molecular weights 125,000, 110,000, 86,000, 58,000, 48,000, 22,000, and 16,000, respectively); peak e (48,000) was the major phosphorylated band. Exogenous protein kinase stimulated the phosphorylation of all peaks. However, the degree of stimulation of the low molecular weight peaks f and g was more marked. Results obtained after treatment of phosphorylated membranes with hydroxylamine at acid pH indicated the absence of any significant amount of acyl phosphate-type incorporation of phosphate. Purified phosphoprotein phosphatase from rabbit liver effected dephosphorylation of previously phosphorylated sarcolemma; this treatment resulted in dephosphorylation of all peaks (a–g). Pretreatment of sarcolemma with trypsin (membrane to trypsin ratio of 100) was found to markedly reduce both the total membrane phosphorylation as well as relative phosphorylation of peaks c, f, and g. On the other hand, pretreatment of sarcolemma with phospholipase c slightly stimulated total membrane phosphorylation with nondiscriminatory enhancement of the phosphorylation of all peaks. Microsomal membrane vesicles (enriched in sarcoplasmic reticulum fragments) isolated from guinea pig heart ventricle also contained endogenous protein kinase activity. Cyclic AMP modestly increased the kinase. Polypeptides of molecular weights 56,000, 22,000, and 16,000 were found to be phosphorylated. Exogenous (purified) cyclic AMP-dependent protein kinase increased the phosphorylation of microsomes and of 22,000 and 16,000 molecular weight polypeptides.     

Characterization of gastric mucosal membranes. IX. Fractionation and purification of K+-ATPase-containing vesicles by zonal centrifugation and free-flow electrophoresis technique

Methods are described for purification of a vesicular membrane fraction of hog gastric mucosa using differential centrifugation, density gradient separation on zonal rotors and free-flow electrophoresis. As a result a fraction is obtained enriched 40-fold in terms of K⁺-ATPase and free of any other enzyme marker other than K⁺-activated $\text{p-}\text{nitrophenyl}$ phosphatase.the 5′-nucleotidase and basal Mg²⁺-ATPase are clearly separated from the latter enzymes.Osmotic shock, Triton X-100 treatment or K⁺ ionophores increased the K⁺-ATPase activity in isotonic conditions, but $\text{K}{}^{\mathrm{+}}\text{-p-}\text{nitrophenyl}$ phosphatase is not affected by these treatments, nor is the ATPase activity in the presence of NH₄⁺. The results suggest that the electrophoretic fraction contains a major population of tight vesicles, whose permeability to K⁺ is rate limiting for the ATPase activity but not for the $\text{p-}\text{nitrophenyl}$ phosphatase activity. It is concluded that K⁺ site for the ATPase is internal whereas the K⁺ site for the $\text{p-}\text{nitrophenyl}$ phosphatase is external, hence, the K⁺ site must be mobile across the membrane.     

Ammonium molybdate-stannous chloride determination of orthophosphate in the presence of triton X-100

A colorimetric method for the determination of orthophosphate in the presence of Triton X-100 and the extent of their mutual interference is presented. Effects of albumin and trichloroacetic acid on the reaction are also examined. The method is based on the very sensitive reaction developed for determination of orthophosphate by complex formation with ammonium molybdate followed by reduction with stannous chloride. The method allows determination of 0.005 μmol of orthophosphate in the presence of up to 0.5% Triton X-100 and as little as 0.3% ($\text{v}\text{v}$) Triton X-100 in the absence of phosphate.     

Role of phospholipid and protein-protein associations in activation and stabilization of soluble Ca2+-ATPase of sarcoplasmic reticulum

The effect of increasing concentrations of the nonionic detergent Triton X-100 on catalytic activity, stability, phospholipid content, and aggregational state of solubilized Ca2+ ion activated adenosinetriphosphatase (Ca2+-ATPase) of sarcoplasmic reticulum has been investigated. Increasing concentrations of Triton X-100 in the range 0.2-0.6% (w/v) inhibited ATP hydrolysis and p-nitrophenyl phosphate hydrolysis in parallel to the extent of 50% and 95%, respectively. Inactivation of p-nitrophenyl phosphate hydrolysis by preincubation in excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) at 25 degrees C was monophasic and first order at all concentrations of Triton X-100. The rate constant for inactivation increased sharply in the range 0.1-0.6% Triton X-100. At higher concentrations, the increase was less marked. Protein-protein associations of the solubilized ATPase were assessed by glutaraldehyde cross-linking and by ultracentrifugation in sucrose gradients. Both methods indicated a decrease in these associations in the 0.1-0.5% range. Cross-linking studies established that above 0.5% Triton X-100 the enzyme is greater than 90% monomeric. The amount of phospholipid associated with the ATPase, recovered from sucrose gradients, decreased from about 50 mol of phospholipid/mol of ATPase at 0.1% Triton X-100 to about 3 mol of phospholipid/mol of ATPase at 0.5% and higher concentrations. Monomeric ATPase and aggregated ATPase isolated from equilibrium mixtures of these components had similar phospholipid/protein ratios. The results indicated that with increasing Triton X-100 concentrations, inhibition of catalysis, destabilization, loss of protein-protein associations, and loss of phospholipid occur concurrently.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variations in Ca(2+)-mediated activation of Ca(2+)-ATPase and its associated inhibitor in erythrocyte membrane.

1. Two distinct patterns of Ca(2+)-mediated activation of Ca(2+)-ATPase were identified in calmodulin-depleted membranes. 2. In membranes showing no activation (type A), preincubation with micromolar concentration of cyclic AMP and ATP made possible stimulation of the enzyme while in membranes already exhibiting activation (type B), preincubation with cyclic AMP and ATP abolished the activation. 3. ATPase stimulation in type A membranes was suppressible by leupeptin. 4. Triton extractable inhibitor isolated from type A membranes was as active as that derived from type B membranes only after preincubating the membranes with cyclic AMP and ATP. 5. The inhibitor could be inactivated by alkaline phosphatase.     

Lysosomal membrane adenosine triphosphatase; solubilization and partial characterization

Membranes prepared from Triton WR-1339-filled lysosomes (tritosomes) contained ATPase activity with a pH optimum of 5–8. These membranes also showed adenosine diphosphatase, adenosine monophosphatase, acid β-glycerol phosphatase, and acid pyrophosphatase activities. The soluble (nonmembrane) fraction of the tritosomes also contained these activities, but the properties of the soluble adenine nucleotide phosphatase activities were different from the membrane-associated enzymes. The pH optimum of tritosomal membrane ATPase changed to 5 after solubilization with Triton X-100, but ADPase and AMPase optima remained at 6–7. The pH optimum of intact membrane ATPase was also 5 when the substrate was α,β-methylene-ATP. Thus, tritosomal membrane ATPase apparently exhibits a pH 8 optimum only when acting in concert with ADPase and AMPase in intact membranes. Rates of ATP hydrolysis to adenosine were also significantly greater in intact membranes than in Triton X-100-solubilized fraction. Centrifugation of Triton X-100-solubilized tritosomal membranes in sucrose density gradients showed that ATPase and ADPase activities sedimented to one peak, and that AMPase, acid phosphatase, and pyrophosphatase were grouped in another peak. Thus, tritosomal membrane ATPase activity was not due to the latter enzymes. The resulting purification was about fourfold for ATPase. The Mr for ATPase and ADPase was estimated to be about 65,000 and for AMPase, acid phosphatase, and pyrophosphatase about 200,000.     

Phosphorylation of external cell-surface proteins by an endogenous ecto-protein kinase of goat epididymal intact spermatozoa

Intact spermatozoa from goat cauda epididymides possess an ecto-(cyclic AMP-independent protein kinase) activity that causes transfer of the terminal phosphate of exogenously added [gamma-32P]ATP to the serine and threonine residues of several endogenous plasma-membrane phosphoproteins located on the external cell surface. Cyclic AMP, cyclic GMP, calmodulin and muscle cyclic AMP-dependent protein kinases I and II had no appreciable effect on the rate of phosphorylation of ecto-proteins by the intact cells. The ecto-enzyme is not derived from the catalytic subunit of a cyclic AMP-dependent kinase. Sperm ecto-kinase activity is not due to contamination of broken cells or any possible cell damage during incubation and isolation of spermatozoa. The phosphorylation reaction was linear for approx. 1 min and there was no detectable uptake of ATP by these cells. The activity of the ecto-kinase was strongly inhibited by proteinases and by the membrane-nonpenetrating surface probes. The products of the reaction were associated with the intact cells and the 32P of the labelled cells was largely lost when treated with Triton X-100 or proteinases: trypsin and pronase. These data are consistent with the view that the observed protein kinase and the phosphoproteins are located on the external surface of spermatozoa. Vigorously forward-motile whole spermatozoa showed a relatively high capacity to phosphorylate ecto-proteins that undergo rapid turnover. The results suggest the occurrence of a novel coupled-enzyme system (ecto-protein kinase and phosphoprotein phosphatase) on the sperm external surface that may modulate sperm physiology by determining the phosphorylated states of the ecto-proteins.     

Activation of chloride conductance in pig jejunal brush border vesicles

Summary Requirements for the activation of Cl conductance have been investigated in pig jejunal brush border vesicles. The stability of ATP as a substrate for protein kinase activity, the stability of the phosphoprotein product of protein kinase action, and the choice of buffer system used for vesicle preparation were studied as variables which affected the outcome of in vitro activation attempts. Arsenate was selected as the most effective agent in protecting ATP from hydrolysis by the phosphatase activity in this vesicle system. Brush border vesicle protein appeared to prevent the accumulation of phosphoprotein in a cAMP-dependent protein kinase reaction, and vesicle protein only had phosphate acceptor activity when KF was added as a presumptive inhibitor of phosphoprotein phosphatase.A Cl conductance response to a potassium gradient and valinomycin was present in vesicles prepared in buffers containing tetramethylammonium. Cl^– conductance activity was not increased in this system by the addition of ATP, dibutyryl cyclic AMP, and cyclic AMP-dependent protein kinase.There was no Cl conductance response to a potassium gradient in vesicles buffered with imidazolium-acetate. Incorporation of ATP, AsO ₄ ^3– , and F^– into these nonconductive vesicles by homogenization, followed by addition of dibutyryl cAMP, produced substantial conductance activity. Maximal activation of Cl^– conductance was obtained with vesicles prepared in imidazolium-acetate buffering, using precautions to stabilize ATP and phosphoprotein prior to conductance measurements.     

The dextro and levorotatory isomers of N-phenylisopropyladenosine: stereospecific effects on cyclic AMP-formation and evoked synaptic responses in brain slices.

The stereoisomers of N⁶-phenylisopropyladenosine elicit accumulations of cyclic AMP in brain slices via interaction with adenosine-receptors. The response in guinea pig cerebral cortical slices and in rat hippocampal slices is blocked by theophylline and potentiated by biogenic amines. A chelator, EGTA, potentiates the response to phenylisopropyladenosine in guinea pig cerebral cortical slices. The $\text{1}$-isomer (EC₅₀ 25 μM) is four- to five-fold more potent than the $\text{d}$-isomer in eliciting accumulations of cyclic AMP in brain slices. In a rat coronal hippocampal slice $\text{in vitro}$, $\text{1}$-phenylisopropyladenosine (IC₅₀ ～ 0.7 μM) reduces the amplitude of evoked synaptic responses generated $\text{via}$ a monosynaptic pathway to the CA1 pyramidal neurons. The $\text{d}$-isomer is nearly one hundred-fold less potent. Thus, the adenosine-receptors involved in the electrophysical response appear much more stereoselective for the $\text{1}$-isomer of phenylisopropyladenosine than the adenosine-receptors involved in cyclic AMP-generation in brain slices.     

Phosphorylation of guinea pig cardiac natural actomyosin and its effect on ATPase activity.

Guinea pig cardiac natural actomyosin incubated with commercial protein kinase, Mg²⁺-ATP, and cyclic AMP produced little or no change in actomyosin ATPase activity. However, addition of sodium fluoride, a known phosphatase inhibitor resulted in a decreased actomyosin ATPase at all measured calcium concentrations. The presence of phosphatase activity in actomyosin and protein kinase was confirmed with ?-nitrophenyl phosphate. These results indicate the importance of inhibiting phosphatase activity, particularly when measuring biological or enzymatic activity as a function of phosphorylation.