Phosphorylation of synaptic-membrane proteins from ox cerebral cortex in vitro. Preparation of fractions enriched in phosphorylated proteins by using extraction with detergents and urea, and gel filtration.

Synaptic-membrane fragments from ox cerebral cortex contain basal and cyclic AMP-stimulated protein kinase(s) that transfer 32P from [gamma-32P]ATP to hydroxyl groups of serine and threonine residues in membrane-protein substrates. In the present work, labelled membrane fragments were partitioned into soluble and insoluble fractions with Triton X-100, Nonidet P. 40, sodium deoxycholate and urea, and the distribution of 32P-labelled protein in the fractions was determined by polyacrylamide-gel electrophoresis and radioautography. A high percentage of phosphorylated protein sustrates remained insoluble, including those whose phosphorylation was most highly stimulated by cyclic AMP. Whole membrane fragments and samples prepared by detergent extraction were fractionated on Sepharose 6B columns in the presence of low concentrations of sodium dodecyl sulphate and pooled fractions were analysed by polyacrylamide-gel electrophoresis and radioautography. Phosphorylated proteins were fractionated on the basis of their molecular weight, but homogeneous protein was not obtained. The results are discussed in relation to the techniques used and the results obtained in other laboratories.     

Phosphorylation of triton X-100 soluble and insoluble protein substrates in a demembranated/reactivated human sperm model

Sperm motility is a process which involves a cascade of events mediated by cAMP and Ca²⁺, cAMP in the initiation of flagellar movement, and Ca²⁺ in the regulation of beat asymmetry, and it has been suggested that these two messengers act through phosphorylation/dephosphorylation of axonemal proteins. Only a few studies on human sperm protein phosphorylation have been reported and no relation of this process with motility or other function has been established. In the present study, phosphorylation of human sperm proteins was performed using detergent-demembranated spermatozoa, in which motility is reactivated by the addition of ATP. This system allows direct accessibility of intracellular kinases to [³²P]-γATP and allows some relation between protein phosphorylation and flagellar movements. After electrophoresis and autoradiography, numerous phosphoproteins were detected. Phosphorylation of 2 proteins (36 and 51 kDa) was stimulated by cAMP in a concentration-dependent manner, and this increase was prevented by inhibitors of cAMP-dependent protein kinase. In order to characterize phosphoproteins originating from the cytoskeleton or axoneme, detergent extracted spermatozoa were also subjected to phosphorylation. Three major phosphorylated proteins (14.8, 15.3, and 16.2 kDa) were detected, the first two expressing cAMP-dependency according to their cAMP concentration-dependent increase in phosphorylation and the reversal of this effect by inhibitors of cAMP-dependent protein kinase. Proteins phosphorylation during the reactivation of demembranated spermatozoa previously immobilized H₂O₂, xanthine + xanthine oxidase-generated reactive oxygen species, or the oxidative phosphorylation uncoupler rotenone, revealed increases in cAMP-independent phosphorylation of proteins of 16.2, 46, and 93 kDa. These results documenting human sperm phosphoproteins form a base for further studies on the role of protein phosphorylation in sperm functions. © 1996 Wiley-Liss, Inc.     

Phosphorylation of ribosomal proteins in rat cerebral cortex in vitro.

Incubation of cerebral cortical tissue from immature rats in the presence of [32P]orthophosphate resulted in similar rates of incorporation of radioactivity into the proteins of free and membrane-bound ribosomes. Incorporation of label into ribosomal proteins of both species continued actively for at least 3 hours. Since recovery of membrane-bound ribosomes from rat cerebral cortex is quite low, further analyses of the radioactive phosphoproteins were restricted to the free ribosome population. A significant fraction of the radioactivity which was precipitated with trichloroacetic acid was not removed by heating in trichloroacetic acid at 90 degrees or extracted with organic solvents and therefore was presumed to be covalently bound to protein. The radioactive phosphoryl groups present in the ribosomal proteins were mainly in ester linkages since they were readily removed by exposure to 1 N NaOH, relatively unaltered by 1N HCl, and unaffected by hydroxylamine. This conclusion was supported by the isolation of labeled o-phosphoserine and o-phosphothreonine residues from hydrolysates of ribosomal proteins. A significant fraction of the labeled phosphoproteins in the purified ribosomes appeared to be bound tightly to the ribosome structure since only 40% of the radioactivity could be removed by extraction of these ribosomes with 1 M KCl. Phosphorylation of proteins of cerebral monoribosomes was more rapid than the same process in polyribosomes from the same source. Eight radioactive phosphoprotein bands could be detected by electrophoresis of proteins obtained from unfractionated cerebral ribosomes on unidimensional polyacrylamide gels containing sodium dodecyl sulfate. The protein nature of these materials was confirmed by pronase digestion. Proteins of subribosomal particles isolated from the total free ribosomal population were labeled differentially. When dissociation was carried out in the presence of EDTA, the small subunit contained four radioactive phosphoprotein bands, whereas the large subunit contained five. Three of the radioactive phosphoprotein components of the small subunit were removed when dissociation of cerebral ribosomes which were previously washed with high salt media was carried out in the presence of puromycin and high salt. However, only the largest labeled phosphoprotein band of the large subunit was removed by this procedure. This component exhibited the same electrophoretic mobility as one of the radioactive phosphoprotein bands which was removed from the small subunit by high salt treatment..     

Assay of mitochondrial membrane-bound enzyme activities in the presence of triton X-100.

Mitochondrial ATPase and cytochrome c oxidase activities are not severely affected by Triton X-100 concentrations between 0.1 and 2.0% (w/v). The former is solubilized by the effect of the detergent, while the latter is not. Succinate: cytochrome c reductase and rotenone-sensitive NADH: cytochrome c reductase activities are destroyed even a low detergent concentrations. Succinate:coenzyme Q oxidoreductase is affected by the surfactant in a more complex way, so that selective solubilization of some subunit(s) could be involved.     

Phosphorylation and protein kinase activities of chromosomal non histone proteins from chick embryo fibroblasts.

Confluent chick embryo fibroblasts were cultured in vitro in (i) medium which prevented the cells from dividing, (ii) medium which stimulated the cells to divide synchronously, (iii) medium without lysine in which the cells were blocked in G1. Chromosomal non histone proteins (NHP) were extracted from cells pulse labelled with 32P phosphate, and the radioactivity analyzed by acrylamide gel electrophoresis. Several radioactive peaks were found all along the gel in the NHP from confluent and stimulated cells. The highest phosphorylation was found in the fast moving proteins, but the stimulation of the cells increases the phosphorylation of the slower moving proteins. In the NHP from cells cultured in the medium without lysine only the slow migrating proteins were phosphorylated. NHP were extracted from unlabelled cell cultures in the three different media, incubated with [gamma-32P] ATP and analyzed by acrylamide gel electrophoresis. Highly labelled peaks were observed in the fast moving proteins from stimulated cells and from cells cultured in a medium deprived from lysine. By comparing in vivo and in vitro phosphorylation, it can be concluded that in confluent cells the turnover of bound phosphate is slow. In stimulated cells there is a fast turnover of the phosphate bound to fast turnover of the phosphate bound to a small group of fast migrating proteins and very little turnover of the phosphate bound to slow migrating proteins. The cells were incubated with labelled lysine and NHP analyzed by gel electrophoresis. The radioactivity of individual NHP varied with the culture conditions, but in all cases, there was little radioactivity in the fast moving proteins. The phosphate groups submitted to a fast turnover are bound to stable proteins. Phosvitin and casein kinase activities were measured in the NHP fractions. Nine-ten peaks of activities were observed with each substrate. Some variations were observed which apparently correlate with the culture conditions.     

Cytoskeletal-associated protein kinase and phosphatase activities from cerebral cortex of young rats

We describe the phosphorylation system associated with the Triton-insoluble cytoskeletal fraction that phosphorylates in vitro the 150 kDa neurofilament subunit (NF-M) and alpha and beta tubulin from cerebral cortex of rats. The protein kinase activities were determined in the presence of 20 M cyclic AMP (cAMP), 1 mM calcium and 1 M calmodulin (Ca²⁺/calmodulin) or 1 mM calcium, 0.2 mM phosphatidylserine and 0.5 M phorbol 12,13-dibutyrate (Ca²⁺/PS/PDBu). Phosphorylation of these cytoskeletal proteins increased approximately 35% and 65% in the presence of cAMP and Ca²⁺/calmodulin, respectively, but was unaffected in the presence of Ca²⁺/PS/PDBu. Basal phosphorylation of these proteins studied increased approximately 35% and 72% in the presence of 0.5 M okadaic acid and 0.01 M microcystin-LR, respectively, suggesting the presence of phosphatase type 1. Results suggest that at least two protein kinases and one protein phosphatase are associated with the Triton-insoluble cytoskeletal fraction from cerebral cortex of rats.     

A simple and rapid procedure for removal of triton X-100 from protein solution

A simple and rapid procedure for removal of Triton X-100 from protein solutions is described. The procedure is based on the extraction of Triton X-100 with chloroform from the protein solution. By the addition of sodium dodecyl sulfate before the extraction, the procedure was improved effectively and the sample thus prepared was used directly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This method can be used for the removal of other nonionic detergents and for samples of larger size.     

Phorbol ester binding and activation of protein kinase C on triton X-100 mixed micelles containing phosphatidylserine

A mixed micellar assay for the binding of phorbol-esters to protein kinase C was developed to investigate the specificity and stoichiometry of phospholipid cofactor dependence and oligomeric state of protein kinase C (Ca2+/phospholipid-dependent enzyme) required for phorbol ester binding. [3H]Phorbol dibutyrate was bound to protein kinase C in the presence of Triton X-100 mixed micelles containing 20 mol % phosphatidylserine (PS) in a calcium-dependent manner with a Kd of 5 X 10(-9) M. The [3H]phorbol dibutyrate X protein kinase C . Triton X-100 . PS mixed micellar complex eluted on a Sephacryl S-200 molecular sieve at an Mr of approximately 200,000; this demonstrates that monomeric protein kinase C binds phorbol dibutyrate. This conclusion was supported by molecular sieve chromatography of a similar complex where Triton X-100 was replaced with beta-octylglucoside. Phorbol dibutyrate activation of protein kinase C in Triton X-100/PS mixed micelles occurred and was dependent on calcium. The PS dependence of both phorbol ester activation and binding to protein kinase C lagged initially and then was highly cooperative. The minimal mole per cent PS required was strongly dependent on the concentration of phorbol dibutyrate or phorbol myristic acetate employed. Even at the highest concentration of phorbol ester tested, a minimum of 3 mol % PS was required; this indicates that approximately four molecules of PS are required. [3H]Phorbol dibutyrate binding was independent of micelle number at 20 mol % PS. The phospholipid dependencies of phorbol ester binding and activation were similar, with PS being the most effective; anionic phospholipids (cardiolipin, phosphatidic acid, and phosphatidylglycerol were less effective, whereas phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin did not support binding or activation. sn-1,2-Dioleoylglycerol displaced [3H]phorbol dibutyrate quantitatively and competitively. The data are discussed in relation to a molecular model of protein kinase C activation.     

Phosphorylation and protein kinase activities of chromosomal non histone proteins from chick embryo fibroblasts

Confluent chick embryo fibroblasts were cultured in vitro in (i) medium which prevented the cells from dividing, (ii) medium which stimulated the cells to divide synchronously, (iii) medium without lysine in which the cells were blocked in G₁.Chromosomal non histone proteins (NHP) were extracted from cells pulse labelled with ³²P phosphate, and the radioactivity analyzed by acrylamide gel electrophoresis. Several radioactive peaks were found all along the gel in the NHP from confluent and stimulated cells. The highest phosphorylation was found in the fast moving proteins, but the stimulation of the cells increases the phosphorylation of the slower moving proteins. In the NHP from cells cultured in the medium without lysine only the slow migrating proteins were phosphorylated.NHP were extracted from unlabelled cell cultures in the three different media, incubated with [γ-³²P] ATP and analyzed by acrylamide gel electrophoresis. Highly labelled peaks were observed in the fast moving proteins from stimulated cells and from cells cultured in a medium deprived from lysine.By comparing in vivo and in vitro phosphorylation, it can be concluded that in confluent cells the turnover of bound phosphate is slow. In stimulated cells there is a fast turnover of the phosphate bound to fast migrating proteins and a slow turnover of the phosphate bound to slow migrating proteins. In cells cultured in a medium without lysine there is a very fast turnover of the phosphate bound to a small group of fast migrating proteins and very little turnover of the phosphate bound to slow migrating proteins.The cells were incubated with labelled lysine and NHP analyzed by gel electrophoresis. The radioactivity of individual NHP varied with the culture conditions, but in all cases, there was little radioactivity in the fast moving proteins. The phosphate groups submitted to a fast turnover are bound to stable proteins.Phosvitin and casein kinase activities were measured in the NHP fractions. Nine-ten peaks of activities were observed with each substrate. Some variations were observed which apparently correlate with the culture conditions.     

Cellular lysis of Streptococcus faecalis induced with triton X-100.

Lysis of exponential-phase cultures of Streptococcus faecalis ATCC 9790 was induced by exposure to both anionic (sodium dodecyl sulfate) and nonionic (Triton X-100) surfactants. Lysis in response to sodium dodecyl sulfate was effective only over a limited range of concentrations, whereas Triton X-100-induced lysis occurred over a broad range of surfactant concentrations. The data presented indicate that the bacteriolytic response of growing cells to Triton X-100: (i) was related to the ratio of surfactant to cells and not the surfactant concentration per se; (ii) required the expression of the cellular autolytic enzyme system; and (iii) was most likely due to an effect of the surfactant on components of the autolytic system that are associated with the cytoplasmic membrane. The possibility that Triton X-100 may induce cellular lysis by releasing a lipid inhibitor of the cellular autolytic enzyme is discussed.     

Effects of triton X-100 treatments on the composition and activities of membrane vesicles from pigeon erythrocytes

Membrane vesicles were prepared from pigeon erythrocytes. The effect of various treatments on the ability of these vesicles to trap and transport glycine was measured. Most of the work concerned the effects of the nonionic detergent, Triton X-100 (Triton).Triton inhibits the capacity of membrane vesicles to trap and transport glycine. The effects of Triton depend on temperaature and pH. At neutral pH and 41°C, the half-inactivating dose of Triton is 6 μl/g wet weight of membrane, while at neutral pH and O°C it is approx. 60 μl/g. At pH 8.5–8.8 and 0°C the half-inactivating dose is 15–20 μl/g. The Triton effect depends on the ratio of Triton to membrane, not the Triton ‘concentration’ in the aqueous phase.Protein is released from the membrane by Triton treatment at 0°C. At pH 8.5–8.8, the dose-response curve for protein release is very similar to the dose-response curve for inhibition of the capacities to trap and take up glycine. All three curves have steep and shallow regions, respectively below and above a Triton dose of 15 μl/g. The protein released by Triton treatment at 0°C is largely the lower molecular weight classes and the maximum protein release is 50–60% of the total membrane protein. The percent of this protein class released by a given Triton dose equals the percent inhibition of the capacity to trap glycine.Triton is bound by the membranes. In the dose range of 0–10 μl Triton/g wet membrane, about half of the added Triton is bound. This bound Triton is not readily removed by washing. Like protein release and inhibition of trapping and transport capacities, the binding process has two phases, one above and one below a dose of 15 μl/g. The membrane is saturated with Triton at a dose of 15 μl/g with 4.5 μl Triton bound/g wet weight, corresponding to 85–90 μl bound/g remaining membrane protein.Bovine serum high density lipoprotein efficiently removes bound Triton from the membrane. By removing Triton bound at 0°C with lipoprotein, the effects of Triton at 0 and 41°C could be distinguished and the time course of Triton action at 0°C could be measured. Triton action was nearly complete by 10 min at 0°C.Possible modes of action of Triton on these membranes are discussed. At least part of the action of Triton can be described as a process in which membrane proteins are partitioned between the membrane phase and an extramembranal Triton micelle phase.     

Removal of triton X-100 from aqueous solution using amberlite XAD-2.

Amberlite XAD-2 beads adsorb the nonionic detergent Triton X-100 over a wide range of conditions with a maximum capacity of 0.475 mmol/g dry weight. After treatment of protein solutions containing Triton X-100 with XAD-2 no interference by Triton with Folin assays was observed. Adsorption of Triton X-100 was favored by a free-energy change of ?835 cal/mol and by a positive entropy value. Adsorbed Triton could be completely desorbed and the XAD-2 regenerated with little loss in capacity by washing with propan-2-ol. Removal of Triton by XAD-2 was also successful in columns or bateh-wise on a pilot-plant scale.     

Phosphorylation of diacylglycerol kinase in vitro by protein kinase C.

We investigated the effects of enzyme phosphorylation in vitro on the properties of diacylglycerol kinase. Diacylglycerol kinase and protein kinase C, both present as Mr-80,000 proteins, were highly purified from pig thymus cytosol. Protein kinase C phosphorylated diacylglycerol kinase (up to 1 mol of 32P/mol of enzyme) much more actively than did cyclic AMP-dependent protein kinase. Phosphorylated and non-phosphorylated diacylglycerol kinase showed a similar pI, approx. 6.8. Diacylglycerol kinase phosphorylated by either protein kinase C or cyclic AMP-dependent protein kinase was almost exclusively associated with phosphatidylserine membranes. In contrast, soluble kinase consisted of the non-phosphorylated form. The catalytic properties of the lipid kinase were not much affected by phosphorylation, although phosphorylation-linked binding with phosphatidylserine vesicles resulted in stabilization of the enzyme activity.     

Evidence that cAMP-dependent protein kinase and a protein factor are involved in reactivation of triton X-100 models of sea urchin and starfish spermatozoa

A fraction obtained from detergent-extract of sea urchin or starfish spermatozoa using DEAE-cellulose chromatography reactivated Triton X-100 models of the spermatozoa in a cAMP-dependent manner. The DEAE fraction contained cAMP-dependent protein kinase with a high level of specific activity. Rabbit muscle inhibitor protein highly specific for cAMP-dependent protein kinases inhibited the ability of the deae fraction to induce reactivation of Triton X-100 models.l This inhibition paralleled inhibition of cAMP-dependent protein kinase activity of the DEAE fraction, suggesting participation of the enzyme in the cAMP-dependent reactivation of Triton X-100 models. However, cAMP-dependent protein kinase further purified from the DEAE fraction was incapable of reactivating these models by itself. A protein factor which was separated from the protein kinase in the course of purification of the enzyme was found to also be necessary for the reactivation. When cAMP-dependent protein kinase was pretreated with protein kinase inhibitor before addition of the protein factor, the reactivation of Triton X-100 models was no longer detected. However, after the protein factor had been incubated with cAMP and cAMP-dependent protein kinase, protein kinase inhibitor did not repress reactivation of Triton X-100 models. We propose that the reactivation needs phosphorylation of the protein factor by cAMP-dependent protein kinase.     

Characterization of triton X-100-solubilized prostaglandin E binding protein of rat liver plasma membranes.

Rat liver plasma membranes bind prostaglandins E1 and E2 (PGE) with high affinity and specificity. We have solubilized plasma membranes, prelabeled with radioactive PGE1, in water solutions of Triton X-100. We sedimented this material into sucrose density gradient containing H2O and D2O. From numerical integration of the sedimentation equation, taking explicitly into account the density and viscosity gradients present during the centrifugation, we have determined a value of s20,w = 5.6 to 5.7 X 10(-13) s and a partial specific volume, v = 0.80 to 0.81 cm3/g, for the PGE binding protein-Triton X-100 composed of 60% (w/w) protein and 40% (w/w) detergent. Gel filtration in water solutions of Triton X-100 gives a Stokes radius of 53 A for the complex. These data imply a molecular weight of 105,000 for the detergent-free binding protein and a frictional ratio of 1.3 for the complex. If the detergent is bound to the protein in a monolayer, about 40% of the PGE binding protein's surface would be covered with detergent. The procedures used in the analysis of the sedimentation behavior of the PGE binding protein-detergent complex, when coupled with a gel filtration measurement of the Stokes radius, allow valid determination of the size, shape, and extent of detergent binding of a wide variety of membrane proteins, even when they are present as minor components of complex mixtures.     

Protein kinase associated with peripheral nerve myelin. 1. Phosphorylation of endogenous myelin proteins and exogenous substrates

When highly purified myelin from rat sciatic nerve was incubated with [γ-³²P]ATP, protein components of the membrane were phosphorylated indicating the presence of both the substrate (receptor protein) and an endogenous kinase in the membrane. Polyacrylamide gel electrophoresis of the phosphorylated membrane proteins followed by scintillation counting of gel slices and autoradiography showed that the polypeptides of molecular weights 28000, 23000 and 19000 were phosphorylated, and ³²P from [γ-³²P]ATP having been incorporated into serine residues of the substrate proteins. Phosphorylation of purified myelin was Mg²⁺-dependent, was optimal at pH 6.5 and was not stimulated by adenosine 3′,5′-monophosphate. We found that proteins other than those in myelin, such as phosvitin, casein, protamine and histones, can also act as a substrate for the membrane associated kinase. Muscle protein kinase inhibitor had no effect on the endogenous phosphorylation of myelin proteins or on the phosphorylation of phosvitin by peripheral nerve myelin protein kinase. However, the phosphorylation of histone by peripheral nerve myelin protein kinase was inhibited by the protein kinase inhibitor. After washing the membrane with 150 mM KCl the protein kinase that utilizes histone as substrate was found in the supernatant. In contrast, the endogenous phosphorylation of membrane proteins or the phosphorylation of phosvitin by the membrane associated kinase was not affected by washing.From these findings we conclude that at least two protein kinase systems exist in purified peripheral nerve myelin. One system is not inhibited by muscle kinase inhibitor, is tightly bound to the membrane and utilizes as its receptor proteins either exogenous phosvitin or endogenous membrane proteins. The second system is inhibited by muscle kinase inhibitor, is removable from the membrane and utilizes histones as its receptor proteins.     

The ribosomal proteins phosphorylated in vitro by protein kinase activities from Krebs II ascites cells

Studies were performed to identify in cytoplasmic extracts of Krebs II ascites cells protein kinase activities that might be responsible for the phosphorylation of the ribosomal proteins previously identified as phosphoproteins in these cells in vivo. Column chromatography resolved a casein kinase activity that could use ATP or GTP as a phosphoryl donor to phosphorylate, in ribosomes, exclusively the acidic 60S phosphoprotein(s) phosphorylated in vivo. A second casein kinase fraction could use ATP, only, in a similar reaction, but also contained protein kinase activity with respect to other ribosomal proteins, including the basic ribosomal protein phosphorylated in vivo, ribosomal protein S6. This latter was also among several proteins phosphorylated by an activity in the cyclic AMP-independent histone kinase fraction.