Studies on the incorporation of [32P]phosphate into pyruvate dehydrogenase in intact rat fat-cells. Effects of insulin.

1. Intact rat epididymal fat-cells were incubated with 32Pi, and the intracellular proteins were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. One of the separated bands of phosphorylated proteins had an apparent subunit mol.wt. of 42 000, which is the same as that of the alpha-subunit of the pyruvate dehydrogenase complex. By using a combination of subcellular fractionation, immunoprecipitation with antiserum raised against pyruvate dehydrogenase complex and two-dimensional electrophoresis it was apparent that the incorporation into alpha-subunits accounted for 35--45% of the total incorporation into this band of phosphoproteins. 2. The increase in the initial activity of pyruvate dehydrogenase that follows brief exposure of fat-cells to insulin was shown to be associated with a decrease in the steady-state incorporation of 32P into the alpha-subunits of pyruvate dehydrogenase. 3. Tryptic peptide analysis of pyruvate dehydrogenase [32P]phosphate, labelled in intact fat-cells, indicated that three serine residues on the alpha-subunit were phosphorylated, corresponding to the three sites phosphorylated when purified pig heart pyruvate dehydrogenase was incubated with [gamma-32P]ATP. The relative phosphorylation of all three serine residues appeared to be similar in 32P-labelled alpha-subunits in both control and insulin-treated fat-cells.     

Control of pyruvate dehydrogenase activity in intact cardiac mitochondria. Regulation of the inactivation and activation of the dehydrogenase.

The control of pyruvate dehydrogenase activity by inactivation and activation was studied in intact mitochondria isolated from rabbit heart. Pyruvate dehydrogenase could be completely inactivated by incubating mitochondria with ATP, oligomycin, and NaF. This loss in dehydrogenase activity was correlated with the incorporation of 32P from [gamma-32P]ATP into mitochondrial protein(s) and with a decrease in the mitochondrial oxidation of pyruvate. ATP may be supplied exogenously, generated from endogenous ADP during oxidative phosphorylation, or formed from exogenous ADP in carbonyl cyanid p-trifluoromethoxyphenylhydrazone-uncoupled mitochondria. With coupled mitochondria the concentration of added ATP required to half-inactivate the dehydrogenase was 0.24 mM. With uncoupled mitochondria the apparent Km was decreased to 60 muM ATP. Inactivation of pyruvate dehydrogenase by exogenous ATP was sensitive to atractyloside, suggesting that pyruvate dehydrogenase kinase acts internally to the atractyloside-sensitive barrier. The divalent cation ionophore, A23187, enhanced the loss of dehydrogenase activity. Pyruvate dehydrogenase activity is regulated additionally by pyruvate, inorganic phosphate, and ADP. Pyruvate, in the presence of rotenone, strongly inhibited inactivation. This suggests that pyruvate facilitates its own oxidation and that increases in pyruvate dehydrogenase activity by substrate may provide a modulating influence on the utilization of pyruvate via the tricarboxylate cycle. Inorganic phosphate protected the dehydrogenase from inactivation by ATP. ADP added to the incubation mixture together with ATP inhibited the inactivation of pyruvate dehydrogenase. This protection may result from a direct action on pyruvate dehydrogenase kinase, as ADP competes with ATP, and an indirect action, in that ADP competes with ATP for the translocase. It is suggested that the intramitochondrial [ATP]:[ADP] ratio effects the kinase activity directly, whereas the cytosolic [ATP]:[ADP] ratio acts indirectly. Mg2+ enhances the rate of reactivation of the inactivated pyruvate dehydrogenase presumably by accelerating the rate of dephosphorylation of the enzyme. Maximal activation is obtained with the addition of 0.5 mM Mg2+..     

Mitochondrial protein phosphorylation and cardiomyopathy in genetically diabetic mice: the effect of estrone treatment

The alpha-subunit of pyruvate dehydrogenase and succinyl-CoA synthetase are phosphorylated after incubation of cardiac mitochondria from genetically diabetic mice with [gamma-32P]ATP. There is significantly increased incorporation of 32P into pyruvate dehydrogenase from diabetic mice when compared to controls. The enhanced rate of pyruvate dehydrogenase phosphorylation correlates well with the previously reported defective oxidative metabolism and decreased activity of this enzyme from diabetic mice. The relationship between abnormal mitochondrial function and development of cardiomyopathy in the diabetic mice has been studied further by in vivo estrone treatment. The results indicate that ultrastructural alterations of myocardium are closely associated with the defective pyruvate oxidation (via phosphorylation of pyruvate dehydrogenase) and both processes can be prevented by 7-12 weeks estrone treatment.     

An electrophoretic study of endogenous phosphorylation in vitro of the polypeptides of microsomal membrane fractions of mouse liver

1. The patterns of phosphopolypeptides produced by endogenous phosphorylation in vitro of rough- and smooth-membrane fractions of the microsomal fraction of mouse liver were studied by radioautographic analysis of sodium dodecyl sulphate/polyacrylamide-gel electrophoretograms. 2. A minimum of 17 polypeptides of both rough- and smooth-microsomal-membrane fractions were phosphorylated by using [gamma-(32)P]-ATP as the phosphate donor; only minor differences in phosphorylation pattern between the two membrane fractions were detected. 3. Phosphorylation in vitro by [gamma-(32)P]ATP was markedly stimulated by Mg(2+), but not by cyclic AMP, cyclic GMP or Ca(2+). The phosphorylation of certain polypeptides was preferentially stimulated by Mg(2+). Addition of cyclic AMP resulted in a decrease in the amount of (32)P detected in one polypeptide of mol.wt. approx. 56000, present in both the rough- and smooth-membrane fractions. 4. [gamma-(32)P]GTP was found to be a relatively poor donor of (32)P as compared with [gamma-(32)P]ATP. However, incubation of rough- and smooth-membrane fractions with this compound resulted in the phosphorylation of one polypeptide of mol.wt. approx. 96000 that was scarcely or not at all phosphorylated by [gamma-(32)P]ATP. 5. Under the conditions of incubation used, appreciable incorporation of (32)P from [gamma-(32)P]ATP occurred into products migrating at the front of the electrophoretograms; these products were identified as being principally comprised of 1-phosphatidylinositol 4-phosphate. Incorporation of (32)P into this lipid was also markedly stimulated by Mg(2+). 6. The overall results show that a considerable number of polypeptides of the rough- and smooth-microsomal-membrane fractions of mouse liver may be phosphorylated in vitro and indicate that the enzymes responsible are principally non-cyclic AMP-dependent protein kinases.     

Evidence for new phosphorylation sites for protein kinase C and cyclic AMP-dependent protein kinase in bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

Bovine heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) was phosphorylated by incubation with [gamma-32P]MgATP and cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). After digestion with chymotrypsin, the phosphorylation sites for the two protein kinases were identified by peptide mapping, and microsequencing. Evidence for new phosphorylation sites for PKA (Ser-483) and PKC (Ser-84 and Ser-466) was obtained.     

Protein phosphorylation by inorganic pyrophosphate in yeast mitochondria.

Inorganic pyrophosphate can function as phosphate donor in protein phosphorylation reactions in yeast mitochondria. It was shown that, when PPi substitutes for ATP as inhibitor of the pyruvate dehydrogenase reaction, maximal activity is reached after a lag-period of 30-60 minutes. 32P-labeling of peptides shows that [32P]PPi gives about 25% of the labeling obtained by [gamma-32P]ATP in the protein kinase reaction. The PPi dependent phosphorylation is increased several fold by the presence of cold ATP.     

Phosphorylation of rat hepatic fructose-1,6-bisphosphatase and pyruvate kinase

Fructose-1,6-bisphosphatase from rat liver was phosphorylated with cyclic AMP-dependent protein kinase and [gamma-32P]ATP. Brief exposure of the 32P-labeled enzyme to trypsin removed all radioactivity from the enzyme core and produced a single-labeled peptide. The partial sequence of the 17-amino acid peptide was found to be Ser-Arg-Pro-Ser(P)-Leu-Pro-Leu-Pro-(Ser2, Glx2, Pro2, Leu, Arg2). The kinetics of cyclic AMP-dependent protein kinase-catalyzed phosphorylation of native fructose bisphosphatase were compared with those of rat liver type L pyruvate kinase where the sequence around the phosphoserine is known (Arg-Arg-Ala-Ser(P)-Val; Hjelmquist, G., Anderson, J., Edlund, B., and Engstrom, L. (1974) Biochem. Biophys. Res. Commun. 61, 559-563). The Km for pyruvate kinase (17 microM) was less than that for fructose bisphosphatase (58 microM); the Vmax was about 3-fold greater with pyruvate kinase as substrate. The relationship between the rates of phosphorylation of these native substrates and the amino acid sequences surrounding the phosphorylated sites is discussed.     

Bovine brain Na+, K+-stimulated ATP phosphohydrolase studied by a rapid-mixing technique. Detection of a transient [32P]phosphoenzyme formed in the presence of potassium ions.

1. Conditions for binding of [gamma-32P]ATP to bovine brain Na+,K+-stimulated ATPase were investigated by the indirect technique of measuring the initial rate of 32P-labelling of the active site of the enzyme. 2. At 100 muM [gamma-32P]ATP in the presence of 3 mM MgCl2, approximately the same very high rate of formation of [32P]phosphoenzyme was obtained irrespective of whether [gamma-32P]ATP was added to the enzyme simultaneously with, or 70 ms in advance of the addition of NaCl. A comparatively slow rate of phosphorylation was obtained at 5 muM[gamma-32P]ATP without preincubation. However, on preincubation of the enzyme with 5 muM[gamma-32P]ATP a rate of formation of [32P]phosphoenzyme almost as rapid as at 100 muM[gamma-32P]ATP was observed. 3. A transient [32P]phosphoenzyme was discovered. It appeared in the presence of K+, under conditions which allowed extensive binding of [gamma-32P]-ATP. The amount of [gamma-32P]ATP that could be bound to the enzyme seemed to equal the amount of [32P] phosphorylatable sites. 4. The formation of the transient [32P] phosphoenzyme was inhibited by ADP. The transient [32P] phosphoenzyme was concluded mainly to represent the K+-insensitive and ADP-sensitive E1-32P. 5. When KCl was present in the enzyme solution before the addition of NaCl only a comparatively slow rate of phosphorylation was observed. On preincubation of the enzyme with [gamma-32]ATP an increase in the rate of formation of [32P] phosphoenzyme was obtained, but there was no transient [32P]-phosphoenzyme. The transient [32P]phosphoenzyme was, however, detected when the enzyme solution contained NaCl in addition to KCl and the phosphorylation was started by the addition of [gamma-32P]ATP.     

A method for the enzymatic synthesis and purification of [alpha-32P] nucleoside triphosphates.

A simplified method is described for the enzymatic synthesis and purification of [alpha-32P]ribo- and deoxyribonucleoside triphosphates. The products are obtained at greater than 97% radiochemical purity with yields of 50--70% (relative to 32Pi) by a two-step elution from DEAE-Sephadex. All reactions are done in one vessel as there is no need for intermediate product purifications. This method is therefore suitable for the synthesis of these radioactive compounds on a relatively large scale. The sequential steps of the method involve first the synthesis of [gamma-32P]ATP and the subsequent phosphorylation of nucleoside 3' monophosphate with T4 polynucleotide kinase to yield nucleoside 3', [5'-32P]diphosphate. Hexokinase is used after the T4 reaction to remove any remaining [gamma-32P]ATP. Nucleoside 3',[5'-32P]diphosphate is treated with nuclease P-1 to produce the nucleoside [5'-32P]monophosphate which is phosphorylated to the [alpha-32P]nucleoside triphosphate with pyruvate kinase and nucleoside monophosphate kinase. Adenosine triphosphate used as the phosphate donor for [alpha-32P]deoxynucleoside triphosphate syntheses is readily removed in a second purification step involving affinity chromatography on boronate-polyacrylamide. [alpha-32P]Ribonucleoside triphosphates can be similarly purified when deoxyadenosine triphosphate is used as the phosphate donor.     

Energy-dependent dissociation of ATP from high affinity catalytic sites of beef heart mitochondrial adenosine triphosphatase

Incubation of [gamma-32P]ATP with a molar excess of the membrane-bound form of mitochondrial ATPase (F1) results in binding of the bulk of the radioactive nucleotide in high affinity catalytic sites (Ka = 10(12) M-1). Subsequent initiation of respiration by addition of succinate or NADH is accompanied by a profound decrease in the affinity for ATP. About one-third of the bound radioactive ATP appears to dissociate, that is, the [gamma-32P]ATP becomes accessible to hexokinase. The NADH-stimulated dissociation of [gamma-32P]ATP is energy-dependent since the stimulation is inhibited by uncouplers of oxidative phosphorylation and is prevented by respiratory chain inhibitors. The rate of the energy-dependent dissociation of ATP that occurs in the presence of NADH, ADP, and Pi is commensurate with the measured initial rate of ATP synthesis in NADH-supported oxidative phosphorylation catalyzed by the same submitochondrial particles. Thus, the rate of dissociation of ATP from the high affinity catalytic site of submitochondrial particles meets the criterion of kinetic competency under the conditions of oxidative phosphorylation. These experiments provide evidence in support of the argument that energy conserved during the oxidation of substrates by the respiratory chain can be utilized to reduce the very tight binding of product ATP in high affinity catalytic sites and to promote dissociation of the nucleotide.     

Occupancy of sites of phosphorylation in inactive rat heart pyruvate dehydrogenase phosphate in vivo.

1. Inactive pyruvate dehydrogenase phosphate complexes were partially purified from hearts of fed, starved or alloxan-diabetic rats by using conditions that prevent phosphorylation or dephosphorylation. 2. Unoccupied sites of phosphorylation were assayed by incorporation of 32P from [gamma-32P]ATP into the complexes. Total sites of phosphorylation were assayed by the same method after complete reactivation, and thus dephosphorylation, of complexes by incubation with pyruvate dehydrogenase phosphate phosphatase. Occupancy is assumed from the difference (total sites--unoccupied sites). Percentage incorporation into individual sites was measured by high-voltage electrophoresis after tryptic digestion. 3. Values (means +/- S.E.M., in nmol of phosphate/unit of inactive complex) for total sites, occupied sites and percentage occupancies, with numbers of observations in parentheses were: fed, 2.1 +/- 0.04, 1.15 +/- 0.04, 54.8 +/- 1.6% (39); starved, 2.05 +/- 0.03, 1.85 +/- 0.03, 90.2 +/- 1.4% (28); alloxan-diabetic, 1.99 +/- 0.03, 1.72 +/- 0.03, 86.4 +/- 1.4% (68%). 4. Values (means +/- S.E.M. for percentage occupancy) for individual sites of phosphorylation in pyruvate dehydrogenase phosphate given in the order sites 1, 2 and 3 were : fed, 100 +/- 2.7, 27.8 +/- 1.6, 33.9 +/- .9; starved, 100 +/- 1.4, 76.2 +/- 2.0, 92.4 +/- 1.5; alloxan-diabetic, 100 +/- 1.2, 64.0 +/- 1.7, 94.6 +/- 1.4. 5. It is concluded that starvation or alloxan-diabetes leads to a 2--3-fold increase in the occupancy of phosphorylation sites 2 and 3 in pyruvate dehydrogenase phosphate in rat heart in vivo.     

Characterization of the kinase activity of bovine adrenal pyruvate dehydrogenase complex

In the presence of [gamma-32P]ATP the bovine adrenal pyruvate dehydrogenase complex accepts the label simultaneously and becomes inactivated. This suggests the existence of kinase in the composition of the complex as is typical of the complexes from other animal sources. The Pi is incorporated into the subunit with molecular weight of 42 000. The kinase activity of the adrenal pyruvate dehydrogenase complex is high: within the first 20 sec of incubation with ATP the inactivation is as high as 60%. The pH optimum for kinase is around 7.3. The apparent Km value for ATP with 50 mM KCl is 7 microM; that in the absence of KCl is 10 microM. ADP is a competitive inhibitor of kinase with respect to ATP (Ki = 100 microM), when K+ are present in the medium. Thiamine pyrophosphate and pyruvate decrease the rate of pyruvate dehydrogenase complex inactivation.     

Protein kinase activity of bovine retina rod outer segments

Dark-adapted pure bovine rod outer segments (ROS) (A280/A500--2.1) can be phosphorylated in the presence of [gamma-32P]ATP and [gamma-32P]GTP. The constant levels of phosphorylation, reached within 10--15 min, are 100 +/- 30 pmol 32P/nmol of rhodopsin for [gamma-32P]ATP and 2--4 pmol 32P/nmol of rhodopsin for [gamma-32P]GTP. These processes are not controlled by 10(-4)--10(-8) cAMP, cGMP or Ca2+, but are inhibited at higher concentrations of these agents. In the presence of histone the constant level of phosphorylation is increased up to 200 +/- 30 pmol 32P/nmol of rhodopsin for [gamma-32P]ATP, but is not changed when [gamma-32P]GTP is used. 10(-5) M cAMP is found to activate the phosphorylation in the presence of histone and [gamma-32P]ATP by 5--6 times. All this evidences that ROS contains cAMP-dependent protein kinase, which utilizes ATP, but not GTP. Moreover, ROS contains cyclic nucleotides- and Ca2+-independent protein kinase. These protein kinases are the ROS endogenous enzymes. This is shown in experiments on separation of pure ROS in a sucrose density gradient.     

Characterization of the major phosphoprotein and its kinase on the surface of the rat adipocyte

Intact rat fat cells exposed to 12.5 microM [gamma-32P]ATP incorporate label into specific proteins within minutes. By solubilizing the reaction mixture with SDS which by passes the subcellular fractionation steps, the labeled proteins can be identified in autoradiographs of SDS-PAGE gels. The most prominently labeled protein has an Mr of 42,000. Localization of this component to the cell surface can be made on the basis of inhibition of phosphorylation by addition of a protein derived from the rat brain with protein kinase inhibitory property, susceptibility of the phosphorylated protein to tryptic digestion, whereas the unphosphorylated protein is unaffected by digestion with trypsin (15 min), inhibition of phosphorylation of this protein after brief exposure to melittin, and the consistent observation that more label is associated with the 42,000 Mr band in homogenates and permeabilized cells than in comparable numbers of intact cells exposed to the same amount of label. A 42,000 Mr phosphoprotein is also present in mitochondria which is most likely the alpha subunit of pyruvate dehydrogenase. To rule out the possibility that the cell surface protein might be a mitochondrial contaminant from broken cells, 32Pi-labeled and [gamma-32P]ATP-labeled cells were solubilized with Triton and chromatographed on a rabbit anti-pyruvate dehydrogenase antibody-Sepharose 4B column. A single labeled peak was detected upon elution of the bound fraction only in the 32Pi-labeled sample, and not in the [gamma-32P]ATP-labeled sample. Subcellular fractionation studies of intact cells labeled with [gamma-32P]ATP showed differences in the recovery of phosphoproteins of 42,000 Mr depending on whether a continuous sucrose gradient (27.6-54.1%, g/ml) or a discontinuous sucrose gradient (16, 35 and 48%, g/ml) was used. Phosphoproteins of 42,000 Mr were located in the mitochondrial and membrane fractions collected by discontinuous sucrose gradient separation, whereas a phosphoprotein of 42,000 Mr was found primarily in the mitochondrial fraction after continuous sucrose gradient separation. By 5'-nucleotidase activity measurements, the latter approach appears to result in the isolation of a heavy fragment of the plasma membrane with the mitochondrial light fraction which is 42,000 in Mr and labeled. Finally, comparison of the autoradiographs of two-dimensional (2D) gels (isoelectric focusing followed by 10% SDS-PAGE) show different isoelectric points for 42,000 Mr components in [gamma-32P]ATP- and 32Pi-labeled cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Topography of the rhodopsin molecule. Identification of the domain phosphorylated.

Studies on the light-stimulated phosphorylation of rod outer segments by [gamma-32P]ATP showed that although nearly 1 mol of [32P]phosphate was incorporated/mol of total opsin, only a small fraction of the molecules were phosphorylated, and these contained at least 2-3 mol of phosphate/mol. Rod outer segments containing the phosphorylated opsin were incubated with 11-cis-retinal to generate phosphorylated rhodopsin and then digested with papain to produce a cleaved complex comprising three fragments, heavy (H), medium (M) and light (L). It was shown that L-fragment of apparent mol.wt. 6000 contained all the phosphorylation sites. This suggests that one specific domain of rhodopsin is susceptible to multiple phosphorylation.     

Substrate phosphorylation catalyzed by the insulin receptor tyrosine kinase. Kinetic correlation to autophosphorylation of specific sites in the beta subunit

The kinetics of insulin-stimulated autophosphorylation of specific tyrosines in the beta subunit of the mouse insulin receptor and activation of receptor kinase-catalyzed phosphorylation of a model substrate were compared. The deduced amino acid sequence of the mouse proreceptor was determined to locate tyrosine-containing tryptic peptides. Receptor was first incubated with unlabeled ATP to occupy nonrelevant autophosphorylation sites, after which [32P]autophosphorylation at relevant sites and attendant activation of substrate phosphorylation were initiated with [gamma-32P]ATP and insulin. Activation of substrate phosphorylation underwent an initial lag of 10-20 s during which there was substantial 32P-autophosphorylation of tryptic phosphopeptides p2 and p3, but not p1. Following the lag, incorporation of 32P into p1 and the activation of substrate phosphorylation increased abruptly and exhibited identical kinetics. The addition of substrate to the receptor prior to ATP inhibits insulin-stimulated autophosphorylation, and consequently substrate phosphorylation. Insulin-stimulated autophosphorylation of the receptor in the presence of substrate inhibited primarily the incorporation of 32P into p1 and drastically inhibited substrate phosphorylation. From Edman radiosequencing of 32P-labeled p1, p2, and p3 and the amino acid sequence of the mouse receptor, the location of each phosphopeptide within the beta subunit was determined. Further characterization of these phosphopeptides revealed that p1 and p2 represent the triply and doubly phosphorylated forms, respectively, of the region within the tyrosine kinase domain containing tyrosines 1148, 1152, and 1153. The doubly phosphorylated forms contain phosphotyrosines either at positions 1148 and 1152/1153 or positions 1152 and 1153. These results indicate that insulin stimulates sequential autophosphorylation of tyrosines 1148, 1152 and 1153, and that the transition from the doubly to the triply phosphorylated forms is primarily responsible for the activation of substrate phosphorylation.     

Use of 8-azidoguanosine 5'-[gamma-32P]triphosphate as a probe of the guanosine 5'-triphosphate binding protein subunits in bovine rod outer segments

In an in vitro incubation, 8-azidoguanosine 5'-[gamma-32P]triphosphate ( [gamma-32P]-8-azido-GTP) labeled bleached rhodopsin independent of ultraviolet light. Characterization of this labeling indicated that rhodopsin was phosphorylated with [gamma-32P]-8-azido-GTP as a phosphate donor. At low concentrations, ATP increased this labeling activity 5-fold. In the same incubation, [gamma-32P]-8-azido-GTP also labeled G alpha (Mr 40 000). This labeling was ultraviolet light dependent. G beta (Mr 35 000) was also labeled dependent for the most part upon ultraviolet light, but a smaller component of labeling appeared to result from phosphorylation. Differential labeling of G alpha and G beta was found to vary intricately with experimental conditions, especially prebleaching of rhodopsin, tonicity of the medium, and the presence or absence of 2-mercaptoethanol. Affinity labeling of G alpha and G beta by [gamma-32P]-8-azido-GTP in competition with ATP or GTP was kinetically complex, consistent with possible multiple binding sites for GTP on both subunits. Independent evidence for two or more binding sites on G alpha has been offered by other laboratories, and recently, at least one binding site on G beta and its analogues among the N proteins of adenylate cyclases has been identified.     

A novel, carbohydrate signal-mediated cell surface protein phosphorylation: ganglioside GQ1b stimulates ecto-protein kinase activity on the cell surface of a human neuroblastoma cell line, GOTO

A ganglioside-stimulated ecto-type protein phosphorylation system (ecto-Gg-kinase) was detected on the cell surface of a human neuroblastoma cell line (GOTO). When intact cells were incubated with [gamma-32P]ATP, at least 28 cell surface proteins were phosphorylated, as evident on SDS-PAGE (4-20%) analysis. Exogenously added gangliosides specifically stimulated the phosphorylation of at least three cell surface associated proteins of Mr = 64,000, 60,000, and 54,000. Phosphorylation was directed toward Thr and Ser residues, respectively, as revealed on acid hydrolysis followed by electrophoresis. GQ1b, at 5 nM, was the most potent among the several gangliosides tested and was more effective when added to cells before [gamma-32P]ATP administration. The simultaneous addition of an excess amount of the saccharide portion of GQ1b (oligo-GQ1b) inhibited the GQ1b-stimulated phosphorylation, indicating the necessity of the sialosaccharide moiety. These results strongly suggest that phosphorylation of the three proteins may be closely associated with the highly specific neuritogenic effect of GQ1b previously reported.     

Phosphorylation and inactivation of the pyruvate dehydrogenase from the anaerobic parasitic nematode, Ascaris suum. Stoichiometry and amino acid sequence around the phosphorylation sites

Tryptic digestion of the fully phosphorylated Ascaris suum pyruvate dehydrogenase complex yielded a single tetradecapeptide containing 2 phosphorylated serine residues. Its amino acid sequence was Tyr-Ser-Gly-His-Ser(P)-Met-Ser-Asp-Pro-Gly-Thr-Ser(P)-Tyr-Arg and was very similar to one of the tryptic phosphopeptides isolated from mammalian and yeast pyruvate dehydrogenases. At partial phosphorylation, three peptides were isolated which corresponded to the monophosphorylated (sites 1 and 2) and diphosphorylated tetradecapeptides. In contrast to results reported from mammalian complexes, phosphorylation of the ascarid complex paralleled inactivation, and no additional phosphorylation occurred after inactivation was complete. Complete inactivation of the complex was associated with the incorporation of 1.7-1.9 mol of phosphoryl groups/mol of alpha-pyruvate dehydrogenase subunit, and the strict preference of the pyruvate dehydrogenase kinase for site 1 was not observed. Whereas site 1 was initially phosphorylated more rapidly than site 2, at 50% inactivation, 41% of the incorporated phosphoryl groups were incorporated into site 2. In addition, substantial amounts of peptide monophosphorylated at site 2 also accumulated, suggesting that prior phosphorylation at site 1 was not necessary for phosphorylation at site 2. Phosphorylation also caused a marked decrease in the mobility of the alpha-pyruvate dehydrogenase subunit on sodium dodecyl sulfate-polyacrylamide gels and the apparent separation of mono- and diphosphorylated forms of the enzyme. The significance of these observations in the regulation of the unique anaerobic mitochondrial metabolism of A. suum is discussed.     

Protein phosphorylation during 1-methyladenine-induced maturation of Asterias oocytes

Maturation was induced in Asterias oocytes with 1-methyladenine (1-MA) at a final concentration of 2 microM. At 5, 10, and 30 min of treatment, oocytes were homogenized and the cytosolic fraction was prepared. The cytosol was incubated with [gamma-32P]ATP and [gamma-32P]GTP. The phosphorylated proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the radioactivity in the gels was determined by autoradiography. The cytosol prepared from 1-MA-treated oocytes incubated with [gamma-32P]ATP showed a marked increase in the radiolabeling of proteins with estimated molecular weights of 70,000 and 62,000 Da. With [gamma-32P]GTP a 56,000-Da protein showed increased radiolabeling. The present finding suggests that an early biochemical event of 1-MA-induced oocyte maturation in Asterias is the stimulation of phosphorylation of specific proteins.