Regulation of C4 photosynthesis: identification of a catalytically important histidine residue and its role in the regulation of pyruvate,Pi dikinase

These studies provide further information regarding the mechanism of the light/dark-mediated regulation of pyruvate,Pi dikinase in leaves. It is shown that a catalysis-linked phosphorylation of pyruvate,Pi dikinase can be demonstrated following incubation of the enzyme with [32P]phosphoenolpyruvate or [beta-32P]ATP plus Pi, that the enzyme-bound phosphate is located on a histidine residue, and that this phosphate is retained during ADP-mediated inactivation. Further evidence is provided that phosphorylation of this histidine is a prerequisite for ADP-mediated inactivation through phosphorylation of a threonine residue from the beta-phosphate of ADP. It is demonstrated that diethylpyrocarbonate (which forms a derivative with histidine residues) prevents [32P]phosphoenolpyruvate-dependent labeling (catalytic labeling) and [beta-32P]ADP-dependent labeling (inactivation labeling) of the enzyme. In addition, it is demonstrated that oxalate, an analog of pyruvate, competitively inhibits ADP-dependent inactivation with respect to ADP. The significance of these results is discussed with regard to the mechanism of regulation of pyruvate,Pi dikinase in vivo.     

Stimulation of phosphate uptake in human platelets by thrombin and collagen. Changes in specific 32P labeling of metabolic ATP and polyphosphoinositides

The uptake of [32P]phosphate by human, gel-filtered blood platelets and its incorporation into cytoplasmic ATP and polyphosphoinositides was studied. In unstimulated platelets, uptake was Na+o-dependent and saturable at approximately 20 nmol/min/10(11) cells with a half-maximal rate at 0.5 mM extracellular phosphate. Upon stimulation with thrombin or collagen, net influx of [32P]Pi was accelerated 5- to 10-fold. With thrombin, [32P]Pi efflux was also increased. After the first 2 min, efflux exceeded influx, resulting in the net release of [32P]Pi from the platelets. Since the stimulus-induced burst in [32P]Pi uptake paralleled the secretory responses, it might be an integral part of stimulus-response coupling in platelets. The stimulus-induced burst in net [32P]Pi uptake led to an enhanced labeling of metabolic ATP, which was already detectable at 5 s after stimulation with thrombin. Concomitantly, the incorporation of [32P]Pi into phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was accelerated. The thrombin-induced increase in specific 32P radioactivity of cytoplasmic ATP fully accounted for the simultaneous increase in specific 32P radioactivity of these phosphoinositides. In studying the extent of 32P labeling of phosphorylated compounds in response to a cellular stimulus, it is therefore essential to measure the effect of the stimulus on the specific radioactivity of cytoplasmic ATP.     

Differential phosphoprotein labeling (DIPPL), a method for comparing live cell phosphoproteomes using simultaneous analysis of (33)P- and (32)P-labeled proteins

We developed a differential method to reveal kinase-specific phosphorylation events in live cells. In this method, cells in which the specified kinase is inactive are labeled with (32)Pi, whereas cells in which the kinase is active are labeled with (33)Pi. The two cell extracts are then mixed, and proteins are separated on a single two-dimensional gel. The dried gel is exposed twice. The first exposure reveals both (32)P- and (33)P-labeled proteins; the kinase-specific spots are revealed because of (33)P labeling. The second exposure is conducted with two acetate sheets intervening between the gel and the detection plate. This maneuver screens out the less energetic (33)P-labeled proteins while allowing the more energetic (32)P-labeled proteins to be detected, thus leaving only those spots that were phosphorylated independently of the specified kinase. We demonstrate the utility of this method for detecting kinase substrates in rare tissue by focusing on extracellular signal-regulated kinase-specific phosphorylation of stathmin/OP18 in primary rat sympathetic neurons.     

Turnover and Storage of Newly Synthesized Adenine Nucleotides in Bovine Adrenal Medullary Cell Cultures

The adenine nucleotide stores of cultured adrenal medullary cells were radiolabeled by incubating the cells with 32Pi and [3H]adenosine and the turnover, subcellular distribution, and secretion of the nucleotides were examined. ATP represented 84-88% of the labeled adenine nucleotides, ADP 11-13%, and AMP 1-3%. The turnover of 32P-adenine nucleotides and 3H-nucleotides was biphasic and virtually identical; there was an initial fast phase with a t1/2 of 3.5-4.5 h and a slow phase with a half-life varying from 7 to 17 days, depending upon the particular cell preparation. The t1/2 of the slow phase for labeled adenine nucleotides was the same as that for the turnover of labeled catecholamines. The subcellular distribution of labeled adenine nucleotides provides evidence that there are at least two pools of adenine nucleotides which make up the component with the long half-life. One pool, which contains the bulk of endogenous nucleotides (75% of the total), is present within the chromaffin vesicles; the subcellular localization of the second pool has not been identified. The studies also show that [3H]ATP and [32P]ATP are distributed differently within the cell; 3 days after labeling 75% of the [32P]ATP was present in chromaffin vesicles while only 35% of the [3H]ATP was present in chromaffin vesicles. Evidence for two pools of ATP with long half-lives and for the differential distribution of [32P]ATP and [3H]ATP was also obtained from secretion studies. Stimulation of cell cultures with nicotine or scorpion venom 24 h after labeling with [3H]adenosine and 32Pi released relatively twice as much catecholamine as 32P-labeled compounds and relatively three times as much catecholamine as 3H-labeled compounds.     

Metabolic Pools of ATP in Cultured Bovine Adrenal Medullary Chromaffin Cells

Cultured bovine adrenal chromaffin cells contain a pool of ATP sequestered within the chromaffin vesicles and an extravesicular pool of ATP. In a previous study it was shown that the turnover of ATP in the extravesicular pool was biphasic. One phase occurred with a t1/2 of 3.5-4.5 h whereas the second phase occurred with a t1/2 of several days. The studies described here were undertaken to characterize further the vesicular and extravesicular pools of ATP by examining the effects of metabolic inhibitors, adenosine, and digitonin on ATP utilization and subcellular localization immediately after and 48 h after labeling with [3H]adenosine and 32Pi. Immediately after labeling a combination of cyanide, 2-deoxy-D-glucose, the beta-glucono-1,5-lactone resulted in a 90-95% depletion of the labeled ATP but only a 25% depletion of the endogenous ATP within 30 min. Forty-eight hours after labeling, addition of the inhibitors resulted in a 70% depletion of the [3H]ATP but only a 25% depletion of the [32P]ATP and endogenous ATP. Addition of 10 microM adenosine to the media resulted in a similar loss of [3H]ATP in cells examined immediately after or 48 h after labeling. Adenosine increased the amounts of [32P]ATP when added immediately after labeling but had no effect on the [32P]ATP content when added 48 h after labeling.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell cycle-specific effects of sodium arsenite and hyperthermic exposure on incorporation of radioactive leucine and phosphate by stress proteins from mouse lymphoma cell nuclei

Cultured mouse lymphoma cells incorporated [3H]leucine and [32P]phosphate into nuclear stress proteins within 3 h after exposure to either elevated temperature (45 degrees C) or sodium arsenite. Radiolabeled proteins were detected by autoradiography after two-dimensional polyacrylamide gel electrophoresis. To determine the cell cycle stage specificity of labeling, nuclei were isolated and sorted into two cell cycle phases using a fluorescent activated cell sorter. After either heat shock or sodium arsenite treatment, the majority of [3H]leucine incorporation into stress proteins occurred during the G0 + G1 phase with minimal labeling in the G2 phase. On the other hand, 32P labeling of stress proteins occurred in both the G0 + G1 and G2 phases after exposure to sodium arsenite, while incorporation of 32P was limited after heat stress. Following sodium arsenite treatment, a distinct set of four stress proteins (80-84 kDa) was detected with [3H]leucine only in G0 + G1 phase, but with [32P]phosphate these stress proteins were labeled in both G0 + G1 and G2. There was differential [32P]phosphate labeling between proteins of the 80-84 kDa set during cell cycling. Individual proteins of this set were isolated from gel plugs after sodium arsenite or heat-shock treatment. Coelectrophoresis of proteins from the two treatment groups showed that they had similar electrophoretic mobilities. All four proteins of the 80-84 kDa set (sodium arsenite induced) possessed similar polypeptide maps after digestion with V8 protease. Cytofluorometric analysis demonstrated a reduction in the number of nuclei in both S and G2 phases of the cell cycle two h after heat shock, but not following sodium arsenite treatment. However, there was a significant depression in the number of nuclei in S and G2 4 h after exposure to sodium arsenite and very modest labeling with 32P of stress proteins was observed at this time.     

4-Azido-2-nitrophenyl phosphate, a new photoaffinity derivative of inorganic phosphate. Study of its interaction with the inorganic phosphate binding site of beef heart mitochondrial adenosine triphosphatase

4-Azido-2-nitrophenyl phosphate (ANPP) was synthesized and characterized. ANPP, unlabeled or labeled by 32P, was used as a photoreactive analogue of Pi to study the Pi binding site(s) in isolated F1-ATPase and inside-out particles from beef heart mitochondria. In the dark, the phosphate bond of ANPP was cleaved by alkaline phosphatase but not by mitochondrial F1-ATPase. ANPP bound reversibly to the phosphate site of F1-ATPase as shown by competitive inhibition of binding of Pi to F1-ATPase by ANPP in the dark; the Ki value was 60 microM. Upon photoirradiation with visible light, [32P]ANPP bound covalently to F1-ATPase and inactivated the enzyme. Part of the added ANPP was, however, photolyzed with release of Pi. By extrapolation, it could be calculated that complete inactivatin of F1-ATPase was accompanied by incorporation of 32P radioactivity corresponding to 1 mol of [32P]ANPP per mol of F1-ATPase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [32P]-ANPP-labeled F1-ATPase revealed only one radioactive peptide with a Mr of 50000. This peptide was characterized as the beta subunit of F1-ATPase by specific labeling with [14C]dicyclohexylcarbodiimide [Pougeois, R., Satre, M., & Vignais, P. V. (1979) Biochemistry 18, 1408-1413]. Photoirradiation of inside-out submitochondrial particles with [32P]ANPP resulted in the labeling of two peptides with a Mr of 50000 and 30000-32000; both labelings were significantly decreased by incubation of the particles with Pi prior to photoirradiation. The Mr 50000 peptide is most probably the beta subunit of F1-ATPase; the other peptide might be the Pi carrier protein.     

Action of insulin on the subcellular metabolism of polyphosphoinositides in isolated rat hepatocytes

The effect of insulin on 32Pi incorporation into phospholipids in various subcellular sites of isolated rat hepatocytes was investigated. After labeling the phospholipids of hepatocytes from rats previously starved for 24 h with 32Pi (10 mu Ci/10(6) cells) for 90 min, either saline or insulin (32 nM) was added. Following incubations of 1, 5, and 30 min, chilled cells were rapidly washed, homogenized in the presence of inhibitors of phospholipid degradation, and fractionated into the major subcellular organelles. Phospholipids were extracted from plasma membranes, microsomes, lysosomes, mitochondria, and nuclei with acidic chloroform:methanol. The aqueous deacylation products were separated by anion exchange high performance liquid chromatography, and the 32Pi incorporated into all the major diacylglycerophospholipids was determined. In parallel experiments, the specific radioactivity of 32Pi and [gamma-32P]ATP was determined. The results revealed that insulin had no effect on the turnover of the major phospholipids, including the polyphosphoinositides, of all subcellular compartments analyzed relative to the control. In addition, there were no significant differences in the amount and 32P labeling of cellular orthophosphate between saline- and insulin-treated cells. The specific radioactivity of [gamma-32P]ATP was increased by 20% after 30-min treatment with insulin, requiring appropriate correction of 32P-labeled phosphatidic acid, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate for estimation of mass changes at near steady-state labeling of cellular ATP.     

Identification of phosphotyrosine in yeast proteins and of a protein tyrosine kinase associated with the plasma membrane

[32P]Phosphotyrosine was detected in a hydrolysate of yeast proteins after in vivo labeling with [32P]phosphoric acid. The phosphoamino acid was present in cells exponentially growing on glucose as well as in cells that had reached the stationary phase of growth. Also, a plasma membrane preparation was shown to phosphorylate casein on tyrosine residues.     

The phosphohydrolase component of the hepatic microsomal glucose-6-phosphatase system is a 36.5-kilodalton polypeptide

The phosphohydrolase component of the microsomal glucose-6-phosphatase system has been identified as a 36.5-kDa polypeptide by 32P-labeling of the phosphoryl-enzyme intermediate formed during steady-state hydrolysis. A 36.5-kDa polypeptide was labeled when disrupted rat hepatic microsomes were incubated with three different 32P-labeled substrates for the enzyme (glucose-6-P, mannose-6-P, and PPi) and the reaction terminated with trichloroacetic acid. Labeling of the phosphoryl-enzyme intermediate with [32P]glucose-6-P was blocked by several well-characterized competitive inhibitors of glucose-6-phosphatase activity (e.g. Al(F)-4 and Pi) and by thermal inactivation, and labeling was not seen following incubations with 32Pi and [U-14C]glucose-6-P. In agreement with steady-state dictates, the amount of [32P]phosphoryl intermediate was directly and quantitatively proportional to the steady-state glucose-6-phosphatase activity measured under a variety of conditions in both intact and disrupted hepatic microsomes. The labeled 36.5-kDa polypeptide was specifically immunostained by antiserum raised in sheep against the partially purified rat hepatic enzyme, and the antiserum quantitatively immunoprecipitated glucose-6-phosphatase activity from cholate-solubilized rat hepatic microsomes. [32P]Glucose-6-P also labeled a similar-sized polypeptide in hepatic microsomes from sheep, rabbit, guinea pig, and mouse and rat renal microsomes. The glucose-6-phosphatase enzyme appears to be a minor protein of the hepatic endoplasmic reticulum, comprising about 0.1% of the total microsomal membrane proteins. The centrifugation of sodium dodecyl sulfate-solubilized membrane proteins was found to be a crucial step in the resolution of radiolabeled microsomal proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Protein phosphorylation in response to stress in Clostridium acetobutylicum

The possible involvement of protein phosphorylation in the clostridial stress response was investigated by radioactively labeling growing cells of Clostridium acetobutylicum with 32Pi or cell extracts with [gamma-32P]ATP. Several phosphoproteins were identified; these were not affected by the growth stage of the culture. Although the extent of protein phosphorylation was increased by heat stress, the phosphoproteins did not correspond to known stress proteins seen in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified clostridial DnaK, a stress protein, acted as a kinase catalyzing the phosphorylation of a 50-kilodalton protein. The phosphorylation of this protein was enhanced in extracts prepared from heat-stressed cells. Diadenosine-5',5"'-P1,P4-tetraphosphate had no influence on protein phosphorylation.     

Protein phosphorylation in response to stress in Clostridium acetobutylicum.

The possible involvement of protein phosphorylation in the clostridial stress response was investigated by radioactively labeling growing cells of Clostridium acetobutylicum with 32Pi or cell extracts with [gamma-32P]ATP. Several phosphoproteins were identified; these were not affected by the growth stage of the culture. Although the extent of protein phosphorylation was increased by heat stress, the phosphoproteins did not correspond to known stress proteins seen in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified clostridial DnaK, a stress protein, acted as a kinase catalyzing the phosphorylation of a 50-kilodalton protein. The phosphorylation of this protein was enhanced in extracts prepared from heat-stressed cells. Diadenosine-5',5"'-P1,P4-tetraphosphate had no influence on protein phosphorylation.     

The preparation and use of pyridoxal [32P]phosphate as a labeling reagent for proteins on the outer surface of membranes.

Pyridoxal [32P] phosphate was prepared using [gamma-32P] ATP, pyridoxal, and pyridoxine kinase purified from Escherichia coli B. The pyridoxal [32P] phosphate obtained had a specific activity of at least 1 Ci/mmol. This reagent was used to label intact influenza virus, red blood cells, and both normal and transformed chick embryo fibroblasts. The cell or virus to be labeled was incubated with pyridoxal [32P] phosphate. The Schiff base formed between pyridoxal [32P] phosphate and protein amino groups was reduced with NaBH4. The distribution of pyridoxal [32P] phosphate in cell membrane or virus envelope proteins was visualized by autoradiography of the proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The labeling of the proteins of both influenza and chick cells appeared to be limited exclusively to those on the external surface of the virus or plasma membrane. With intact red blood cells the major portion of the probe was bound by external proteins, but a small amount of label was found associated with the internal proteins spectrin and hemoglobin.     

Phosphorylation of phosphoprotein phosphatase inhibitor-2 (I-2) in rat fat cells

Fat cells were incubated with 32Pi for 2 h before the [32P]I-2 was immunoprecipitated, subjected to SDS/PAGE, and detected by autoradiography. [32P]I-2 (Mr = 32,000) was not recovered when excess purified I-2 was added with the antiserum or when nonimmune serum was used. Immunoprecipitated I-2 was heat-stable, inhibited phosphatase activity, and could be synergistically phosphorylated by casein kinase II and FA/GSK-3. Several times more [32P]phosphoserine than [32P]phosphothreonine was found in I-2 from 32P-labeled cells. Insulin increased the 32P-content of I-2 by as much as 40%, suggesting that phosphorylation of I-2 might be involved in the effect of insulin on stimulating protein dephosphorylation.     

Nucleophile in the active site of Escherichia coli galactose-1-phosphate uridylyltransferase: degradation of the uridylyl-enzyme intermediate to N3-phosphohistidine.

The [32P]uridylyl-enzyme intermediate form of Escherichia coli galactose-1-P uridylyltransferase can be converted to a [32P]phosphoryl-enzyme by first cleaving the ribosyl ring with NaIO4 and then heating at pH 10.5 and 50 degrees C for 1 h. After alkaline hydrolysis of the [32P]phosphoryl-enzyme the major radioactive product is N3-[32P]phosphohistidine. A lesser amount of 32Pi is also produced as a side product of the hydrolysis of N3-[32P]phosphohistidine. No N1-phosphohistidine, N-phospholysine, or phosphoarginine can be detected in these hydrolysates. It is concluded that the nucleophile in galactose-1-P uridylyltransferase to which the uridylyl group is bonded in the uridylyl-enzyme intermediate is imidazole N3 of a histidine residue. This degradation procedure should have general applicability in the degradation and characterization of nucleotidyl-proteins.     

Blot overlays with 32P-labeled fusion proteins

Proteins labeled with 32P can be used as sensitive "prime" in blot overlays to detect binding proteins or domains. Small G-protein Ras can bind GTP with extremely high affinity (Kd approximately 10(-11)-10(-12) M) in the presence of Mg2+. We have taken advantage of this property of Ras to develop a vector that expresses proteins of interest such as glutathione S-transferase (GST)/Ras fusion proteins for noncovalent labeling with [gamma-32P]GTP. The labeling efficiency of this method is >60% and involves a single short incubation step. We have previously identified several binding proteins for the second SH3 domain of the adaptor Nck using this method. Here we illustrate the overlay method using the GST/Ras system and compare results with the SH3 domain labeled by phosphorylation with [gamma-32P]ATP. Both methods are similarly specific and sensitive; however, we show that signals are dependent primarily on GST-mediated probe dimerization. These dimeric probes allow a more stable probe-target complex similar to immunoglobulin interactions, thus significantly improving the sensitivity of the technique.     

Phosphorylation of class I histocompatibility antigens in human B lymphocytes. Regulation by phorbol esters and insulin.

Phosphorylation of membrane proteins is one of the earliest steps in cell activation induced by growth-promoting agents. Since MHC (major histocompatibility complex) class I molecules are known to contain phosphorylation sites in their C-terminal intracellular domain, we have studied the regulation of HLA (human leucocyte antigen) phosphorylation in intact cells by two mitogens, namely TPA (12-O-tetradecanoylphorbol 13-acetate), a phorbol ester, and insulin, which are thought to exert their mitogenic effects through the stimulation of different protein kinases (protein kinase C and a tyrosine kinase respectively). Human B lymphoblastoid cells (526 cell line) were pulsed with [32P]Pi to label the intracellular ATP pool. Cells were then stimulated for 10 min with TPA, insulin, cyclic AMP or EGF (epidermal growth factor). The reaction was stopped by cell lysis in the presence of kinase and phosphatase inhibitors, and class I HLA antigens were immunoprecipitated with monoclonal antibodies. Analysis of labelled proteins by gel electrophoresis and autoradiography revealed that TPA increased the phosphorylation of the 45 kDa class I heavy chain by 5-7-fold, and insulin increased it by 2-3-fold. Cyclic AMP and EGF had no stimulatory effect. Analysis of immunoprecipitated HLA molecules by two-dimensional gel electrophoresis showed that TPA and insulin stimulated the incorporation of 32P into different 45 kDa molecular species, suggesting that different sites were phosphorylated by two agents. Moreover, incubation of purified class I MHC antigens with partially purified insulin-receptor tyrosine kinase and [gamma-32P]ATP revealed that class I antigens could also be phosphorylated in vitro by this tyrosine kinase. Altogether, these results therefore confirm that insulin receptors and HLA class I molecules are not only structurally [Fehlmann, Peyron, Samson, Van Obberghen, Brandenburg & Brossette (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 8634-8637] but also functionally associated in the membranes of intact cells.     

Mechanism of endogenous phosphorylation of microtubule proteins during GTP-induced microtubule assembly and implications for stability of the assembled structures

Cycle-purified microtubule protein from mammalian brain incorporated [32P]Pi upon incubation with [gamma-32P]GTP under the conditions used to promote assembly. This phosphorylation also occurred in the same proteins when phosphorylated with [gamma-32P]ATP and was only slightly stimulated by cAMP. GTP was a much less effective substrate than ATP. The transfer of phosphoryl groups from [gamma-32P]GTP to endogenous proteins followed a linear time-course and was stimulated by low concentrations of ATP and, more efficiently, by ADP. These data are in agreement with the predictions derived from a mechanism of phosphorylation by which [gamma-32P]GTP does not act as a phosphoryl donor for the protein kinase activity but, instead, only as a repository of high group transfer potential phosphoryl groups used to make [gamma-32P]ATP, from contaminating ADP, by means of the nucleoside diphosphate kinase activity. Using 100 mM fluoride, which suppressed protein phosphorylation without inhibiting the nucleoside diphosphate kinase activity, formation of [gamma-32P]ATP was detected. Fluoride was also able to protect microtubules from a slow depolymerization which was found to occur during long-term incubation of microtubules. This indicates that the phosphorylation observed in the presence of GTP is sufficient to destabilize microtubules.     

Regulation of the phosphate (Pi) concentration in UMR 106 osteoblast-like cells: Effect of Pi,Na+ and K+

Osteoblast-like cells possess Na-dependent transporters which accumulate orthophosphate (Pi) from the extracellular medium. This may be important in bone formation. Here we describe parallel measurements of Pi uptake and cellular [Pi] in such cells from the rat (UMR 106–01 and UMR 106–06) and human (OB), and in non-osteoblastic human fibroblasts (Detroit 532 (DET)). In UMR 106–01, cellular [Pi] was weakly dependent on extracellular [Pi] and higher than expected from passive transport alone. [³²Pi]-uptake was inhibited by Na deprivation, but paradoxically increased on K deprivation. With Na, 87 per cent of cellular ³²P was found in organic phosphorus pools after only 5 min. Na deprivation also decreased cellular [Pi], in both UMR 106–01 and DET, but the decrease was smaller than that in [³²Pi]-uptake. Ouabain decreased [³²Pi]-uptake and cellular [Pi] in DET, but not in UMR 106–01. Regulation of cellular [Pi] is therefore at least partly dependent on Na/Pi co-transport, but this does not seem to be an exclusive property of osteoblasts.