Inhibition of mouse brain synaptosomal gamma-aminobutyric acid transport by pyridoxal 5'-phosphate

Crude lysates from a strain of enterotoxigenic $\text{E. coli}$ have been shown to catalyse the incorporation of [³²P] from [adenylate-³²P] NAD⁺ into an 11,000 dalton protein in rat liver membranes. [³²P] incorporation paralleled adenylate cyclase activation and the results suggest that the mechanism of action of the heat-labile $\text{E. coli}$ enterotoxin may involve ADP-ribosylation of an intracellular acceptor protein.     

An enzymatic method for [32P]phosphoenolpyruvate synthesis

A convenient method for the enzymatic synthesis of [³²P]phosphoenolpyruvate from [γ-³²P]ATP using partially pufified phosphoenolpyruvate carboxykinase from Escherichia coli is described. The synthesis was shown to convert essentially all the [γ-³²P]ATP to [³²P]phosphoenolpyruvate, which was subsequently separated from residual [γ-³²P]ATP and $\text{[}{}^{32}\text{P}\text{]P}{}_{\mathrm{<mtext>i</mtext>}}$ by chromatography on AG-1-X8-bicarbonate resin.     

Differential phosphorylation of histone H1 subfractions in vivo.

A study was made of the phosphorylation of chromatographically purified histone H1 subfractions from the liver of premetamorphic tadpoles ($\text{Rana}\text{catesbeiana}$). Two H1 subfractions were obtained which differed in terms of net incorporation of [³²P]phosphate $\text{in}\text{vivo}$. Analysis of N-bromosuccinimide cleavage products further revealed that the two subfractions also differed in the relative distribution of [³²P]phosphate in N- and C-terminal regions of the molecule. Incorporation of [³²P]phosphate into both regions of the molecule occurred virtually exclusively in serine residues.     

Phosphorylation of high mobility group proteins 14 and 17 by nuclear protein kinase NII in rat O6 glioma cells

High mobility group (HMG) proteins 14 and 17 of rat C₆ glioma cells are phosphorylated $\text{in}\text{vivo}$ on both serine and threonine. In HMG 14 about 60% of the total [³²P]phosphate was identified as phosphoserine and 40% as phosphothreonine. In HMG 17, there was 88% phosphoserine and 12% phosphothreonine. Glioma cell nuclear protein kinase NII phosphorylates HMG 14 and 17 $\text{in}\text{vitro}$ on serine as well as threonine and the relative percentages of [³²P]phosphoamino acid are similar to those seen $\text{in}\text{vivo}$. Nuclear protein kinase NI and the type I and II cAMP-dependent protein kinases exhibit only minor phosphorylating activity towards HMG 14 and 17. We conclude that nuclear protein kinase NII is responsible for the phosphorylation of HMG 14 and 17 $\text{in}\text{vivo}$.     

Evidence for phosphoryl moieties in a proteinase from Dictyostelium discoideum.

A proteinase (called Proteinase I) present in myxamoebae of the cellular slime mold, $\text{Dictyostelium}\text{discoideum}$, was labeled $\text{in}\text{vivo}$ with [³²P] by growth of cells on media containing [³²P] orthophosphate. The labeled proteinase was purified to apparent homogeneity and characterized by dissociation chromatography and quantitative immune-precipitin analysis. Based upon the results of these studies it was concluded that phosphoryl moieties were tightly associated (presumably covalently bonded) with the polypeptide subunits of Proteinase I.     

Acetylcholine stimulates hydrolysis of 32 P-labeled phosphatidic acid in guinea pig synaptosomes

[³H]-inositol or [³H]-arachidonate was injected intracerebrally into guinea pigs. Labeled nerve endings were incubated with Ach¹ or CCh, both of which stimulate labeling of PhA and PhI from ³²P_i by > 100% and 70% respectively. Their addition did not affect the $\text{in}$$\text{vivo}$ labeled phosphatidyl-[³H]-inositol or [³H]-arachidonyl-diglyceride and -PhI. Enhanced hydrolysis of [³H]-inositol-PhiP and -PhIP₂ in the presence of ACh, CCh or choline was not reversed by atropine. In a two-step experiment, PhA was labeled with ³²P_i, and DNP was added to block further $\text{γ-[}{}^{32}\text{P]-ATP}$ formation. Addition of ACh stimulated an atropine-sensitive $\text{decrease}$ in [³²P]-PhA.     

Mechanism of interferon action partial purification and characterization of a low-molecular-weight interferon-mediated inhibitor of translation with nucleolytic activity

A low-molecular-weight interferon-mediated ribosome-associated inhibitor of reovirus mRNA translation was purified from the 0.5 $\text{M}$ KCl ribosomal salt-wash fraction of mouse L₉₂₉ cells. The inhibitor possessed nucleolytic activity with reovirus [³H]mRNA as a substrate. Loss of translational inhibitory activity correlated with the thermal inactivation of the nuclease. A low-molecular-weight ($\text{<}$10K) component present in the Bio-Gel P150 chromatography fractions which contained the interferon-mediated nucleolytic activity was labeled in vivo with [¹⁴C]valine; a smaller component present in the same fractions was phosphorylated in vitro with [γ-³²P]ATP. The $\text{<}$10K components were resolved from ～50K, ～30K and ～20K phosphorylatable proteins associated with ribosomes that possess the interferon-mediated inhibitor(s) of viral mRNA translation.     

Anomalies in 5'-end [32P] labelling of transfer RNA and partial sequence of a tRNAGlu from Scenedesmus obliquus

A chloroplast tRNA_m^Met species from $\text{Scenedesmus}\text{obliquus}$ is very poorly 5′-end [${}^{32}\text{P}$] labelled using [γ-³²P]ATP and T4 polynucleotide kinase. In sequencing the tRNA using standard 5′-labelled methods a very minor contaminating tRNA is preferentially labelled. The partial tRNA sequence determined by this method has an anticodon (CUC) for tRNA^Glu.     

Thylakoid membrane protein phosphorylation in correlation with photosynthetic membrane activation

The phosphorylation of five $\text{E.}\text{gracilis}$ thylakoid membrane polypeptides was studied, in isolated chloroplasts. Using [³²P] labelling, in the light, we found that phosphorylation was inhibited by ethanol and DCMU. Inhibition curves were characteristic of photosynthetic inhibition. [γ-³²P] ATP labelling was used to distinguish between two groups of phosphoproteins: the first one, includes protein I, II, V which require only ATP for phosphorylation while the second one includes protein III and IV whose phosphorylation is light-requiring. Phosphorylation of protein III and IV was inhibited by CCCP, NH₄Cl and DCMU, and was reversible in the dark.     

Evidence for a 5' leads to 3' direction of hydrolysis by a 5' mononucleotide-producing exoribonuclease from Saccharomyces cerevisiae.

[³H]rRNA labeled at the 5′ terminus with ³²P and [³H]rRNA labeled at the 3′ end with [¹⁴C] (pA)_n have been degraded at 0° with a highly purified exoribonuclease from $\text{Saccharomyces cerevisiae}$. The results show that with the [³²P, ³H] substrate, the ³²P label is rendered acid-soluble at a much faster rate than the ³H label. Both acid-soluble labels are found in 5′ mononucleotide. With the [¹⁴C, ³H]rRNA, the ³H label is hydrolyzed at a faster rate than the ¹⁴C label. The exoribonuclease hydrolyzes in the 5′ → 3′ direction.     

Isoproterenol-induced selective phosphorylation invivo of the 214,000 dalton subunit of rat C6 glioma cell RNA polymerase II

The phosphorylation $\text{in}\text{vivo}$ of RNA polymerase II after isoproterenol stimulation of confluent rat C6 glioma cell cultures has been investigated. Glioma cells were incubated in the presence of Na₂H³²PO₄ and stimulated for 1 hour with the β-adrenergic agonist isoproterenol. The phosphorylation pattern was analyzed after purification of RNA polymerase II by immunoprecipitation, SDS-polyacrylamide gel electrophoresis and autoradiography. Isoproterenol markedly increased [³²P]phosphate incorporation into the 214,000 dalton RNA polymerase subunit. Analysis of the phosphate acceptor amino acid revealed the presence of only [³²P]phosphoserine. The data demonstrates an isoproterenol-induced structural modification of RNA polymerase II.     

Proteins and glycoproteins in plasma membranes and in the membrane lamellae produced by purified oligodendroglia in culture

Oligodendroglial plasma membranes are complex structures composed of a heterogeneous mixture of proteins and glycoproteins. The Coomassie stained gel patterns showed a maximum of 40 proteins with molecular weights ranging from $\text{> 200 000}\text{to}\text{12 500}$. Autoradiography was used to detect binding of radioiodinated lectins to glycoproteins. With concanavalin A, 5 major glycoproteins were seen; with wheat germ agglutinin, 2 major glycoproteins with approximate molecular weights of 95 000 and 78 000 were found; with Ulex europaeus, 7 major glycoproteins were observed. Additional minor bands were also seen. The impermeant probe diazodi[¹²⁵I]iodosulfanilic acid was used to radiolabel intact cells. It was found that 5 major proteins were radiolabeled in the plasma membranes. In all cases, the whorls of membrane lamellae produced in culture by oligodendroglia tend to have a somewhat less complicated pattern with fewer proteins and glycoproteins than the plasma membranes. However, the whorls of membrane lamellae have far more complicated protein patterns than myelin.     

Isolation of an androgen acceptor from salt extract of rat prostatic chromatin

The soluble androgen acceptor has been isolated from 0.35 M NaCl extract of rat prostatic chromatin by affinity chromatography on DNA-cellulose. The acceptor activity was assayed by interaction with 5α-dihydrotestosterone-receptor. Native DNA enhances this interaction. Polyacrylamide gel electrophoresis of the acceptor under denaturing conditions reveals a single polypeptide of molecular weight of 14,000. Amino acid analysis shows that the acceptor protein contains a higher content of acidic amino acid residues than basic amino acid residues. In an $\text{in}\text{vitro}$ RNA synthesizing system catalyzed by rat RNA polymerase II, addition of the acceptor stimulates RNA synthesis. Based on incorporation of [γ-³²P]ATP and [γ-³²P]GTP, the stimulation by the acceptor is mainly on the initiation of RNA chains.     

Properties of the 5'-terminus of tRNAHis: kinetics of polynucleotide kinase catalyzed exchange and effect of dephosphorylation on the aminoacylation reaction.

Bacteriophage T₄ induced polynucleotide kinase was found to be ineffective in transferring ³²P from [γ-³²P]ATP to the 5′-terminus of 5′-phosphorylated $\text{E. coli}$ tRNA^His using the ADP mediated exchange reaction. However, prior dephosphorylation with alkaline phosphatase allowed polynucleotide kinase catalyzed phosphorylation of tRNA^His. Contrary to reports for other tRNA species, alkaline phosphatase catalyzed 5′-terminus dephosphorylation destroys the amino acid accepting ability of tRNA^His. Aminoacylation competency of the tRNA^His is restored after phosphorylation with polynucleotide kinase.     

Identical 110,000 dalton phosphoproteins in nuclei and polyribosomes of a rapidly growing rat hepatoma

Nuclei and free polyribosomes from rat hepatoma tissue were incubated $\text{in vitro}$ in the presence of [³²P] ATP. 0.2 N HCl-soluble nuclear proteins and polyribosomal proteins binding to oligo (dT) cellulose were prepared. A 110 kd phosphoprotein was separated by SDS polyacrylamide gel electrophoresis from each of the samples. The phosphoprotein was analysed using limited digestion with protease V8 in a second SDS gel. The patterns obtained for stained peptides and phosphopeptides were completely identical for both preparations. Thus significant amounts of the 110 kd protein appear to be present, as genuine constituents, in both nuclei and polyribosomes.     

Identification of ubiquinone binding proteins in ubiquinol-cytochrome c reductase by arylazido ubiquinone derivative

A functionally active $\text{arylazido-1-[}{}^{14}\text{C]-β-alanine}$ ubiquinone derivative has been synthesized for the identification of the ubiquinone binding protein in ubiquinol-cytochrome $\text{c}$ reductase. After photolysis, the ¹⁴C activity was found to be specifically associated to proteins with mobilities relative to cytochrome $\text{c}$ of 0.841 and 0.475 in the sodium dodecylsulfate polyacrylamide gel electrophoresis of the Weber and Osborn system. These two proteins have previously been identified as $\text{b}$ cytochromes. The ¹⁴C activity distribution pattern was observed to be identical in the presence or absence of phospholipids during the photolysis. Antimycin A also produces no change in the ¹⁴C activity distribution among the proteins of this enzyme complex.     

Mitochondrial phosphate transport and the N-ethylmaleimide binding proteins of the inner membrane

Mitochondria have been prepared from the flight muscles of mature blowflies (Sarcophaga bullata). Phosphate transport by these mitochondria, determined by rates of passive swelling in ammonium phosphate, is sensitive to inhibition by N-ethylmaleimide. 20 nmol of N-ethylmaleimide/nmol cytochrome a inhibit the swelling by 90%. When the mitochondria are inhibited by $\text{N-[}{}^3\text{H}\text{]}\text{ethylmaleimide}$, then solubilized in dodecyl sulfate/mercaptoethanol at 100°C and then electrophoresed on dodecyl sulfate-polyacrylamide gels, many labeled protein bands can be detected, including a large labeled peak that has the same mobility as the tracking dye, bromophenol blue. Sonic submitochondrial particles that are prepared from the $\text{N-[}{}^3\text{H}\text{]}\text{ethylmaleimidelabeled}$ mitochondria, solubilized, and electrophoresed on dodecyl sulfatepolyacrylamide gels, possess only seven major labeled protein bands with no radioactive peak at the tracking dye. These labeled proteins have molecular weights of 71, 68, 64, 45, 32, 30, and approx. 10 · 10³. The nmol $\text{N-[}{}^3\text{H}\text{]-}\text{ethylmaleimide}$ bound to each of these proteins per nmol cytochrome a are 0.15, 0.19, 0.35, 0.45, 0.87, 0.10, and 0.17, respectively, when the mitochondria are inhibited with 21.5 mol $\text{N-[}{}^3\text{H}\text{]}\text{ethylmaleimide/mol}$ cytochrome a at 10 μM cytochrome a. Coty and Pedersen ((1975) J. Biol. Chem. 250, 3515–3521) sensitized rat liver mitochondria to $\text{N-[}{}^3\text{H}\text{]}\text{ethylmaleimide}$ and identified five labeled proteins. Only the labeled 32 · 10³ dalton and the 45 · 10³ dalton proteins are common to both systems     

A novel method for measurement of the specific radioactivity of gamma-32P ATP.

The specific radioactivity of the γ-phosphorus of ATP has been determined by an indirect method. Galactokinase is employed to transfer the terminal phosphate group of [γ-³²P] ATP to [1-³H] galactose. The doubly labeled galactose-1-phosphate is purified by ion exchange chromatography on QAE Sephadex. The specific radioactivity of the phosphorus is calculated from the ${}^{32}\text{P}{}^3\text{H}$ ratio. The method is extremely sensitive, requiring only 0.005 μmoles of ATP with a specific radioactivity of 1 μCi/μmole, and the chromatographic isolation of galactose-1-phosphate is simple and reproducible. The method is directly applicable to the determination of the specific radioactivity of [γ-³²P] ATP in biological samples.     

Phosphatidylinositol 4,5-bisphosphate: Light-mediated breakdown in the vertebrate retina

Isolated frog ($\text{Rana}\text{Pipiens}$) retinas were labeled in the dark with either [³²P]PO₄-orthophosphate or $\text{myo}$-[2-³H]inositol for 2.5–4 hrs. After washing the retinas with cold buffer, they were exposed to brief flashes of light (5 secs or 15 secs) and their rod outer segments isolated. Upon separation of labeled phospholipids, a specific decrease in label in phosphatidylinositol 4,5-bisphosphate was observed, whereas there was no significant effect on the labeling of phosphatidylinositol 4-phosphate, phosphatidylinositol, or phosphatidic acid. These results are indicative of a light-activated phosphatidylinositol 4,5-bisphosphate-specific phospholipase C in frog rod outer segments.     

Characterization of native 40S particles from krebs II mouse ascites tumor cells. I. Phosphorylation in vitro of non ribosomal proteins by a cAMP independent protein kinase.

In the presence of [γ³²-P] ATP or [γ³²-P] GTP 4 non ribosomal proteins (M_r 110,000; 105,000; 89,000 and 25,000) of the native 40S subunit became phosphorylated. The protein kinase responsible for this phosphorylation could be removed by treatment with 0.5$\text{M}$ KCl. Sucrose density gradient analysis showed that the endogenous enzyme activity sedimented with approx. 7.5S.