Phosphorylation of muscle plasma membrane protein by a membrane-bound protein kinase

Protein kinase activity has been demonstrated in purified plasma membranes from rat diaphragm by measuring the incorporation of ³²P from [³²P]-ATP into endogenous membrane proteins and into histone, in vitro. Histone appears to be a better substrate than the endogenous membrane proteins; however, the properties of the enzyme are similar when phosphorylating endogenous or exogenous proteins. The activity of this membrane-associated protein kinase is not significantly affected by cyclic adenosine 3′,5′-monophosphate or by cyclic guanosine 3′,5′-monophosphate, but is inhibited by theophylline. The ³²P incorporated into membrane proteins is alkali-labile and is released from the membrane by protease digestion, but it is not removed by phospholipase C, by hydroxylamine, or by chloroform—methanoll extraction. Solubilization of ³²P-labeled membranes by sodium dodecylsulfate and fractionation by sodium dodecylsulfate polyacrylamide gel electrophoresis reveals that the radioactivity is predominantly associated with a single protein band with an apparent molecular weight of about 51 000. The phosphoprotein is a minor membrane component as judged by Coomassie blue staining.     

Polyamine-like effects of cobalt(III) hexaammine on various cyclic nucleotide-independent protein phosphokinase reactions

The chemically inert trivalent ion cobalt(III) hexaammine, Co³⁺(NH₃)₆, was found to exert polyamine-like effects in enhancing certain cyclic nucleotide-independent protein kinase reactions catalyzed by nuclear enzyme preparations from rat ventral prostate or liver. At 1 mM, Co³⁺(NH₃)₆ stimulated chromatin- and also non-histone-protein-associated kinase activities with partially dephosphorylated phosvitin as substrate by 38% and 72% respectively, whereas chromatin-associated kinase-catalyzed phosphorylation of lysine-rich histones was not affected under the same conditions. ³²P incorporation (from γ-³²P-ATP) into endogenous protein substrates of chromatin or non-histone protein fractions catalyzed by their erdogencus kinase activity was increased by 47% and 153%, respectively. These effects of Co³⁺(NH₃)₆ were similar to those produced by 1mM spermine. Autoradiographic analysis of endogenous ³²P-labelled nonhistone proteins revealed similar enhancements of the phosphorylation of several of the same proteins, induced by 1mM spermine or 1 mM Co³⁺(NH₃)₆ or 2mM spermidine. The stimulatory actions of polyamines or Co³⁺(NH₃)₆ were not mimicked by raising the ionic strength by addition of comparable concentrations of NaCl. The effects of 1 mM spermine and of 1 mM Co³⁺(NH₃)₆ tested separately were not additive. Phosphorylation of lysine-rich histones by beef heart cyclic AMP-dependent protein kinase was not affected by polyamines or Co³⁺(NH₃)₆ Various findings hint that the enhancement of cyclic nucleotide-independent kinase-catalyzed phosphorylation of certain protein substrates by spermidine, spermine and Co³⁺(NH₃)₆ is primarily due to interaction of these cations with appropriate protein substrates affecting their conformational status. Further, these effects of polyamines may be a reflection of their cationic charge properties rather than being dependent on any particular conformations assumed by the polyamines.     

Cyclic adenosine monophosphate-dependent and -independent protein kinase activity of renal brush border membranes.

Kinase(s) in brush border membranes, isolated from rabbit renal proximal tubules, phosphorylated proteins intrinsic to the membrane and exogenous proteins. cAMP stimulated phosphorylation of histone; phosphorylation of protamine was cAMP independent. cAMP-dependent increases in phosphorylation of endogenous membrane protein were small, but highly reproducible. Most of the ³²P incorporated into membranes represented phosphorylation of serine residues, with phosphorylthreonine comprising a minor component. cAMP did not alter the electrophoretic pattern of ³²P-labeled membrane polypeptides. The small cAMP-dependent phosphorylation of brush border membrane proteins was not due to membrane phosphodiesterase or adenylate cyclase activities. Considerable cAMP was found “endogenously” bound to the membranes as prepared. However, this did not result in preactivation of the kinase since activity was not inhibited by a heat-stable protein inhibitor of cAMP-dependent protein kinases. With intrinsic membrane protein as phosphate acceptor, the relationship between rate of phosphorylation and ATP concentration appeared to follow Michaelis-Menton kinetics. With histone the relationship was complex. cAMP did not affect the apparent K_m for histone. One-half maximal stimulation of the rate of histone phosphorylation was obtained with 7 × 10^?8m cAMP. The K_a values for dibutyryl cAMP, cIMP, and cGMP were one to two orders of magnitude greater. Treatment of brush border membranes with detergent greatly increased the dependency of histone phosphorylation on cAMP. Phosphorylations of intrinsic membrane protein and histone were nonlinear with time, due in part to the lability of the protein kinase, the hydrolysis of ATP, and minimally to the presence of phosphoprotein phosphatase in the border membrane. The membrane phosphoprotein phosphatase was unaffected by cyclic nucleotides. Protein kinase activity was also found in cytosolic and crude particulate fractions of the renal cortex. Activity was enriched in the brush border membrane relative to that in the crude membrane preparation. The kinase activities in the different loci were distinct both in relative activities toward different substrates and in responsiveness to cAMP.     

Endogenous phosphorylation of platelet membrane proteins.

The characteristics of the phosphorylating activity of platelet membranes have been studied. Plasma membranes of human platelets isolated by the glycerol lysis technique were shown to incorporate significant amounts of [³²P]phosphate into specific membrane proteins. This activity was only partially cyclic 3′:5′-monophosphate (cyclic AMP)-dependent but had most of the other characteristics of protein kinases derived from other sources. Maximal stimulation of endogenous phosphorylation was obtained at 1 × 10^?7, m cyclic AMP and exceeded by approximately 30% the [³²P]phosphate incorporation in the absence of this cyclic nucleotide. The platelet membrane protein kinase was able to phosphorylate exogenous proteins, e.g., histone, fibrinogen etc., as well as endogenous membrane proteins. The latter solubilized by sodium dodecyl sulfate and separated by dodecyl sulfate-polyacrylamide gel electrophoresis incorporated [³²P]phosphate into three polypeptides of apparent molecular weights 52,000, 31,000, and 20,000. The phosphorylation of the polypeptide of molecular weight 52,000 was cyclic AMP-dependent.     

Stimulation of protein phosphorylation during fertilization-induced maturation of Urechis caupo oocytes

Protein phosphorylation was studied during fertilization of Urechis caupo oocytes both in vivo, by measuring [³²P]phosphate incorporation into ³²P preloaded oocytes and in vitro, by measuring endogenous protein kinase and phosphatase activities in homogenates. During fertilization (and maturation) the rate of protein phosphorylation is dramatically increased. No change in the [³²P]phosphate uptake, or the nucleotide levels was observed at fertilization, so the increase cannot be attributed to changes in substrate availability. In vitro enzyme assays showed changes in protein kinase activity which approximately mirrored the changes in the in vivo phosphorylation pattern. As there were no changes in protein phosphatase activity, these results suggest the phosphorylation change results from an increase in protein kinase activity. The pattern of change, investigated by SDS-polyacrylamide gel electrophoresis, shows that proteins that were phosphorylated in the unfertilized egg become phosphorylated to a greater degree after fertilization. One protein (48 kd) undergoes an increase followed by a decrease of its phosphorylation level.     

Cholera-toxin-dependent ADP-ribosylation of the adenylate cyclase regulatory protein in turkey erythrocyte membranes

Incubation of turkey erythrocyte membranes with cholera toxin and [³²P]NAD caused toxin-dependent incorporation of ³²P into a 42,000 M_r peptide which could be distinguished from toxin-independent ³²P incorporation into other membrane proteins. The radiolabeled 42,000 M_r peptide could be extracted from the membranes using Lubrol PX. When toxin-treated membranes were incubated with isoproterenol and GMP before detergent solubilization, the 42,000 M_r labeled peptide was adsorbed by GTP-γ-agarose which, with the same conditions, adsorbed the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide and guanine nucleotide regulatory protein activity were coeluted from the affinity matrix by guanylyl-β,γ-imidodiphosphate, GDP, and GMP. Guanosine 5′-O-(2-thiodiphosphate), an analog of GDP which blocks guanine nucleotide- and fluoride-stimulated adenylate cyclase activity, caused elution of labeled peptide which exhibited no regulatory protein activity. Our data support the view that the 42,000 M_r peptide is part of the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide allows identification of both active and inactive regulatory protein and should be useful in monitoring the purification of the regulatory protein from turkey erythrocytes.     

Studies on chromatin-associated protein phosphokinase of submandibular gland from isoproterenol-treated rats

Water-soluble chromatin from rat submandibular gland nuclei was isolated, and had a DNA: RNA:protein ratio of 8:1:20. The spectral properties of this preparation were similar to those described for chromatins from other tissues. The rat submandibular gland chromatin possessed protein phosphokinase activity. It was able to incorporate ³²P from [γ-³²P]-ATP into chromatin proteins, and into dephospho-phosvitin. The chromatin-associated protein phosphokinase activity (measured with dephospho-phosvitin as substrate) required Mg²⁺, Na⁺ or K⁺ and dithiothreitol for optimal activity. A single injection of isoproterenol influenced the activity of this enzyme system, so that it was decreased at 2 h, showed a transient increase at 4 h, and a large increase at 10–16 h after the injection. This event appears to precede the increase in ribosomal RNA induced by Ipr [13]. By 48 h the chromatin-associated protein kinase returned to the normal control levels. These changes appeared to be commensurate with the corresponding alterations in the non-histone acidic protein complement of these chromatins. Actinomycin D or cycloheximide, when administered 30 min prior to isoproterenol, blocked the increase in chromatin-associated protein kinase at 4 as well as 10 h after the injection of isoproterenol. Injection of pilocarpine did not influence the chromatin-associated protein phosphokinase activity. Dichloroisoproterenol appeared to be antagonistic to the influence of isoproterenol in mediating changes in chromatin-associated protein kinase. The results suggest that the isoproterenol-induced increase in chromatin-bound protein phosphokinase which precedes the increase in RNA synthesis is related to the eventual onset of DNA synthesis in rat submandibular gland stimulated by isoproterenol. The chromatin-bound protein phosphokinase activity (or activities) may have a regulatory role on gene action, mediated through the control of phosphorylation of nuclear non-histone acidic proteins [26].     

A study of adenosine 3′–5′ cyclic monophosphate binding sites of human erythrocyte membranes using 8-azidoadenosine 3′–5′ cyclic monophosphate,a photoaffinity probe

An earlier report (1a) has shown the utility of 8-N₃cAMP (8-azidoadenosine-3′, 5′-cyclic monophosphate) as a photoaffinity probe for cAMP binding sites in human erythrocyte membranes. The increased resolution obtained using a linear-gradient SDS polyacrylamide gel system now shows that: (1) both cAMP and 8-N₃cAMP stimulate the phosphorylation by [γ-³²P]-ATP of the same red cell membrane proteins; (2) the protein of approximately 48,000 molecular weight whose phosphorylation by [γ-³²P]-ATP is stimulated by cAMP and 8-N₃cAMP migrates at a solwer rate than the protein in the same molecular weight range which is heavily photolabeled with [³²P]-8-N₃cAMP; (3) other cyclic nucleotide binding sites exist besides those initailly reported; (4) the variation in the ratio of incorporation of ³²P-8-N₃cAMP into the two highest affinity binding sites appears to be the result of a specific proteolysis of the larger protein.     

Evolutionary analysis of prokaryotic heat-shock transcription regulatory protein σ³²

Heat-stress to any living cell is known to trigger a universal defense response, called heat-shock response, with rapid induction of tens of different heat-shock proteins. Bacterial heat-shock genes are transcribed by the σ³²-bound RNA polymerase instead of the normal σ⁷⁰-bound RNA polymerase. In this study, the diversity in sequence, variation in secondary structure and function amongst the different functional regions of the proteobacterial σ³² family of proteins, and their phylogenetic relationships have been analyzed. Bacterial σ³² proteins can be subdivided into different functional regions which are referred to as regions 2, 3, and 4. There is a great deal of sequence conservation among the functional regions of proteobacterial σ³² family of proteins though some mutations are also present in these regions. Region 2 is the most conserved one, while region 4 has comparatively more variable sequences. In the present work, we tried to explore the effects of mutations in these regions. Our study suggests that the sequence diversities due to natural mutations in the different regions of proteobacterial σ³² family lead to different functions. So far, this study is the first bioinformatic approach towards the understanding of the mechanistic details of σ³² family of proteins using the protein sequence information only. This study therefore may help in elucidating the hitherto unknown molecular mechanism of the functionalities of σ³²family of proteins.     

Inhibition of α-aminoisobutyric acid transport in membrane vesicles from mouse fibroblasts after phosphorylation by cyclic AMP-dependent protein kinase

Cyclic AMP-dependent protein kinases from several mammalian sources inhibit Na⁺-dependent α-aminoisobutyric acid transport by membrane vesicles isolated from 3T3 cells. Evidence is provided that phosphorylation of membrane proteins by the enzyme is responsible for the inhibition. Lysis of the vesicles, or a reduction in the intravesicular volume is not the cause of reduced transport.The cyclic AMP-dependent protein kinase and its catalytic subunit phosphorylate a number of membrane proteins. Most of these proteins are phosphorylated, but to a lesser extent in the absence of protein kinase or cyclic AMP. The phosphorylated proteins remain associated with the membranes during hypotonic lysis treatments, which would be expected to release intra-vesicular contents and loosely associated membrane proteins. ³²P-labeled bands detected on sodium dodecyl sulfate polyacrylamide gels after phosphorylation of membranes by the catalytic subunit of the cyclic AMP-dependent kinase are eliminated by treatment with either pronase or 1 N NaOH, but not by ribonuclease nor by phospholipase C. The stability of the incorporated radioactivity to hot acid and hydroxylamine relative to hot base suggests that most of the ³²P from [γ-³²P]ATP is incorporated into protein phosphomonoester linkages.     

Tyrosine kinases and calcium dependent activation of endothelial cell phospholipase D by diperoxovanadate

Reactive oxygen species (ROS) mediated modulation of signal transduction pathways represent an important mechanism of cell injury and barrier dysfunction leading to the development of vascular disorders. Towards understanding the role of ROS in vascular dysfunction, we investigated the effect of diperoxovanadate (DPV), derived from mixing hydrogen peroxide and vanadate, on the activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). Addition of DPV to BPAECs in the presence of .05% butanol resulted in an accumulation of [32P] phosphatidylbutanol (PBt) in a dose- and time-dependent manner. DPV also caused an increase in tyrosine phosphorylation of several protein bands (Mr 20-200 kD), as determined by western blot analysis with antiphosphotyrosine antibodies. The DPV-induced [³²P] PBt-accumulation was inhibited by putative tyrosine kinase inhibitors such as genistein, herbimycin, tyrphostin and by chelation of Ca²⁺ with either EGTA or BAPTA, however, pretreatment of BPAECs with the inhibitor PKC bisindolylmaleimide showed minimal inhibition. Also down-regulation of PKC and , the major isotypes of PKC in BPAECs, by TPA ( 100 nM, 18 h) did not attenuate the DPV-induced PLD activation. The effects of putative tyrosine kinase and PKC inhibitors were specific as determined by comparing [³²P] PBt formation between DPV and TPA. In addition to tyrosine kinase inhibitors, antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate also attenuated DPV-induced protein tyrosine phosphorylation and PLD stimulation. These results suggest that oxidation, prevented by reduction with thiol compounds, is involved in DPV-dependent protein tyrosine phosphorylation and PLD activation.     

Occurrence of phosphorylated proteins and kinase activity in coconut tissues cultured in vitro in a mediumthat induces somatic embryogenesis

The presence of tyrosine kinase and tyrosine-phosphorylated proteins was investigated in coconut tissues cultured in vitro. In order to study this phenomenon, plumular explants were taken from mature zygotic embryos and cultured in a medium that induces somatic embryogenesis. Immunoblot analyses of soluble proteins of coconut cultured tissues with a recombinant monoclonal antibody against phosphotyrosine detected protein bands with molecular masses ranging from 170 to 27 kDa. The highest response was exhibited by plumule-forming callus, which decreased both in number and intensity of bands with a longer time of in vitro culture. The specific immunodetection was corroborated by incubating the membranes with anti-phosphotyrosine antibody in the presence of 1 mM phosphotyrosine. Tyrosine phosphorylated proteins was also suggested by the presence of phosphoproteins resistant to alkaline treatment. In plumule, plumular callus and callus with globular embryos and shoots, a 41-kDa protein remained phosphorylated after alkaline treatment. In plumule, most [³²P]-proteins remained phosphorylated after alkaline treatment. Phosphoaminoacid analysis in protein hydrolysates from [³²P]-labelled 41-kDa protein showed the presence of [³²P]-tyrosine and [³²P]-threonine. Evaluation of tyrosine kinase activity in these tissues by the use of RR-SRC, a synthetic peptide substrate (derived from the amino acid sequence surrounding the phosphorylation site), showed that the activity was highest in plumule forming callus and initial explant, whereas in other tissues, tyrosine kinase activity decreased to values close to zero. Genistein, a specific tyrosine kinase inhibitor, diminished the ability of soluble extracts from coconut tissues cultured in vitro to incorporate ³²P into RR-SRC. These results suggest the presence of tyrosine phosphorylated proteins and tyrosine kinase activity in coconut tissues that have been cultured in vitro.     

Protein phosphorylation in intact lymphocytes stimulated by Concanavalin A

The phosphorylation of proteins in intact mouse spleen lymphocytes was monitored following mitogenic activation. Little change in the autoradiographic patterns of phosphorylated protein fractionated by polyacrylamide gel electrophoresis occurred during the first 8 h after Concanavalin A (conA) treatment. The intensity of ³²P incorporation into two proteins of 135 000 and 150 000 mol. wt began to increase, relative to control cells, 10 h after conA treatment and was maximal at 50 h. This increased phosphorylation followed the rise in RNA synthesis but preceded the onset of DNA synthesis. In addition to this temporal link between enhanced phosphorylation of these proteins and the initiation of DNA synthesis, various agents which inhibited the onset of S phase also blocked the phosphorylation of both proteins. Such treatments included the displacement of conA from its surface receptors by α-methyl-mannoside (αMM), the omission of serum from the culture medium, and the presence of indomethacin. The similar time courses of phosphorylation and responses to various proliferation inhibitors supports the idea that the 135 000 and 150 000 mol. wt proteins have a common physiological function. These proteins may be involved in the progression of stimulated lymphocytes toward S phase, and their phosphorylation may be an important regulatory event in this sequence.     

Altered phosphorylation of lung proteins following administration of butylated hydroxytoluene (BHT) to mice.

The incorporation of P³² from (γ?³²P)ATP into lung proteins was examined using lung extracts from mice which had been injected with BHT. Increased phosphorylation, relative to mice not treated with BHT, was associated with proteins of 87,000 M.W. (5–12 fold) and 135,000 M.W. (40–70%). These changes in phosphorylation correlated in time with the transient lung enlargement induced by BHT and were dependent upon the dose of BHT. Antioxidants and structural derivatives of BHT which did not affect lung size also had no effect on lung protein phosphorylation. BHT slightly increased cyclic AMP-dependent protein kinase activity but had no effect on cyclic AMP-binding activity.     

Benzo(a)pyrene quinone metabolism in tracheal organ cultures.

The C^? methyl group of methionine-29 of RNAase was enriched with ¹³C. The synthesis involved the reaction of RNAase with ¹³CH₃I at pH 4. S-Methylmethionine-29 RNAase was recovered in 80% yield. This sulfonium derivative was subsequently demethylated with 0.1 M mercaptoethanol at pH 8.5, 25°C for 4 days. These conditions allowed the demethylation reaction to successfully compete with the reaction of the thiol with the four disulfide bridges in RNAase. After dialysis, concentration and chromatography, native RNAase with approx. 50% of its Met²⁹ methyl groups enriched in ¹³C was recovered as was unreversed S-Methylmethionine-29 RNAase. Both proteins showed full enzymatic activity toward cytidine 2′:3′-cyclic monophosphate. ¹³C-methyl signals from enriched RNAase and the sulfonium derivative were observed at 13.8 and 26.7 ppm from TMS respectively. Preliminary denaturation studies with the methylated protein suggest that ¹³C enrichment of methionine methyl groups in RNAase will be a useful technique for following the unfolding transition at these sites of the protein.     

Protein phosphorylation in a dopamine-sensitive homogenate of rat caudate: Effect of adenosine 3′,5′-cyclic monophosphate and putative neurotransmitters

Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) and its 8-methylthio derivative stimulate the incorporation of ³²P into proteins endogenous to a homogenate of rat caudate nucleus when 4 μM [γ?³²P] ATP is usedas substrate. Higher concentrations of ATP reduced the effect of the cyclic nucleotide until at 400 μM no significant increase in protein phosphorylation was seen.Incubation of the homogenate with 400 μM ATP and 100 μM dopamine resulted in an approx. 2-fold increase in cyclic AMP but did not alter caudate protein phosphorylation suggesting that the catecholamine could not stimulate protein phosphorylation under the experimental conditions used in the present study.     

Effects of cyclic adenosine 3': 5'-monophosphate on phosphoprotein kinase and phosphatase fractions prepared from rat liver nuclei.

A soluble rat liver nuclear extract containing total RNA polymerase activities also exhibits appreciable amounts of protein kinase activity. This unfractionated protein kinase catalyzes the phosphorylation of both endogenous proteins and exogenous lysine-rich histone in the presence of [γ-³²P]ATP and Mg²⁺. The optimal concentration of Mg²⁺ is 5 mm for histone phosphorylation and 25 mm for the phosphorylation of endogenous proteins. Cyclic AMP has no effect on the phosphorylation of lysine-rich histone by this unfractionated nuclear protein kinase. However, addition of cyclic AMP causes a reduction in the ³²P-labeling of an endogenous protein (CAI) which can be characterized by its mobility during SDS-acrylamide gel electrophoresis and elution in the unbound fraction of a DEAESephadex column. If CAI is first labeled with ³²P and then incubated with 10^?6m cyclic AMP under conditions where protein kinase activity is inhibited, the presence of the cyclic nucleotide causes a loss of the ³²P-labeling of this protein, implying the activation of a substrate-specific protein phosphatase. When rat liver RNA polymerases are purified by DEAE-Sephadex chromatography, protein kinase activity is found in the unbound fraction and in those column fractions containing RNA polymerase I and II. The fractionated protein kinases exhibit different responses to cyclic AMP, the unbound protein kinase being stimulated and the RNA polymerase-associated protein kinases being dramatically inhibited. A second protein (CAII) whose phosphorylated state is modified by cyclic AMP is found within the DEAE-Sephadex column fractions containing RNA polymerase II. The cyclic nucleotide in this case appears to reduce labeling of CAII by inhibition of the protein kinase activity which co-chromatographs with both CAII and RNA polymerase II. Based on molecular weight estimates, neither CAI nor CAII appears to be an RNA polymerase subunit. The identity of CAI as a protein factor whose phosphorylated state influences nuclear RNA synthesis is suggested by the fact that addition of fractions containing CAI to purified RNA polymerase II inhibits the activity of this enzyme, but only if CAI has been previously incubated in the presence of cyclic AMP.     

External cell surface protein phosphorylation in normal and Rous sarcoma virus transformed chick embryo fibroblasts

Normal and Rous sarcoma virus (RSV)-transformed chick embryo fibroblasts growing on plastic dishes were incubated with ATP (γ³²P) in situ to detect external cell surface protein kinase activity. Under the conditions employed, ³²P was incorporated exclusively into proteins, specifically those at the external cell surface, as radioactivity was removed by tryspin treatment of labeled whole cells. In addition, exogenous histones were phosphorylated when added to the reaction mixture. Cyclic nucleotides had virtually no effect on ³²P incorporation, suggesting that little or no cyclic nucleotide-dependent protein kinase activity was present at the external cell surface. Cell surface protein kinase activity was higher in transformed than in normal cells, and, using a temperature-sensitive RSV src mutant, this difference was shown to be transformation-specific. Several differences were observed in the cell surface proteins phosphoryllated in normal and transformed cells and at least two of these were transformation-specific. These data suggest that changes in external cell surface protein physphorylation are associated with RSV transformation and thus could play a role in the formation of the transformed cell phenotype.     

Interactions of acetylcholine receptor and acetylcholinesterase with lipid monolayers

The interaction of acetylcholine receptor and acetylcholinesterase with lipid monolayers was followed by measuring changes in surface pressure.When injected into the subphase of a lipid monolayer, the proteins caused increases in surface pressure from 5 to 10 dynes/cm, indicating a penetration of protein into the monolayer. At pH values below the isoelectric point of the proteins the incorporation was improved. The same was observed when Ca²⁺ (2 mM) was added.The presence of the enzyme in the mixed film could be demonstrated by using diiso[³H]propyl fluorophosphate-labelled acetylcholinesterase as well as by measuring enzyme activity. Acetylcholine receptor was shown to be present in the mixed film by using a complex made of the receptor and α-[³H]neurotoxin.     

Orchestration of Haemophilus influenzae RecJ Exonuclease by Interaction with Single-Stranded DNA-Binding Protein

RecJ exonuclease plays crucial roles in several DNA repair and recombination pathways, and its ubiquity in bacterial species points to its ancient origin and vital cellular function. RecJ exonuclease from Haemophilus influenzae is a 575-amino-acid protein that harbors the characteristic motifs conserved among RecJ homologs. The purified protein exhibits a processive 5′-3′ single-stranded-DNA-specific exonuclease activity. The exonuclease activity of H. influenzae RecJ (HiRecJ) was supported by Mg^2 + or Mn^2 + and inhibited by Cd^2 +, suggesting a different mode of metal binding in HiRecJ as compared to Escherichia coli RecJ (EcoRecJ). Site-directed mutagenesis of highly conserved residues in HiRecJ abolished enzymatic activity. Interestingly, substitution of alanine for aspartate 77 resulted in a catalytically inactive enzyme that bound to DNA with a significantly higher affinity as compared to the wild-type enzyme. Noticeably, steady-state kinetic studies showed that H. influenzae single-stranded DNA-binding protein (HiSSB) increased the affinity of HiRecJ for single-stranded DNA and stimulated its exonuclease activity. HiSSB, whose C-terminal tail had been deleted, failed to enhance RecJ exonuclease activity. More importantly, HiRecJ was found to directly associate with its cognate single-stranded DNA-binding protein (SSB), as demonstrated by various in vitro assays. Interaction studies carried out with the truncated variants of HiRecJ and HiSSB revealed that the two proteins interact via the C-terminus of SSB protein and the core-catalytic domain of RecJ. Taken together, these results emphasize direct interaction between RecJ and SSB, which confers functional cooperativity to these two proteins. In addition, these results implicate SSB as being involved in the recruitment of RecJ to DNA and provide insights into the interplay between these proteins in repair and recombination pathways.