Affinity of Synthetic Peptides for the HSV-2 Ribonucleotide Reductase R1 Subunit Measured with an Iodinated Photoaffinity Peptide

The herpes simplex virus (HSV) ribonucleotide reductase comprises two nonidentical subunits, R1 and R2, which associate to form the active holoenzyme. A sensitive binding assay was developed to measure the affinity of inhibitory peptides for the HSV R1 subunit. The assay involved the use of a photoreactive radioligand [4′-azido-Phe³²⁸,3′,5′-¹²⁵I-Tyr³²⁹] HSV R2-(328-337), an analogue of the decapeptide Ser-Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu which corresponds to the C-terminal sequence (328-337) of the HSV R2 protein. As the radioligand binds covalently to the HSV R1 subunit upon uv irradiation, the affinity of peptide inhibitors can be easily determined by measuring their ability to compete with this highly specific binding. The method, which did not require any pure preparation of R1, was tested at 25 and 4°C and showed a significant increase in the affinity of the peptide inhibitors at 4°C. The relative affinity of these peptides was in agreement with their relative potency to inhibit reductase activity. The affinity of R2 subunit for R1 was also determined, and an IC₅₀ of 0.05 μM was measured. Altogether, this assay represents a precise and reliable tool with which to study more potent HSV ribonucleotide reductase peptide inhibitors, and the method could be applied to the study of other protein-protein and peptide-protein interactions.     

An autophosphorylating but not transphosphorylating activity is associated with the unique N terminus of the herpes simplex virus type 1 ribonucleotide reductase large subunit.

We report on a protein kinase function encoded by the unique N terminus of the herpes simplex virus type 1 (HSV-1) ribonucleotide reductase large subunit (R1). R1 expressed in Escherichia coli exhibited autophosphorylation activity in a reaction which depended on the presence of the unique N terminus. When the N terminus was separately expressed in E. coli and partially purified, a similar autophosphorylation reaction was observed. Importantly, transphosphorylation of histones and of proteins in HSV-1-infected cell extracts was also observed with purified R1 and with truncated R1 mutants in which most of the N terminus was deleted. Ion-exchange chromatography was used to separate the autophosphorylating activity of the N terminus from the transphosphorylating activity of an E. coli contaminant protein kinase. We propose a putative function for this activity of the HSV-1 R1 N terminus during the immediate-early phase of virus replication.     

Characterization of the novel protein kinase activity present in the R1 subunit of herpes simplex virus ribonucleotide reductase.

We have compared the protein kinase activities of the R1 subunits from herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) ribonucleotide reductase following expression in Escherichia coli. Autophosphorylation activity was observed when kinase assays were performed with immunoprecipitated R1 or proteins purified to homogeneity, and the activity was stimulated by the basic protein protamine. Transphosphorylation of histones or calmodulin by purified or immunoprecipitated HSV-1 and HSV-2 R1 was not observed, and our results suggest that the activities of these two proteins are similar. We further characterized the protein kinase activity of HSV-1 R1 by producing insertion and deletion mutants constructed with a plasmid expressing R1 amino acids 1 to 449. C-terminal deletion analysis identified the catalytic core of the enzyme as comprising residues 1 to 292, and this polypeptide will be useful for structural determinations by X-ray crystallography. Insertion of a 4-amino-acid sequence at sites within the protein kinase domain identified regions essential for activity; insertions at residues 22 and 112 completely inactivated activity, and an insertion at residue 136 reduced activity sixfold. Similar insertions at residues 257, 262, 292, and 343 had no effect on activity. The ATP analog 5'-fluorosulfonylbenzoyladenosine, which covalently modifies conventional eukaryotic kinases at an essential lysine residue within the active site, did label HSV R1, but this labelling occurred outside the N-terminal domain. These data indicate that the HSV R1 kinase is novel and distinct from other eukaryotic protein kinases.     

Herpes simplex virus ribonucleotide reductase: expression in Escherichia coli and purification to homogeneity of a tyrosyl free radical-containing, enzymatically active form of the 38-kilodalton subunit.

Infection of mammalian cells with herpes simplex virus (HSV) induces a virus-encoded ribonucleotide reductase which is different from the cellular enzyme. This essential viral enzyme consists of two nonidentical subunits of 140 and 38 kilodaltons (kDa) which have not previously been purified to homogeneity. The small subunit of ribonucleotide reductases from other species contains a tyrosyl free radical essential for activity. We have cloned the gene for the small subunit of HSV-1 ribonucleotide reductase into a tac expression plasmid vector. After transfection of Escherichia coli, expression of the 38-kDa protein was detected in immunoblots with a specific monoclonal antibody. About 30 micrograms of protein was produced per liter of bacterial culture. The 38-kDa protein was purified to homogeneity in an almost quantitative yield by immunoaffinity chromatography. It contained a tyrosyl free radical which gave a specific electron paramagnetic resonance spectrum identical to that we have observed in HSV-infected mammalian cells and clearly different from that produced by the E. coli and mammalian ribonucleotide reductases. The recombinant 38-kDa subunit had full activity when assayed in the presence of HSV-infected cell extracts deficient in the native 38-kDa subunit.     

The R1 subunit of herpes simplex virus ribonucleotide reductase has chaperone-like activity similar to Hsp27

HSV-2 R1, the R1 subunit of herpes simplex virus (HSV) ribonucleotide reductase, protects cells against apoptosis. Here, we report the presence in HSV-2 R1 of a stretch exhibiting similarity to the alpha-crystallin domain of the small heat shock proteins, a domain known to be important for oligomerization and cytoprotective activities of these proteins. Also, the HSV-2 R1 protein, which forms multimeric structures in the absence of nucleotide, displayed chaperone ability as good as Hsp27 in a thermal denaturation assay using citrate synthase. In contrast, mammalian R1, which does not contain an alpha-crystallin domain, has neither chaperone nor anti-apoptotic activity. Thus, we propose that the chaperone activity of HSV-2 R1 could play an important role in viral pathogenesis.     

The role of herpes simplex virus ribonucleotide reductase small subunit carboxyl terminus in subunit interaction and formation of iron-tyrosyl center structure.

Herpes simplex virus ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, which are required together for activity. Active R2 protein contains a tyrosyl free radical and a binuclear iron center. A truncated form of the R2 subunit, lacking 7 amino acid residues in the carboxyl terminus, was constructed, overexpressed in Escherichia coli and purified to homogeneity. In the presence of ferrous iron and oxygen, the truncated protein readily generated similar amounts of tyrosyl free radical as the intact protein. However, the radical showed differences in EPR characteristics in the truncated protein compared with the normal one, indicating an altered structural arrangement of the radical relative to the iron center. The truncated R2* protein was completely devoid of binding affinity to the R1 protein, demonstrating that the subunit interaction is totally dependent on the 7 outermost carboxyl-terminal amino acids of protein R2.     

Interactions between regulatory and catalytic subunits of the Candida albicans cAMP-dependent protein kinase are modulated by autophosphorylation of the regulatory subunit

The cAMP-dependent protein kinase (PKA) from Candida albicans is a tetramer composed of two catalytic subunits (C) and two type II regulatory subunits (R). To evaluate the role of a putative autophosphorylation site of the R subunit (Ser(180)) in the interaction with C, this site was mutated to an Ala residue. Recombinant wild-type and mutant forms of the R subunit were expressed in Escherichia coli and purified. The wild-type recombinant R subunit was fully phosphorylated by the purified C subunit, while the mutant form was not, confirming that Ser(180) is the target for the autophosphorylation reaction. Association and dissociation experiments conducted with both recombinant R subunits and purified C subunit showed that intramolecular phosphorylation of the R subunit led to a decreased affinity for C. This diminished affinity was reflected by an 8-fold increase in the concentration of R subunit needed to reach half-maximal inhibition of the kinase activity and in a 5-fold decrease in the cAMP concentration necessary to obtain half-maximal dissociation of the reconstituted holoenzyme. Dissociation of the mutant holoenzyme by cAMP was not affected by the presence of MgATP. Metabolic labeling of yeast cells with [(32)P]orthophosphate indicated that the R subunit exists as a serine phosphorylated protein. The possible involvement of R subunit autophosphorylation in modulating C. albicans PKA activity in vivo is discussed.     

Protein kinase activity associated with the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10).

The large subunit of the herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (RR1) is demonstrated to possess serine/threonine-specific kinase activity. Computer-assisted sequence analysis identified regions within the amino terminus of ICP10 that are homologous to the catalytic domain of known protein kinases (PKs). An in vitro kinase assay confirmed the ability of ICP10, immunoprecipitated from either HSV-2-infected cells or from cells transfected with an ICP10 expression vector, to autophosphorylate and transphosphorylate exogenous substrates in the presence of ATP and Mg2+. The HSV-1 RR1 was shown to be negative for PK activity under these conditions. PK activity was localized to a 57-kDa amino-terminal region within ICP10 that lacked RR activity.     

PKA from Mucor circinelloides: model to study the role of linker I in the interaction between R and C subunits

Protein kinase A from the fungus Mucor circinelloides shows high affinity interaction between regulatory (R) and catalytic (C) subunits. Its R subunit shows a differential presence of several acidic residues in linker I region, in the amino terminus. Mutants R1, lacking the N-terminal region, and R2, lacking the acidic cluster, were used to analyze its effect on the interaction with the C subunit, assessed through inhibition of catalytic activity and cAMP activation of reconstituted holoenzyme. A similar decrease in the interaction was obtained when using R1 and R2 with the homologous C subunit; however when using heterologous bovine C, only R1 had a decreased interaction. The results show the general importance of linker I region in the R-C interaction in protein kinases A and point to the importance of the acidic cluster present in the N-terminus of M. circinelloides R subunit in the high affinity interaction between R and C in this holoenzyme.     

Cloning of the vaccinia virus ribonucleotide reductase small subunit gene. Characterization of the gene product expressed in Escherichia coli.

During its infectious cycle, vaccinia virus expresses a virus-encoded ribonucleotide reductase which is distinct from the host cellular enzyme (Slabaugh, M.B., and Mathews, C.K. (1984) J. Virol. 52, 501-506; Slabaugh, M.B., Johnson, T.L., and Mathews, C.K. (1984) J. Virol. 52, 507-514). We have cloned the gene for the small subunit of vaccinia virus ribonucleotide reductase (designated VVR2) into Escherichia coli and expressed the protein using a T7 RNA polymerase plasmid expression system. After isopropyl beta-D-thiogalactopyranoside induction, accumulation of a 37-kDa peptide was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this peptide reacted with polyclonal antiserum raised against a TrpE-VVR2 fusion protein. The 37-kDa protein was purified to homogeneity, and gel filtration of the purified protein revealed that the recombinant protein existed as a dimer in solution. Purified recombinant VVR2 protein was shown to complement the activity of purified recombinant ribonucleotide reductase large subunit, with a specific activity that was similar to native VVR2 from a virus-infected cell extract. A CD spectrum of the recombinant viral protein showed that like the mouse protein, the vaccinia virus protein has 50% alpha-helical structure. Like other iron-containing ribonucleotide reductase small subunits, recombinant VVR2 protein contained a stable organic free radical that was detectable by EPR spectroscopy. The EPR spectrum of purified recombinant VVR2 was identical to that of vaccinia virus-infected mammalian cells. Both the hyperfine splitting character and microwave saturation behavior of VVR2 were similar to those of mouse R2 and distinct from E. coli R2. By using amino acid analysis to determine the concentration of VVR2, we determined that approximately 0.6 radicals were present per R2 dimer. Our results indicate that vaccinia virus small subunit is similar to mammalian ribonucleotide reductases.     

Studies on in vitro proteolytic sensitivity of peptides inhibiting herpes simplex virus ribonucleotide reductases lead to discovery of a stable and potent inhibitor.

The nonapeptide, HSV R2-(329-337), corresponding to the subunit 2 (R2) carboxyl terminus of herpes simplex virus (HSV) ribonucleotide reductases, specifically inhibits this enzyme activity. We report here that under standard reductase assay conditions, this peptide was rapidly degraded by proteases present in the partially purified enzyme extract. The main process of proteolysis involves the successive removal of Tyr329 and Ala330, which corresponds to an aminopeptidase activity. Determination of the proteolytic susceptibility of HSV R2-(329-337) analogs showed that natural modifications which are present in the homologous varicella zoster virus (VZV) nonapeptide decreased its susceptibility to protease action 1.5-fold. Nx-acetylation, a modification known to protect peptides against aminopeptidase attacks, greatly improved the proteolytic resistance of HSV and VZV nonapeptides. Moreover, Ac-VZV R2-(298-306) exhibited a 15-fold higher potency on reductase inhibition than HSV R2-(329-337). The degradation process of HSV R2-(329-337) was partially inhibited by amastatin, bestatin, and leupeptin whereas it was completely abolished by bacitracin, suggesting a combined action of more than one aminopeptidase activity. Moreover, bacitracin protected most of these nonapeptide analogs from proteolysis, although it was less effective in preventing HSV R2-(332-337) degradation. Our results indicate that it is possible to determine, in the presence of bacitracin, the relative inhibitory potencies of HSV R2-(329-337) analogs with minimal error due to proteolytic susceptibility. Moreover, HSV R2-(329-337) modifications that were found to protect the peptide against degradation might be useful to increase its efficacy in vivo.     

Cloning and characterization of ribonucleotide reductase from Chlamydia trachomatis

In all organisms the deoxyribonucleotide precursors required for DNA synthesis are synthesized from ribonucleotides, a reaction catalyzed by ribonucleotide reductase. In a previous study we showed that Chlamydia trachomatis growth was inhibited by hydroxyurea, an inhibitor of ribonucleotide reductase, and a mutant resistant to the cytotoxic effects of the drug was isolated. Here we report the cloning, expression, and purification of the R1 and R2 subunits of the C. trachomatis ribonucleotide reductase. In comparison with other ribonucleotide reductases, the primary sequence of protein R1 has an extended amino terminus, and the R2 protein has a phenylalanine where the essential tyrosine is normally located. Despite its unusual primary structure, the recombinant enzyme catalyzes the reduction of CDP to dCDP. Results from deletion mutagenesis experiments indicate that while the extended amino terminus of the R1 protein is not required for enzyme activity, it is needed for allosteric inhibition mediated by dATP. Results with site-directed mutants of protein R2 suggest that the essential tyrosine is situated two amino acids downstream of its normal location. Finally, Western blot analysis show that the hydroxyurea-resistant mutant C. trachomatis isolate overexpresses both subunits of ribonucleotide reductase. At the genetic level, compared with wild type C. trachomatis, the resistant isolate has a single base mutation just upstream of the ATG start codon of the R2 protein. The possibility that this mutation affects translational efficiency is discussed.     

Stimulation of cellular signaling and G protein subunit dissociation by G protein betagamma subunit-binding peptides

We previously developed peptides that bind to G protein betagamma subunits and selectively block interactions between betagamma subunits and a subset of effectors in vitro (Scott, J. K., Huang, S. F., Gangadhar, B. P., Samoriski, G. M., Clapp, P., Gross, R. A., Taussig, R., and Smrcka, A. V. (2001) EMBO J. 20, 767-776). Here, we created cell-permeating versions of some of these peptides by N-terminal modification with either myristate or the cell permeation sequence from human immunodeficiency virus TAT protein. The myristoylated betagamma-binding peptide (mSIRK) applied to primary rat arterial smooth muscle cells caused rapid activation of extracellular signal-regulated kinase 1/2 in the absence of an agonist. This activation did not occur if the peptide lacked a myristate at the N terminus, if the peptide had a single point mutation to eliminate betagamma subunit binding, or if the cells stably expressed the C terminus of betaARK1. A human immunodeficiency virus TAT-modified peptide (TAT-SIRK) and a myristoylated version of a second peptide (mSCAR) that binds to the same site on betagamma subunits as mSIRK, also caused extracellular signal-regulated kinase activation. mSIRK also stimulated Jun N-terminal kinase phosphorylation, p38 mitogen-activated protein kinase phosphorylation, and phospholipase C activity and caused Ca2+ release from internal stores. When tested with purified G protein subunits in vitro, SIRK promoted alpha subunit dissociation from betagamma subunits without stimulating nucleotide exchange. These data suggest a novel mechanism by which selective betagamma-binding peptides can release G protein betagamma subunits from heterotrimers to stimulate G protein pathways in cells.     

Isolation and characterization of recombinant human casein kinase II subunits alpha and beta from bacteria

cDNA encoding the casein kinase II (CKII) subunits alpha and beta of human origin were expressed in Escherichia coli using expression vector pT7-7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most of the expressed proteins were produced in an insoluble form. The recombinant CKII alpha subunit was purified by DEAE-cellulose chromatography, followed by phosphocellulose and heparin-agarose chromatography. The recombinant CKII beta subunit was extracted from the insoluble pellet and purified in a single step on phosphocellulose. From 10 g bacterial cells, the yield of soluble protein was 12 mg alpha subunit and 5 mg beta subunit. SDS/PAGE analysis of the purified recombinant proteins indicated molecular masses of 42 kDa and 26 kDa for the alpha and beta subunits, respectively, in agreement with the molecular masses determined for the subunits of the native enzyme. The recombinant alpha subunit exhibited protein kinase activity which was greatest in the absence of monovalent ions. With increasing amounts of salt, alpha subunit kinase activity declined rapidly. Addition of the beta subunit led to maximum stimulation at a 1:1 ratio of both subunits. Using a synthetic peptide (RRRDDDSDDD) as a substrate, the maximum protein kinase stimulation observed was fourfold under the conditions used. The Km of the reconstituted enzyme for the synthetic peptide (80 microM) was comparable to the mammalian enzyme (40-60 microM), whereas the alpha subunit alone had a Km of 240 microM. After sucrose density gradient analysis, the reconstituted holoenzyme sedimented at the same position as the mammalian CKII holoenzyme.     

Improvement of a simple method to purify ribonucleotide reductase

The use of an ATP-agarose column to purify ribonucleotide reductase from human D-98 cells was recently reported. The column selectively retains greater than 99.9% of the contaminating nucleoside diphosphate (NDP) kinase from crude preparations of ribonucleotide reductase. It was presently found, however, that extending the length of the column caused the ribonucleotide reductase to dissociate into subunits. One subunit appeared in the low ionic strength buffer wash while the other required 0.5 M KCl for elution. The enzyme could also be recovered intact (non-dissociated) by equilibrating the enzyme preparation and the column with 0.5 M KCl prior to chromatography. Either method greatly improved the overall yield and the specific activity of the ribonucleotide reductase because it prevented the binding and subsequent loss of any of the subunits. In addition, the use of a larger column permitted the gel-filtration properties of the ATP-agarose to separate the bulk of the residual (not bound) NDP kinase from the ribonucleotide reductase.     

Phosphorylation and modulation of the enzymic activity of native and protease-cleaved purified hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase by a calcium/calmodulin-dependent protein kinase

A Ca2+/calmodulin-dependent kinase has been purified which catalyzed the phosphorylation and concomitant inactivation of both the microsomal native (100,000 Da) and protease-cleaved purified 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) (53,000 Da) fragments. This low molecular weight brain cytosolic Ca2+/calmodulin-dependent kinase phosphorylates histone H1, synapsin I, and purified HMG-CoA reductase as major substrates. The kinase, purified by sequential chromatography on DEAE-cellulose, calmodulin affinity resin, and high performance liquid chromatography (TSKG 3000 SW) is an electrophoretically homogeneous protein of approximately 110,000 Da. The molecular weight of the holoenzyme, substrate specificity, subunit protein composition, subunit autophosphorylation, subunit isoelectric points, and subunit phosphopeptide analysis suggest that this kinase of Mr 110,000 may be different from other previously reported Ca2+/calmodulin-dependent kinases. Maximal phosphorylation by the low molecular form of Ca2+/calmodulin-dependent kinase of purified HMG-CoA reductase revealed a stoichiometry of approximately 0.5 mol of phosphate/mol of 53,000-Da enzyme. Dephosphorylation of phosphorylated and inactivated native and purified HMG-CoA reductase revealed a time-dependent loss of 32P-bound radioactivity and reactivation of enzyme activity. Based on the results reported here, we propose that HMG-CoA reductase activity may be modulated by yet another kinase system involving covalent phosphorylation. The elucidation of a Ca2+/calmodulin-dependent HMG-CoA reductase kinase-mediated modulation of HMG-CoA reductase activity involving reversible phosphorylation may provide new insights into the molecular mechanisms involved in the regulation of cholesterol biosynthesis.     

Improvement of a Simple Method to Purify Ribonucleotide Reductase

Abstract

The use of an ATP-agarose column to purify ribonucleotide reductase from human D-98 cells was recently reported.¹ The column selectively retains < 99.9% of the contaminating nucleoside diphosphate (NDP) kinase from crude preparations of ribonucleotide reductase. It was presently found, however, that extending the length of the column caused the ribonucleotide reductase to dissociate into subunits. One subunit appeared in the low ionic strength buffer wash while the other required 0.5 M KC1 for elution. The enzyme could also be recovered Intact (non-dissociated) by equilibrating the enzyme preparation and the column with 0.5 M KC1 prior to chromatography. Either method greatly improved the overall yield and the specific activity of the ribonucleotide reductase because it prevented the binding and subsequent loss of any of the subunits. In addition, the use of a larger column permitted the gel-filtration properties of the ATP-agarose to separate the bulk of the residual (not bound) NDP kinase from the ribonucleotide reductase.     

Purification and characterization of Snf1 kinase complexes containing a defined Beta subunit composition

The Snf1 kinase complex of Saccharomyces cerevisiae contains one of three possible beta subunits encoded by either SIP1, SIP2, or GAL83. Snf1 kinase complexes were purified from cells expressing only one of the three beta subunits using a tandem affinity purification tag on the C terminus of the Snf1 protein. The purified kinase complexes were enzymatically active as judged by their ability to phosphorylate a recombinant protein containing the Snf1-responsive domain of the Mig1 protein. The Snf1 kinase complexes containing Gal83 or Sip2 as the beta subunit showed comparable and high levels of activity, whereas the Sip1-containing enzyme was significantly less active. Examination of the protein composition of the purified Snf1 enzyme complexes indicated that the Sip1 protein was present in substoichiometric levels. Increased gene dosage of SIP1 rescued the ethanol growth defect observed in cells expressing Sip1 as their only beta subunit and increased the in vitro activity of Snf1 kinase purified from these cells. Our studies indicate that the reduced activity of Snf1-Snf4-Sip1 kinase is due to low level of Sip1 accumulation rather than a limited ability of the Sip1 form of the enzyme to direct phosphorylation of specific substrates.     

The PK Domain of the Large Subunit of Herpes Simplex Virus Type 2 Ribonucleotide Reductase (ICP10) Is Required for Immediate-Early Gene Expression and Virus Growth

The large subunit of herpes simplex virus (HSV) ribonucleotide reductase (RR), RR1, contains a unique amino-terminal domain which has serine/threonine protein kinase (PK) activity. To examine the role of the PK activity in virus replication, we studied an HSV type 2 (HSV-2) mutant with a deletion in the RR1 PK domain (ICP10ΔPK). ICP10ΔPK expressed a 95-kDa RR1 protein (p95) which was PK negative but retained the ability to complex with the small RR subunit, RR2. Its RR activity was similar to that of HSV-2. In dividing cells, onset of virus growth was delayed, with replication initiating at 10 to 15 h postinfection, depending on the multiplicity of infection. In addition to the delayed growth onset, virus replication was significantly impaired (1,000-fold lower titers) in nondividing cells, and plaque-forming ability was severely compromised. The RR1 protein expressed by a revertant virus [HSV-2(R)] was structurally and functionally similar to the wild-type protein, and the virus had wild-type growth and plaque-forming properties. The growth of the ICP10ΔPK virus and its plaque-forming potential were restored to wild-type levels in cells that constitutively express ICP10. Immediate-early (IE) genes for ICP4, ICP27, and ICP22 were not expressed in Vero cells infected with ICP10ΔPK early in infection or in the presence of cycloheximide, and the levels of ICP0 and p95 were significantly (three- to sevenfold) lower than those in HSV-2- or HSV-2(R)-infected cells. IE gene expression was similar to that of the wild-type virus in cells that constitutively express ICP10. The data indicate that ICP10 PK is required for early expression of the viral regulatory IE genes and, consequently, for timely initiation of the protein cascade and HSV-2 growth in cultured cells.     

Chromatographic behavior of cyclic AMP dependent protein kinase and its subunits from Dictyostelium discoideum

An adenosine cyclic 3',5'-monophosphate (cAMP) dependent protein kinase has recently been shown to exist in Dictyostelium discoideum and to be developmentally regulated. In this report we have followed the chromatographic behavior of both the holoenzyme and its subunits. A cAMP-dependent holoenzyme could be obtained from the 100000 g soluble fraction after passage through DE-52 cellulose (pH 7.5) and Sephacryl S300. Under conditions of low pH the holoenzyme could be further purified by flat-bed electrofocusing (pI = 6.8). Application of the holoenzyme to electrofocusing at high pH resulted in dissociation of the holoenzyme into a cAMP binding component (pI = 6.1) and a cAMP-independent catalytic activity (pI = 7.4). Dissociation of the holoenzyme into subunits also occurred during histone affinity chromatography and gel filtration chromatography (S300) in the presence of a dissociating buffer. Although the subunit structure was clearly evident during chromatography, the holoenzyme could not be dissociated by simple addition of cAMP to the extract. The catalytic subunit could be purified further by CM-Sephadex, DE-52 cellulose (pH 8.5), histone affinity, and hydrophobic chromatography. The regulatory subunit was further purified by DE-52 cellulose (pH 8.5) and cAMP affinity chromatography. Proof that the cAMP binding activity and the cAMP-independent catalytic activity were in fact the regulatory and catalytic subunits was shown by reconstitution of the cAMP-dependent holoenzyme from the purified subunits. By using these separation procedures, one can obtain from extracts of Dictyostelium the subunits that are free of each other as well as free of any endogenous protein substrates.