Deoxythymidine kinase induced in HeLa TK- cells by herpes simplex virus type I and type II. II. Purification and characterization.

Deoxythymidine kinase activities were induced in HeLa TK- (deoxythymidine kinase-deficient) cells infected with either herpes simplex virus type I or herpes simplex virus type II. The herpes simplex virus type I-induced enzyme was found in the cytoplasmic and nuclear fractions of the infected cells, whereas the herpes simplex type II-induced deoxythymidine kinase could only be found in the cytoplasm. Herpes simplex virus type I and II specific deoxythymidine kinases were purified by affinity column chromatography. Both purified deoxythymidine kinases retained the deoxycytidine kinase activity present in the crude preparation. The purified herpes simplex virus type I deoxythymidine kinase had a different mobility on electrophoresis, but the same sedimentation rate on a glycerol gradient as the corresponding unpurified enzyme, whereas the purified herpes simplex virus type II deoxythymidine kinase had the same mobility and sedimentation rate as the corresponding unpurified enzyme. In the presence of Mg2+ATP and dithiothreitol, herpes simplex virus type II deoxythymidine kinase was more stable than herpes simplex virus type I deoxythymidine kinase at both 45 degrees and 4 degrees. The deoxycytidine kinase activity present in the purified preparations was inactivated at the same rate as the deoxythymidine kinase activity. In the presence of the other substrate, deoxythymidine, herpes simplex virus type I deoxythymidine kinase was more stable than herpes simplex virus type II kinase. The purified herpes simplex virus type I and II deoxythymidine kinase had different activation energies when Mg2+ATP and deoxythymidine were used as substrates, but showed the same sensitivity toward ammonium sulfate inhibition.     

Human deoxythymidine kinase II: substrate specificity and kinetic behavior of the cytoplasmic and mitochondrial isozymes derived from blast cells of acute myelocytic leukemia.

Cytoplasmic and mitochondrial deoxythymidine kinase isozymes derived from the blast cells of acute myelocytic leukemia differ in their substrate specificity and kinetic behavior. These enzymes require divalent cations for their activity. The data suggest that the major role of idvalent cations is to chelate with ATP; the complex thus formed serves as the phosphate donor for the reaction. The activity of various triphosphate nucleosides as a phosphate donor for cytoplasmic deoxythymidine kinase is as follows: ATP = dATP greater than ara-ATP greater than GTP greater than CTP greater than dGTP = dCTP greater than dUTP, whereas for mitochondrial deoxythymidine kinase, the order of activity is ATP greater than CTP greater than UTP = dATP greater than ara-ATP greater than dGTP = dCTP greater than dUTP. Neither IdUTP nor dTTP could serve as a phosphate donor in the reaction catalyzed by either isozyme. From the many pyrimidine analogues tested for their binding affinity to each of these isozymes, I-dUrd and Br-dUrd had high good affinity which was equivalent to that of deoxythymidine. 5-Allyl-dUrd, 5-ethyl-dUrd, and 5-propyl-dUrd were only weakly bound to each isozyme. 5-I-dCyd, 5-Br-dCyd, dCyd, and 5-vinyl-dUrd were tightly bound to mitochondrial deoxythymidine kinase but not to the cytoplasmic isozyme. dTTP and I-dUTP are potent inhibitors of the reaction catalyzed by both isozymes. In contrast, dCTP and ara-CTP are potent inhibitors only of the mitochondrial isozyme, but not of the cytoplasmic isozyme. ATP-MG2+ acts as a sigmoidal substrate of the cytoplasmic isozyme with a"Km" of 0.22 mM, and as a regular substrate of the mitochondrial isozyme with a Km of 0.1 mM. Deoxythymidine acts as a regular substrate for both cytoplasmic and mitochondrial isozyme with a Km of 2.6 and 5.2 muM, respectively. Initial velocity as well as product inhibition studies suggest that the cytoplasmic isozyme catalyzes the reaction via a "sequential" mechanism. In contrast, mitochondrial deoxythymidine kinase catalyzes the reaction via a "ping-pong" mechanism.     

Adenosine diphosphate: thymidine 5'-phosphotransferase, a new enzyme activity, associated with the Herpes simplex virus-induced deoxypyrimidine kinase

The deoxypyrimidine kinase induced in mouse fibroblasts, strain CLID (TK-) infected with either herpes simplex virus (HSV) type 1 or type 2, possesses besides deoxypyrimidine kinase (ATP:dThd/dCyd phosphotransferase) two further enzyme activities: an AMP:dThd phosphotransferase and an ADP:dThd phosphotransferase. The latter enzyme activity, described in this report, was found to be inhibited by antiserum against the HSV deoxypyrimidine kinase and to be absent after infection with TK- mutant MDK 10 (B 2006). The ADP:dThd phosphotransferase, which had been purified approx. 340-fold, differs by a series of physicochemical properties from the viral AMP:dThd- and ATP:dThd phosphotransferase.     

Identification of purine deoxyribonucleoside kinases from human leukemia cells: substrate activation by purine and pyrimidine deoxyribonucleosides

Cell extracts from human leukemic T lymphoblasts and myeloblasts were chromatographed on DEAE-cellulose columns to separate purine deoxyribonucleoside, deoxyadenosine (dAdo) and deoxyguanosine (dGuo), phosphorylating activities. Three distinct purine deoxyribonucleoside kinases, a deoxycytidine (dCyd) kinase, an adenosine (Ado) kinase, and a deoxyguanosine (dGuo) kinase (the latter appears to be localized in mitochondria), were resolved. dCyd kinase contained the major phosphorylating activity for dAdo, dGuo, and 9-beta-D-arabinofuranosyladenine (ara-A). Ado kinase represented a second kinase for dAdo and ara-A while a third kinase for dAdo was found in mitochondria. dCyd kinase was purified about 2000-fold with ion-exchange, affinity, and hydrophobic chromatographies. On gel electrophoresis, both dCyd and dAdo phosphorylating activities comigrated, indicating that the activities are associated with the same protein. The enzyme showed a broad pH optimum ranging from pH 6.5 to pH 9.5. Divalent cations Mg2+, Mn2+, and Ca2+ stimulated dCyd kinase activity; Mg2+ produced the maximal activity. dCyd kinase from either lymphoid or myeloid cells showed broad substrate specificity. The enzyme used several nucleoside triphosphates, but ATP, GTP, and dTTP were the best phosphate donors. dCyd was the best nucleoside substrate, since dCyd kinase had an apparent Km of 0.3, 85, 90, and 1400 microM for dCyd, dAdo, dGuo, and ara-A, respectively. The enzyme exhibited substrate activation with both pyrimidine and purine deoxyribonucleosides, suggesting that there is more than one substrate binding site on the kinase. These studies show that, in lymphoblasts and myeloblasts, purine deoxyribonucleosides and their analogues are phosphorylated by dCyd kinase, Ado kinase, and dGuo kinase.     

Adenosine triphosphatases associated with bovine brain microtubules. II. Properties of ATPases I and II

The catalytic properties of two ATPases which had been purified from bovine brain microtubules (Tominaga, S. & Kaziro, Y. (1983) J. Biochem. 93, 1085-1092) were studied. ATPase I, which had a molecular weight of 33,000, required the presence of 1.0 microM tubulin, 0.2 mM Mg2+, and 10 mM Ca2+ for maximal activity. The activation of ATPase I by tubulin was specific to the native form of tubulin, which could not be replaced by F-actin or tubulin denatured either by heat or more mildly by dialysis in the absence of glycerol. ATPase I was not specific to ATP, and GTP, and to a lesser extent, UTP and CTP were also hydrolyzed. Km for ATP of ATPase I was about 0.04 mM. ATPase I was inhibited by 5 mM Mg2+, 0.04 M K+, 10(-3) M vanadate, 10 mM N-ethylmaleimide, or 20% (v/v) glycerol. ATPase II, which was associated with membrane vesicles, required the presence of 0.2-2.0 mM Mg2+ and 20 mM KCl for activity. Tubulin stimulated the reaction of ATPase II only partially, and the addition of Ca2+ was rather inhibitory. ATPase II was specific to ATP with a Km value of 0.14 mM. It was inhibited by 1.6 mM N-ethylmaleimide and 20% (v/v) glycerol, but was not very sensitive to vanadate. Instead, ATPase II was inhibited by trifluoperazine, chlorpromazine, and nicardipin at 10(-3) M.     

Substrate Specificity of Three Viral Thymidine Kinases (TK): Vaccinia Virus TK,Feline Herpesvirus TK,and Canine Herpesvirus TK

In search of novel suicide gene candidates we have cloned and characterized thymidine kinases from three viruses; vaccinia virus TK (VVTK), feline herpesvirus TK (FHV-TK), and canine herpesvirus TK (CHV-TK). Our studies showed that VVTK primarily is a thymidine kinase, with a substrate specificity mainly restricted to dThd and only minor affinity for dCyd. VVTK also is related closely to mammalian thymidine kinase 1 (TK1), with 66% identity and 75% general homology. Although CHV-TK and FHV-TK are sequence related to herpes simplex virus types 1 thymidine kinase (HSV1-TK), with 31% and 35% identity and a general similarity of 54%, the substrate specificity of these enzymes was restricted to dThd and thymidine analogs.     

Human deoxycytidine kinase. Purification and characterization of the cytoplasmic and mitochondrial isozymes derived from blast cells of acute myelocytic leukemia patients.

A procedure for purifying human cytoplasmic and mitochondrial deoxycytidine kinase (NTP:deoxycytidine 5'-phosphotransferase, EC 2.7.1.74) was developed. Both purified isozymes have a similar molecular weight, activation energy and catalyze the reaction by a sequential mechanism. These two isozymes differ with respect to their substrate specificities. With cytoplasmic deoxycytidine kinase, ATP, GTP and TTP have the highest reaction velocity. Pyrimidine nucleoside triphosphates have higher affinity but lower V than purine nucleoside triphosphates. Cytidine and arabinosylcytidine can serve as substrates. With mitochondrial isozyme only ATP gives the highest reaction velocity. ATP and dATP have the same Km but different V values. Besides deoxycytidine, also deoxythymidine but not cytidine or arabinosylcytidine can serve as substrates. There are also differences between these two isozymes with respect to their sensitivity to inhibition. For cytoplasmic enzyme, Br5dCyd and Iodo5dCyd are not inhibitory. Both dCTP and UTP are competitive inhibitors (Ki 0.25 and 0.5 micronM, respectively) with respect to ATP. For mitochondrial isozyme both Br5dCyd and Iodo5dCyd are inhibitory and dCTP and TTP are competitive inhibitors (Ki 2 and 10 micronM, respectively) with respect to ATP.     

Characterization of a 67-kDa S6 protein kinase from rat liver.

Fractionation of rat liver cytosol on DEAE-cellulose resolved two S6 kinases eluting at 25 mM KCl (peak I) and 100 mM KCl (peak II). The apparent molecular weights of the peak I and peak II kinases are 26,300 and 67,000, respectively. The peak II kinase was further purified and characterized. Incubation of the kinase with [gamma-32P] ATP and Mg2+ resulted in the incorporation of 32P predominantly into a 67-kDa band. Optimal activity of the kinase was observed in the presence of 5 mM Mg2+ and in the pH range of 8.0-8.5. The Km for ATP and 40S subunit were 7.3 microM and 1.5 microM, respectively. The Mg(2+)-stimulated kinase activity was inhibited by various divalent metals, NaF, and polyamines. The properties of the peak II S6 kinase are very similar or identical to the previously described mitogen-activated S6 protein kinase and may represent the nonactivated form of this enzyme.     

Partial purification and properties of rat-heart adenosine kinase.

The activity of myocardial adenosine kinase (E.N. 2.7.1.20) in a number of species was assayed. Rat heart contained the highest specific activity. From this source adenosine kinase was purified in a simple way 80-fold, until it was free of adenosine deaminase activity. A molecular weight of about 39 000 was measured. NSC 113939 (1), NSC 113940 and 8-azaadenosine inhibited myocardial adenosine kinase. Dipyridamole stimulated the enzyme at high adenosine levels, and inhibited at low substrate concentrations. A number of divalent cations could (partially) substitute for Mg2+. The optimal concentration of MgCl2 or MnCl2 was about 0.5 mM; concentrations exceeding 1 mM inhibited severely. An apparent Km for ATP of 0.1 mM was measured, whereas an apparent Km for adenosine of 0.5 muM was was found. The latter increased to 3.3 muM, when dipyridamole was added. Replacement of ATP by GTB or ITP increased the activity, and UTP and CTP were inferior as a phosphate donor.     

Purification and properties of a nucleoside triphosphatase from Sinclair swine

A nonspecific nucleoside triphosphatase was partially purified from skin and cutaneous melanoma tumors from Sinclair swine using chloroform precipitation, hydrophobic, ion-exchange and affinity chromatography techniques. The enzyme was not stimulated by Na+, K+ or Mg2+ but it was inhibited by EDTA. The enzyme was not inhibited by quercetin, proflavin, azide or ovabain. The enzyme exhibited optimal activity over a pH range of 8-9 and the activation energy was 10.4 and 9.8 kcal/mol for dUTP and ATP, respectively. The apparent Km of the enzyme for dUTP and dTTP was approximately 20 mumol/l while the apparent Km for dATP, ATP, dCTP, CTP and UTP was in the range of 65-80 mumol/l.     

Protein kinases of the chick oviduct: a study of the cytoplasmic and nuclear enzymes.

Subcellular fractionation of oviduct tissue from estrogen-treated chicks indicated that the bulk of the protein kinase activity of this tissue is located in the cytoplasmic and nuclear fractions, DEAE-cellulose chromatography of cytosol revealed a major peak of cAMP stimulatable activity eluting at 0.2 M KCl. This peak was further characterized and found to exhibit properties consistent with cytoplasmic cAMP dependent protein kinases isolated from other tissues; it had a Km for ATP of 2 X 10(-5) M, preferred basic proteins such as histones, as substrate, and had a M of 165 000. Addition of 10(-6) M cAMP caused the holoenzyme to dissociate into cAMP binding regulatory subunit and a protein kinase catalytic subunit. Extraction of purified oviduct nuclei with 0.3 M KCl released greater than 80% of the kinase activity in this fraction. Upon elution from phospho-cellulose, the nuclear extract was resolved into two equal peaks of kinase activity (designated I and II). Peak I had a sedimentation coefficient of 3S and a Km for ATP of 13 muM. while peak II had a sedimentation coefficient of 6S and a Km for ATP of 9 muM. Both enzymes preferred alpha-casein as a substrate over phosvitin or whole histone, although they exhibited different salt-activity profiles. The cytoplasmic and nuclear enzymes were well separated on phospho-cellulose and this resin was used to quantitate the amount of cAMP dependent histone kinase activity in the nucleus and the amount of casein kinase activity in the cytosol. Protein kinase activity in nuclei from estrogen-stimulated chicks was found to be 40% greater than hormone-withdrawn animals. This increase in activity was not due to translocation of the cytoplasmic protein kinase in response to hormone, but to an increase in nuclear (casein) kinase activity. During the course of this work, we observed small but significant amounts of cAMP binding activity very tightly bound to the nuclear fraction. Solubilization of the binding activity by sonication in high salt allowed comparison studies to be performed which indicated that the nuclear binding protein is identical with the cytoplasmic cAMP binding regulatory subunit. The possible role of the nuclear binding activity is discussed.     

2',3'-didehydro-2',3'-dideoxy-5-chlorocytidine is a selective anti-retrovirus agent

2',3'-Didehydro-2',3'-dideoxy-5-chlorocytidine (D4CC) is, in contrast with 2',3'-dideoxy-5-chlorocytidine (ddClCyd) and 2',3'-didehydro-2',3'-dideoxy-5-chlorouridine (D4CU), a potent and selective inhibitor of the replication of human immunodeficiency virus (HIV) types 1 and 2, simian immunodeficiency virus (SIV) and simian AIDS related virus (SRV). D4CC is a poor inhibitor of the phosphorylation of [5-3H]2'-deoxycytidine (dCyd) by partially purified MT-4 cell dCyd kinase (Ki: 612 microM). The findings that (i) D4CC has little, if any, affinity for MT-4 cell Cyd/dCyd deaminase, (ii) D4CU is not antivirally active and (iii) the antiretroviral action of D4CC can be reversed by dCyd, but not dThd, indicate that D4CC is antivirally active as its Cyd metabolite (D4CC 5'-triphosphate) and does not need to be deaminated (to the corresponding Urd metabolite) to exert its antiretroviral action.     

Deoxycytidine kinase from rabbit kidney cells infected with herpes simplex virus type 1 or 2.

When rabbit kidney cells were infected with herpes simplex virus type 1 (strain Seibert) or herpes simplex virus type 2 (strain 316D), deoxycytidine kinase (CdR kinase) activity, assayed at 38 degrees, increased 5- to 15-fold relative to controls. The CdR kinase activity induced by type 2 virus was more thermolabile than the enzyme activity induced by type 1 virus. When CdR kinase activity was assayed at various temperatures between 0.5 and 38 degrees, maximum activity for type 1 enzyme was obtained at 16 degrees while maximum activities for host and type 2 enzymes were obtained at 38 degrees. Both type 1 and type 2 induced CdR kinase activities eluted at the same positions as deoxythymidine kinase activities on a Sephadex G-100 column. The estimated mol wt for HSV-1 (Seibert) and HSV-2 (316D) induced CdR kinases are 67,000 and 60,000, respectively.     

Evidence for the identity of nuclear and cytoplasmic adenosine-3':5'-monophosphate-dependent protein kinase from porcine ovaries and nuclear translocation of the cytoplasmic enzyme.

Protein phosphokinase activity from a 0.5 M NaCl extract of purified porcine ovary nuclei has been resolved by Sephadex G-200 gel filtration into three forms of kinase, protein kinase I and III, both independent of adenosine 3':5'-monophosphate (cyclic AMP), and cyclic-AMP-dependent protein kinase II. Cyclic AMP-binding activity was associated with protein kinase II but not with protein kinases I and III. Protein kinases I, II, and III exhibited different cyclic nucleotide dependency and substrate specificity. Protein kinase II was inhibited by a heat-stable protein from rabbit skeletal muscle, whereas protein kinases I and III were not inhibited. According to previously established criteria [Traugh, J.A., Ashby, C.D. and Walsh, D.A. (1974) nuclear protein kinase II can be classified as cyclic-AMP-dependent protein kinase consisting of regulatory and catalytic subunits. Nuclear protein kinases I and III are cyclic-AMP-independent enzymes. Evidence for the identity of nuclear cyclic-AMP-dependent protein kinase II with cytosol (105 000 X g supernatant fraction) cyclic-AMP-dependent protein kinase was obtained in several ways. Nuclear and cytosol cyclic-AMP-dependent protein kinases exhibited identical elution characteristics on DEAE-cellulose and Sephadex G-200 indicating that both kinases are of similar molecular size and possess similar ionic charge. Both kinases exhibited an identical Km for ATP of 8 muM, showed similar substrate specificity, and revealed similar antigenic properties. Cyclic-AMP-dependent protein kinase II was also identified in nuclei isolated in nonaqueous media, eliminating the possibility that the cyclic-AMP-dependent protein kinase activity identified in nuclei isolated in aqueous media may have arisen as the result of cytoplasmic contamination. After incubation of neonatal porcine ovaries which lack nuclear cyclic-AMP-dependent protein kinase with 0.1 muM 8-p-chlorophenylthio cyclic AMP, considerable cyclic-AMP-dependent protein kinase II activity was identified in nuclei isolated in nonaqueous media. From these data it is concluded that the nuclear cyclic-AMP-dependent protein kinase II is related to or identical with the ovary cytoplasmic cyclic-AMP-dependent protein kinase, supporting the concept that nuclear cyclic-AMP-dependent protein kinase is of cytoplasmic origin.     

Cyclic nucleotide phosphodiesterases of Hyalophora cecropia silkmoth fat body.

Two soluble forms of 3':5'-cyclic-nucleotide phosphodiesterase (o':5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) were found in the larval fat body of the silkmoth Hyalophora cecropia. These differ in elution profile on Sephadex G-200, solubility in ammonium sulfate, metal ion requirements and kinetic properties. Phosphodiesterase I has Km values of 11 muM and 1.8 muM for cyclic AMP and cyclic GMP, respectively, has 5-fold greater maximal activity with cyclic AMP than with cyclic GMP, and is activated by Mg2+ and Co2+, and inhibited by EDTA. phosphodiesterase II has Km values of 625 muM and 125 muM for cyclic AMP and cyclic GMP, respectively, has similar maximal activity with both substrates, and is not activated by divalent metal ions or inhibited by EDTA. Cyclic nucleotides and methylxanthines competitively inhibit both enzymes. Phosphodiesterase is found in both soluble and particulate fractions of homogenates. Total activity is highest during the larval stage of the insect, drops markedly following pupation, and rises again during pharate adult development.     

A plasmodium protein kinase that is developmentally regulated, stimulated by spermine, and inhibited by quercetin

Plasmodium berghei-infected murine red cells possess protein kinase activity that is associated with the isolated parasites. Schizonts contain significantly higher levels of this protein kinase than the more immature forms, suggesting a relationship between this enzyme activity and parasite development. Partially purified protein kinase has a Km for ATP of approximately 30 microMs, whereas the Km for GTP is approximately 300 microMs and the substrate preference is phosvitin greater than casein much greater than histone greater than protamine. The Mg2+ optimum is 10-20 mM, and the protein kinase activity is stimulated by the polyamines spermine and spermidine. The flavone, quercetin, inhibits the protein kinase activity in a competitive manner with respect to ATP (Ki approximately 3 microMs), and P chabaudi also has a very similarly regulated protein kinase. Protein kinases from both species are very similar to the type I casein kinase.     

Cloning and characterization of the multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster.

A Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) was reported to phosphorylate all four natural deoxyribonucleosides as well as several nucleoside analogs (Munch-Petersen, B., Piskur, J., and Sondergaard, L. (1998) J. Biol. Chem. 273, 3926-3931). The broad substrate specificity of this enzyme together with a high catalytic rate makes it unique among the nucleoside kinases. We have in the present study cloned the Dm-dNK cDNA, expressed the 29-kDa protein in Escherichia coli, and characterized the recombinant enzyme for the phosphorylation of nucleosides and clinically important nucleoside analogs. The recombinant enzyme preferentially phosphorylated the pyrimidine nucleosides dThd, dCyd, and dUrd, but phosphorylation of the purine nucleosides dAdo and dGuo was also efficiently catalyzed. Dm-dNK is closely related to human and herpes simplex virus deoxyribonucleoside kinases. The highest level of sequence similarity was noted with human mitochondrial thymidine kinase 2, and these enzymes also share many substrates. The cDNA cloning and characterization of Dm-dNK will be the basis for studies on the use of this multisubstrate nucleoside kinase as a suicide gene in combined gene/chemotherapy of cancer.     

Purification and properties of cyclic AMP-dependent protein kinase from rat epididymis

Two protein kinases (I and II: EC 2.7.1.37) that show a high degree of substrate specificity for protamine rather than histones, phosvitin and casein were partly purified from rat epididymal tissue. The enzymes were present in the cytosol because greater than 80% of the enzymic activity was recovered in the soluble fraction. The kinases required Mg2+ for activity although Co2+ and Mn2+ were partial substitutes. Zn2+ (1 mM) inhibited nearly completely the activity of the enzymes. Both the kinases showed high affinity for activation with cyclic AMP compared to other cyclic nucleotides. Amino acid analysis of 32P-labelled protamine product revealed that the kinases transfer the terminal phosphate of ATP to serine residues of the protein. The isoenzymes I and II showed certain differences in relation to their hydroxyapatite-chromatography profiles, pH activation profiles, heat sensitivity and Km for ATP and cyclic AMP.     

The structures of thymidine kinase from herpes simplex virus type 1 in complex with substrates and a substrate analogue.

Thymidine kinase from Herpes simplex virus type 1 (TK) was crystallized in an N-terminally truncated but fully active form. The structures of TK complexed with ADP at the ATP-site and deoxythymidine-5'-monophosphate (dTMP), deoxythymidine (dT), or idoxuridine-5'-phosphate (5-iodo-dUMP) at the substrate-site were refined to 2.75 A, 2.8 A, and 3.0 A resolution, respectively. TK catalyzes the phosphorylation of dT resulting in an ester, and the phosphorylation of dTMP giving rise to an anhydride. The presented TK structures indicate that there are only small differences between these two modes of action. Glu83 serves as a general base in the ester reaction. Arg163 parks at an internal aspartate during ester formation and binds the alpha-phosphate of dTMP during anhydride formation. The bound deoxythymidine leaves a 35 A3 cavity at position 5 of the base and two sequestered water molecules at position 2. Cavity and water molecules reduce the substrate specificity to such an extent that TK can phosphorylate various substrate analogues useful in pharmaceutical applications. TK is structurally homologous to the well-known nucleoside monophosphate kinases but contains large additional peptide segments.     

Evidence for two distinct phosphatidylinositol kinases in fibroblasts. Implications for cellular regulation.

Phosphatidylinositol (PtdIns) kinase activities from non-transformed and polyoma-middle-T-transformed murine fibroblasts were examined. Both normal and transformed 3T3 fibroblasts have two PtdIns kinases, which can be separated by anion-exchange chromatography. One of these activities (Type I) has a Km for ATP of 10 microM, is resistant to inhibition by adenosine, AMP or ADP, and is inhibited by non-ionic detergents. The other activity (Type II) has a somewhat higher Km for ATP (35 microM) and is inhibited competitively by ADP, AMP and adenosine at concentrations suggesting regulation of this activity by the energy charge of the cell. The Type II PtdIns kinase is activated by non-ionic detergents. We have previously reported the specific association of a PtdIns kinase activity with polyoma-middle-T immunoprecipitates [Whitman, Kaplan, Schaffhausen, Cantley & Roberts (1985) Nature (London) 315, 239-242; Kaplan, Whitman, Schaffhausen, Raptis, Garcea, Pallas, Roberts & Cantley (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3624-3628]. Comparison of the immunoprecipitated PtdIns kinase with the activities identified by ion-exchange chromatography indicates that it is the Type I enzyme which specifically associates with the middle-T/pp60c-src complex. This PtdIns kinase activity is separable from both middle T and pp60c-src. Type I PtdIns kinase also associates with pp60v-src immunoprecipitates from Rous-sarcoma-virus-transformed cells. Furthermore, this PtdIns kinase appears to co-precipitate with partially purified platelet derived growth factor (PDGF) receptor. The amount of this activity found in anti-phosphotyrosine immunoprecipitates or in wheat-germ-lectin-agarose precipitates is increased 50-fold by stimulation of quiescent Balb/C 3T3 fibroblasts with PDGF. These results suggest that the Type I PtdIns kinase is regulated by agents which affect cell growth and transformation, whereas the Type II PtdIns kinase may be regulated by the local [ATP]/[ADP] ratio.