Search

Chick-mouse heterokaryons were obtained by UV-Sendai virus-induced fusion of chick erythrocytes with thymidine (dT) kinase-deficient mouse fibroblast [LM(TK(-))] cells. Autoradiographic studies demonstrated that 1 day after fusion, [(3)H]dT was incorporated into both red blood cell and LM(TK(-)) nuclei of 23% of the heterokaryons. Self-fused LM(TK(-)) cells failed to incorporate [(3)H]dT into nuclear DNA. 15 clonal lines of chick-mouse somatic cell hybrids [LM(TK(-))/CRB] were isolated from the heterokaryons by cultivating them in selective hypoxanthine-aminopterin-thymidine-glycine medium. LM(TK(-)) and chick erythrocytes exhibited little, if any, cytosol dT kinase activity. In contrast, all 15 LM(TK(-))/CRB lines contained levels of cytosol dT kinase activity comparable to that found in chick embryo cells. Disk polyacrylamide gel electrophoresis and isoelectric focusing analyses demonstrated that the LM(TK(-))/CRB cells contained chick cytosol, but not mouse cytosol dT kinase. The LM(TK(-))/CRB cells also contained mouse mitochondrial, but not chick mitochondrial dT kinase. Hence, the clonal lines were somatic cell hybrids and not LM(TK(-)) cell revertants. The experiments demonstrate that chick erythrocyte cytosol dT kinase can be activated in heterokaryons and in hybrid cells, most likely as a result of functions supplied by mouse fibroblast cells.

The cell line LM(TK - ) Cl 1D, a derivative of mouse L fibroblasts deficient in thymidine kinase (EC 2.7.1.21) that shows very little thymidine kinase activity in extracts of whole cells as compared to the parental line, and that does not incorporate thymidine or 5-bromodeoxyuridine into nuclear DNA, has maintained the capacity to incorporate these precursors into mitochondrial DNA at a substantial rate. The amount of [ methyl - 3 H]thymidine incorporated into mitochondrial DNA of LM(TK - ) Cl 1D cells after long-term labeling has been conservatively estimated in different experiments to be between 30 and 60% of that incorporated into mitochondrial DNA or nuclear DNA of strain A9, an L-cell derivative without any thymidine-kinase dificiency; by contrast, the incorporation of thymidine into nuclear DNA of Cl 1D cells is less than 1% of that in A9 cells. These results strongly suggest that the loss of thymidine kinase activity in the extramitochondrial compartment of LM(TK - ) Cl 1D cells has not been accompanied by the loss of the mitochondria-associated enzyme activity, pointing to a different genetic or epigenetic control of the extramitochondrial and mitochondrial enzymes.

Cytoplasmic thymidine kinase from cardiac muscle of the rat has been characterized. It has a pH optimum of 9.0 and a Km value for thymidine of 1.6μm. The sedimentation coefficient of this enzyme in sucrose gradients is 4.5S, which represents a molecular weight of approx. 69000. Thymidine kinase prepared from cardiac muscle of foetal, neonatal and adult rats is inhibited by dTTP and dTDP; there is neither inhibition nor stimulation by dTMP, dCTP, dATP, dGTP or cyclic AMP. The activity of thymidine kinase in differentiating cardiac muscle of foetal and neonatal rats declines progressively with development, reaching adult values of almost zero by the fifteenth to seventeenth day of postnatal development. This represents a 70-fold decrease in enzyme activity from 3 days before birth to 17 days after birth. The loss of thymidine kinase activity in differentiating cardiac muscle correlates temporally with the cessation of DNA biosynthesis and the loss of cytoplasmic DNA polymerase activity in this tissue.

A protein kinase which is intimately associated with equine herpesvirus (equine abortion virus) was found by using adenosine triphosphate-γ- 32 P as a phosphate donor and virus protein as an acceptor. Consistent demonstration of the activity requires prior removal of phosphohydrolase. The kinase activity requires Mg 2+ , is not stimulated by cyclic adenosine monophosphate, but is enhanced by added protamine or arginine-rich histone. The labeled product is resistant to ribonuclease, deoxyribonuclease, and chloroform-methanol but is sensitive to Pronase. Other tests suggest that serine and threonine residues are the acceptor sites. In the in vitro reaction, the incorporation represents an average of approximately 4,500 phosphate residues per virion, and all 17 virus protein bands resolved by polyacrylamide gel electrophoresis appear to be labeled.

Nuclei isolated from glucagon-stimulated perfused rat livers contained 2–3 times as much protein kinase activity as did nuclei from control animals. In the presence of either the heat-stable inhibitor or the protein kinase regulatory subunit the elevated cyclic AMP-independent enzyme activity from stimulated nuclei was inhibited to an activity equivalent to that found in controls.

Summary Genetical and biochemical evidence indicates that herpes simplex virus (types 1 and 2) specifies a unique kinase activity which is able to phosphorylate both thymidine and deoxycytidine. Pseudorabies virus specifies an enzyme which has only thymidine kinase activity and so does vaccinia virus, while equine abortion virus does not induce either activity. It is shown that the HSV specified deoxypyrimidine kinase is indispensable for virus growth in serum starved cells but not important for virus replication in actively growing culture cells.

The role of cAMP in the regulation of nuclear protein kinase activity was investigated. Acidic nuclear proteins prepared from rat liver nuclei were separated by phosphocellulose chromatography into four peaks of protein kinase activity and two peaks of cAMP-binding activity. A fraction which bound cAMP also inhibited the most active nuclear protein kinase, K IV, and the inhibition was diminished in the presence of 5 μM cAMP. Further support for the regulation of nuclear kinases by cAMP was obtained using a regulatory subunit prepared from rabbit muscle protein kinase. The muscle regulatory subunit markedly inhibited liver nuclear kinase activities. The addition of cAMP partially restored the activities.

1. Protein kinase activity was measured in islets of Langerhans that had been incubated in the presence of agents known to affect insulin release. 2. Glucagon, theophylline, caffeine and 3-isobutyl-1-methylxanthine, agents that raise cyclic AMP concentrations in islet cells and stimulate insulin release, increased protein kinase activity. Adrenaline and diazoxide, agents that decrease cyclic AMP concentrations and inhibit insulin secretion, decreased the activity. 3. The increase in protein kinase activity produced by different concentrations of 3-isobutyl-1-methylxanthine was apparently related to the increase in intracellular concentrations of cyclic AMP. 4. The sulphonylureas, tolbutamide and glibenclamide, agents that increase insulin release, also increased the protein kinase activity; however, leucine, arginine and xylitol, which also stimulate insulin release, were without effect on the kinase activity. 5. Increasing the glucose concentration of the incubation medium from 2 to 20mm had no effect on protein kinase activity. Further, the ability of 3-isobutyl-1-methylxanthine to increase the protein kinase activity was not affected by the glucose concentration of the incubation medium. 6. These results suggest that agents which affect insulin secretion by altering cyclic AMP concentrations may exert their effects on hormone release by altering the activity of a cyclic AMP-dependent protein kinase in islet cells.

A quantitative fluorimetric method for determination of creatine kinase activity in isolated single nerve cells (2-10 ng dry weight) has been developed. Enzyme activity was measured in the direction of adenosine triphosphate and creatine formation. The creatine generated was measured by the fluorescent compound formed with ninhydrin in alkali. The coefficient of variation for the assay of enzyme activity in brain homogenate (47 ng wet tissue/sample) was 3.17%. The coefficient of variation for single neurons of the lateral vestibular nucleus (nucleus Deitersi) in rabbit was about 15%. The creatine kinase activity in these cell bodies was 342.0 ± 14.2 moles/kg dry tissue/hr, at 38°C.

In earlier studies it could be shown that most agents which stimulate RNA synthesis in animal cells affect initially the synthesis of pre-rRNA in the nucleolus [ 1,2] . The rates of phosphorylation of nuclear proteins are increased in cells undergoing such gene activation [3-71. Whether, however, the regulation of tRNA synthesis is accompanied by reversible phosphorylation of specific nucleolar proteins is a still unresolved problem. This paper describes a protein kinase activity which is associated with highly purified nucleoli. This enzyme catalyzes the phosphorylation of both salinesoluble and chromosomal nucleolar proteins.

1. Properties of the stimulation by cyclic AMP of the intrinsic protein kinase activity of membrane fragments from ox brain were studied. 2. Stimulation of activity declined from about 100% at 1min to less than 20% at 10min. The time-course was explained by the observation that cyclic AMP did not stimulate turnover of protein-bound serine phosphate once the membrane protein was fully phosphorylated. 3. Cyclic AMP accelerated the activity of a component of the basal activity rather than activating a different kinase. 4. The pH optimum for both the stimulated and basal activities was 7.2–7.4. NaCl (100mm) and KCl (10–100mm) inhibited the stimulated activity but did not affect the basal activity. 5. Strychnine and theophylline inhibited both activites equally, but the stimulated activity was more sensitive to inhibition by adenosine, bicuculline, vinblastin, veratrine, N-ethylmaleimide and cysteine. 6. No firm evidence for a role for endogenous cyclic AMP in the basal activity was found, but the possibility was not excluded. 7. Some 90% of both the stimulated and basal activities remained in an insoluble form after treatment of the membrane fragments with Triton X-100 (0.5%).

Mouse somatic cells lacking thymidine kinase were mixed in culture with human diploid cells lacking hypoxanthine guanine phosphoribosyl transferase, and hybrid cells were isolated and maintained in a selective medium containing hypoxanthine, aminopterin, and thymidine. The hybrid cells at the time of isolation had karyotypes consisting predominantly of mouse chromosomes but with one human chromosome, a submetacentric member of group E, apparently giving thymidine kinase to the hybrid cell. However, after long-term propagation in the selective medium this chromosome has been lost, although cells continue to show thymidine kinase activity as demonstrated by the incorporation of (3)H-thy-midine into DNA in the hybrid cell. The hybrid cells have only mouse electro-phoretic variants for glucose-6-phosphate dehydrogenase, lactate dehydrogenase, and malate dehydrogenase, suggesting that the human genetic loci for these enzymes are not represented in the hybrid genome and may be unlinked to that for thymidine kinase.

Cells deficient in the enzyme thymidine kinase were derived from transplantable SV40-transformed hamster cells. The resultant cell lines were less transplantable when inoculated into hamsters. Tumors which did arise from such cells had prolonged latent periods and were found to contain a mixture of enzyme-containing and enzyme-deficient cells. Revertant cell lines obtained either spontaneously or after mutagenesis in vitro contained intermediate levels of thymidine kinase activity and displayed an oncogenic potential which was intermediate between the wild type and enzyme-deficient cells. It is postulated that salvage pathway enzymes may play a rate-limiting role in tumorigenesis.

The regulation of the activity of blowfly flight-muscle phosphorylase b kinase by P(i) and Ca(2+) was studied, and the actions of these effectors on the kinases from insect flight and rabbit leg muscles were compared. Preincubation of blowfly kinase with P(i) increased activity severalfold. The effect was concentration-dependent, with an apparent K(m) of about 20mm, and time-dependent, requiring at least 10min for maximal activation. Neither ATP nor cyclic AMP was needed, suggesting that a protein kinase may not be involved. Maximal activation of the insect kinase required Mg(2+) in addition to P(i). The apparent K(m) for Mg(2+) was 3mm. Rabbit leg-muscle phosphorylase b kinase was slightly inhibited, rather than stimulated, by P(i), and was strongly inhibited by K(+), Na(+) and Li(+). At physiological concentrations, Ca(2+) activated the phosphorylase b kinases from both blowfly flight and rabbit leg muscles. However, the responses to Ca(2+) of the enzymes from the two tissues were different. The mammalian kinase had virtually no activity in the absence of Ca(2+), and showed a large increase in activity over a narrow range of Ca(2+) concentrations. Flight-muscle kinase had appreciable activity in the absence of Ca(2+), and had a smaller increase over a wide range of Ca(2+) concentration. The concentrations of Ca(2+) required for half-activation were 0.1 and 1mum for the blowfly and rabbit enzymes respectively. The pH-activity profiles of the non-activated, phosphate- and Ca(2+)-activated kinase revealed considerable enhancement of activity with little, if any, increase in the ratio of activities at pH6.8 to those at 8.2. These results are discussed in relation to the mechanism coupling contraction to glycogenolysis and to the biochemical distinction between asynchronous and synchronous types of muscle.

A comparison was made of the inhibition of deoxythymidine kinase derived froin Ehrlich ascites carcinoma cells by 5-iodo-2'-deoxyuridine 5'-triphosphate and deoxythymidine 5'-triphosphate. The inhibition produced by the halogenated triphosphate derivative was observed to be qualitatively similar to deoxythymidine 5'-triphosphate over a rangs of pH, temperature and substrate concentrations; however, 5-iodo-2'-deoxyuridine 5'-triphosphate was quantitatively more inhibitory. Although the apparent K m for thymidine was not significantly different at pH 5.0, 6.5 and 7.5, the inhibitory effect of deoxythymidine 5'-triphosphate and 5-iodo-2'-deoxyuridine 5'-triphosphate was 43- and 26-fold larger respectively at pH 5.0 compared to pH 6.5; whereas the inhibitory effect for both compounds at pH 6.5 was about 4-fold greater than that at pH 7.5. In the presence or absence of the inhibitors, analysis indicated “allosteric” kinetics prevailed at pH 5.0 and 6.5, whereas Michaelis competitive kinetics occurred at pH 7.5 for both. These triphosphate derivatives protected thymidine kinase froin thermal inactivation (65°, 30 min); however, the halogenated derivative was effective at lower concentrations. Thus the halogenated triphosphate not only exerted a greater inhibitory effect than deoxythymidine triphosphate, but also afforded greater protection to thermal inactivation. A Hill plot indicated cooperative interactions at pH 5.0 and 6.5 but not at pH 7.5, and the presence of either triphosphate derivative increased the Hill coefficient ( n ) at pH 5.0 and 6.5 but not at pH 7.5. The order of addition of the substrate (thymidine) or of the inhibitory effectors had no significant effect on the amount of inhibition observed.

IN the course of treatment of leukaemia L1210 in mice using an antifolate compound, methotrexate (MTX), the leukaemic cells are affected early during treatment and later, while becoming resistant1, show impaired retention and decreased permeability to the drug2–4. Some antifolate-resistant sub-lines of leukaemia .L1210 have substantial dihydrofolate reductase (1.5.1.3) (DHR) activities5–7 and use NADPH to reduce folate to tetrahydrofolate8,9. Some authors have therefore considered the possibility of using the level of DHR activity to measure the effectiveness of the action of a drug on MTX-resistant leukaemic cells10,11. The extent to which the capacities to synthesize NADP and NADPH are influenced by the antifolate treatment is, however, unknown. Waravdekar et al.12 reported that livers of mice bearing various transplantable tumours showed impaired capacities to synthesize NAD. Furthermore, differences in the ratio of NAD to NADH2 are known to reflect the extent of oxidative metabolism and reductive synthesis in the livers of different species13. In the present experiments, the effects of daily treatment with MTX on hepatic and splenic NAD kinase (2.7.1.23) were examined in mice implanted with leukaemia L1210.

Isoproterenol stimulates cell proliferation in mouse salivary glands. Prior to the stimulation of DNA synthesis, (3)H-uridine incorporation into RNA is decreased. This decreased incorporation results from a depression of uridylate kinase activity.

Commercially available crystalline yeast alcohol dehydrogenase contained protein kinase activity. Casein and phosvitin were readily phosphorylated, but whole calf thymus histone was not. The protein kinase activity was inhibited by KCl, was not stimulated by cyclic AMP and could be separated from the alcohol dehydrogenase activity by sucrose density centrifugation.

A colonial variant of a virulent Shigella flexneri 2a has lost both virulence and glycerol kinase activity. It also has several other altered characteristics: lowered ability to oxidize tricarboxylic acid cycle intermediates, increased electrophoretic mobility, and decreased sensitivity to sodium lauryl sulfate. Genetic analysis has revealed that the gene governing glycerol kinase activity in Shigella has a different chromosomal locus than that from Escherichia coli. Furthermore, transduction of the Shigella glycerol kinase gene ( glp K ) into the avirulent Shigella strain can restore the ability to penetrate HeLa cells, whereas the gene from E. coli cannot. About half of the glp K mutants lose this ability, and only about half of the revertants of an avirulent glp K mutant regain it. This indicates that more than one gene affects glycerol kinase activity in Shigella, only one of which is associated with penetration. Glycerol kinase activity is closely correlated with changes in electrophoretic mobility, but does not appear to have any relationship to sodium lauryl sulfate sensitivity nor to the oxidation of tricarboxylic acid cycle intermediates.

1. The modification of pyruvate kinase activity in vitro was examined by altering the environmental [Mg2+]/[Ca2+] ratio with EDTA on the one hand and isolated rat liver mitochondria on the other. 2. Controlled additions of Ca2+ and EDTA caused pyruvate kinase activity to be alternately and rapidly switched on and off. 3. By being able to accumulate Ca2+ in preference to Mg2+ rat liver mitochondria were able to alter the [Mg2+]/[Ca2+] ratio in the vicinity of pyruvate kinase and thereby modify the activity of this enzyme. 4. The possible role of mitochondria in modifying pyruvate kinase and other ion-sensitive cytoplasmic enzyme activities is discussed.

The pyruvate kinase (PK) activity of mouse liver increases after injection of endotoxin. It decreases in animals given cortisone alone and remains essentially unchanged in those given cortisone and endotoxin at the same time. CCl 4 causes an increase in liver PK activity, but neither it nor endotoxin changes the activity of muscle PK. Addition of octonoate to liver homogenates inhibits the activity of liver PK. These results suggest that the rapid depletion in liver glycogen after administration of endotoxin or CCl 4 may be related to increased PK activity. Induction of tolerance does not prevent the increase in liver PK activity in challenged animals.

Deoxythymidine kinase (TdR kinase) from rabbit kidney cells infected in vitro with herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) was further investigated. Enzyme activity induced by HSV-2 was more heat-labile and more sensitive to inhibition by deoxythymidine nucleotides than HSV-1 induced enzyme activity in both crude extracts and partially purified preparations. The thermostability of TdR kinase activity from cells coinfected with HSV-1 and HSV-2 was intermediate between enzymes induced separately. The difference in thermostability between the two enzyme activities was also observed in extracts from 1-β-D-arabinofuranosylcytosine (ara-C) treated infected cells. HSV-1 and HSV-2 induced enzymes also differed in their response to substrate concentration. Gel filtration with Sephadex G-100 revealed the presence of monomer, dimer and aggregated forms of HSV-1 induced enzyme. The molecular weights of the monomer and dimer were estimated to be 58,000 and 97,000, respectively. Such multiple forms were not observed with HSV-2; the molecular weight of the HSV-2 induced enzyme was estimated to be 45,000 to 60,000, depending upon the eluting buffers. HSV-1 induced TdR kinase activity showed a different electrophoretic activity pattern in polyacrylamide gel electrophoresis than did the enzyme induced by HSV-2.

The P3HR-1 line of human lymphoblastoid cells that is Epstein-Barr virus positive was made resistant to 5-bromodeoxyuridine. Epstein-Barr virus-associated antigens, but not virus particles, were produced in P3HR-1(BU) cells maintained on 5-bromodeoxyuridine. However, virus particles did appear within 4 days after removal of the drug. Thymidine kinase activity was limited to P3HR-1(BU) cells producing viral antigen, whereas all control P3HR-1 cells showed thymidine kinase activity regardless of viral antigen synthesis. Cellular DNA in most P3HR-1(BU) cells was made via pathways that did not involve thymidine kinase. In cells having a pathway that involved thymidine kinase, a second DNA of density 1.71 g/cm 3 , corresponding to Epstein-Barr virus, was detected. It was concluded that: ( a ) a repressed Epstein-Barr virus genome persists in P3HR-1(BU) cells that do not contain thymidine kinase, with activation of the viral genome being accompanied by productive infection and the appearance of enzyme, and ( b ) thymidine kinase activity in P3HR-1(BU) cells could be used as a marker for viral genome expression.

DNA synthesis in regenerating liver was studied to determine whether the onset of stimulated DNA synthesis preceded the onset of increased incorporation of thymidine into DNA. Thymidine incorporation into hepatic DNA was not stimulated 15 h after operation, but was stimulated after 18 h; peak stimulation occurred 30 h after operation. Thymidine kinase activity was stimulated 24 h after operation; highest kinase activity was observed at 36 h. The onset of stimulated DNA synthesis was estimated by following the incorporation of labeled aspartic acid, sodium formate, adenine or orotic acid into appropriate DNA bases, viz. , thymine, adenine, adenine or cytosine, respectively. Incorporation of adenine and orotic acid was stimulated between 15 h and 18 h after operation; incorporation of aspartic acid and sodium formate was stimulated between 18 h and 21 h after operation. The incorporation of thymidine into DNA was accelerated by stress stimulus and was inhibited by hydrocortisone. Changes in thymidine kinase activity also were correspondingly accelerated or delayed. Incorporation of labeled thymidine, adenine, formate, orotic acid or thymine into appropriate DNA bases, viz. , thymine, adenine, adenine, cytosine or thymine, respectively, was stimulated by stress stimulus or was inhibited by hydrocortisone. It was concluded from these data that stimulation of DNA synthesis and of thymidine incorporation into DNA was essentially synchronized in regenerating rat liver. Results from this study were compared with results from similar studies in 2 other tissues, and the limitations, attendant with using thymidine incorporation into DNA as an indicator of stimulated DNA synthesis, were discussed.

1. Pig lymphocytes were transformed by dibutyryl cyclic AMP (6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate) at concentrations of 0.01–0.1μm. The pattern of incorporation of label from [5-3H]uridine and [6-3H]thymidine into RNA and DNA respectively was identical with that obtained with unpurified phytohaemagglutinin. 2. Chlorpromazine (0.1μm) prevented the stimulation of [5-3H]uridine incorporation into RNA by phytohaemagglutinin, but only slightly lowered the lymphocyte response to dibutyryl cyclic AMP. 3. An increase in the size and specific radioactivity of the intracellular Pi pool was found immediately after stimulation by both phytohaemagglutinin and dibutyryl cyclic AMP. This was followed after some 30min by a rise in the specific radioactivity and concentration of ATP. 4. There was an immediate increase in the specific radioactivity of phosphate groups of histones; by about 45min after stimulation only the histones remaining after extraction of histone fraction F1 continued to incorporate 32P from [32P]Pi. 5. Histone kinase activity increased in the first 30min after stimulation; subsequently histone F1 kinase activity decreased, but activity with the other histones as substrate continued to increase for a further 30min. Kinase activation was effected by cyclic AMP (adenosine 3′:5′-cyclic monophosphate). 6. Histone phosphatase activity behaved similarly to that of the kinase.

The intracellular distribution of thymidine phosphorylating kinases has been studied in rat liver at different times after partial hepatectomy. After conventional fractionation in 0.25 M sucrose, thymidine kinase is present in all fractions, the highest proportion being found, however, in the supernatant; phosphorylation of thymidine to TDP and TTP is detected only in the supernatant fraction from 24 h onwards after hepatectomy. Nuclei prepared in 2.2 M sucrose or in non-aqueous media have the same level of thymidine kinase activity and can never phosphorylate thymidine to TDP and TTP. In the nuclear fractions, the absolute activity of thymidine kinase increases from 16 to 46 h after hepatectomy but represents a decreasing proportion of the activity of the homogenate. At the same time, the proportion of kinase activity rises markedly in the supernatant fraction with a maximum of 83% in 40 h regenerating livers. The implications of these results are discussed.

A continuous line of rodent cells carrying the genome of the Rous sarcoma virus was stably transformed by exposure to viable herpes simplex virus of either subtype 1 or 2. Transformation was accompanied by alteration of morphology, growth characteristics, acquisition of a thymidine kinase activity (EC 2.7.1.21) resembling the corresponding specific activity of the enzyme in the herpes simplex virus, and capacity to express continuously some antigens specific for herpes simplex virus.

Thymidine kinase activity is known to increase in BHK 21 cells infected with herpes simplex virus type 1 (Klemperer et al. 1967). Moreover, there is now evidence showing that this is a virus-specified enzyme (Klemperer et al. 1967; Buchan & Watson 1969; Buchan et al. 1970). We now report a similar increase in thymidine kinase activity in BHK 21 cells infected with herpes simplex virus type 2. The pH optima are the same for both enzymes. The growth kinetics are similar and the type 2 virus always induces at least as much enzyme activity as the type 1 and usually up to twice as much. There are striking differences in the stability and serological specificity of the two enzymes. Infected BHK cell extracts were prepared and the thymidine kinase activity measured by the method of Klemperer et al. (1967). Briefly, BHK cells were infected with herpes simplex virus at a multiplicity of infection of 10 to 20 p.f.u./cell.

Total adenylate kinase activity was determined in developing rat liver. The activity was 18 units/g wet weight of tissue in foetal liver; this increased to 41 units/g immediately after birth and continued increasing until adult activities of 150 units/g were reached after two weeks. The adenylate kinase activity was separated into four isoenzymes. Only isoenzymes II and III were observed in foetal rat liver. Isoenzyme II activity was 2 units/g in the foetal liver and increased to 25 units/g in adult liver. Adenylate kinase III activity was 20 units/g in the foetal liver and increased to 118 units/g in adult liver. The possible role that adenylate kinase might have in regulating the energy flow in the developing liver cell is discussed.

Cultured Chinese hamster ovary cells showed no significant change in generation time or fraction in the S-phase in the presence of 1 mM N 6 , O 2′ -dibutyryl adenosine 3′:5′-cyclic monophosphate. Growth continued for at least two generations after expression of the morphological transformation induced by this cyclic AMP analog. Despite identical growth rates, apparent rates of DNA and RNA synthesis (incorporation of [ 3 H]-thymidine or [ 3 H]uridine) were reduced up to 15-fold in log phase by 1 mM cyclic nucleotide. [ 3 H]Deoxycytidine incorporation was much less sensitive to dibutyryl cyclic AMP. Uptake studies with [ 3 H]thymidine demonstrated an inhibition of transport rate dependent on the concentration of dibutyryl cyclic AMP in the growth medium. The rate of thymidine uptake at 1° was decreased 21-fold by 1 mM cyclic nucleotide; half-maximal inhibition occurred at 6 μM. At 37°, the pool size of acid-soluble thymidylate was strongly reduced by 1 mM cyclic nucleotide, and synergistic reduction of the pool size was found with 0.5 mM aminophylline. Phosphorylation of the acid-soluble intracellular label was unaffected by dibutyryl cyclic AMP. Inhibition of thymidine uptake is attributed to an observed decrease in thymidine kinase activity caused by growth in 1 mM dibutyryl cyclic AMP, and possibly to a simultaneous alteration in membrane permeability. Kinase-facilitated uptake of other metabolites may be regulated in a similar fashion by cyclic AMP.

Summary Antisera produced in rabbits against extracts of cells infected with type 1 or type 2 herpes simplex virus neutralized thymidine kinase activities of six type 1 and six type 2 herpes virus strains. There was intertypic neutralizing activity, suggesting some common antigenic determinants, but much greater intratypic activity, suggesting that the enzyme bears type-specific determinants. The effects of some anti-type 1 sera were complicated since they also stabilized the type 2 enzyme activity. When anti-type 1 and anti-type 2 sera were absorbed with extracts of cells infected with thymidine-kinase deficient mutants of homologous type they retained their power to neutralize thymidine kinase activity but completely lost their ability to neutralize infectivity. Each absorbed serum gave a single precipitin line against the parent strain and none against the parent of the other type. It was surmised that these precipitin lines were associated with type-specific thymidine kinase antigens.

Following SV40 infection of CV-1 monkey cells, cytosol thymidine (dT) kinase activity increased 4- to 10-fold. This enzyme was not significantly different from the cytosol dT kinase of uninfected cells with respect to mobility, isoelectric point, sedimentation coefficient, and phosphate donor specificity. Monkey mitochondria contain a distinctive dT kinase isozyme localized in the mitochondrial matrix. The distinctive mitochondrial isozyme had a higher electrophoretic mobility, lower isoelectric point, and smaller sedimentation coefficient than the cytosol dT kinase, and could utilize either ATP or UTP as phosphate donor; it did not increase after SV40 infection. Mitochondria also contained a cytosol-like dT kinase which was enhanced in infected cells. These findings suggest that SV40 infection derepresses the normal cytosol dT kinase of host cells.

1. Adenosine kinase was measured in dialysed extracts from Ehrlich ascites-tumour cells by a chromatographic procedure. 2. In the absence of added Mg(2+) the K(m) values for ATP and adenosine were 0.22mm and 2.8mum respectively. 3. The maximum velocity of adenosine kinase with free ATP was about three times that with the Mg(2+)-ATP complex. Free Mg(2+) was a non-competitive inhibitor of the reaction. A small amount of added Mg(2+), Mn(2+) or Ca(2+) was required for maximum adenosine kinase activity after cation bound to the enzyme had been released by treatment with p-chloromercuribenzoate and then removed by dialysis. 4. GTP, ITP, deoxy-ATP, deoxy-GTP, CTP, xanthosine triphosphate, UTP and thymidine triphosphate could partially or completely replace ATP as a phosphate donor. 5. The reaction of ATP with adenosine kinase was competitively inhibited by AMP, GMP, IMP, ADP, deoxy-ADP and IDP (K(i) 0.2, 1.1, 5.9, 1.2, 0.5 and 0.78mm respectively). Enzymic activity was markedly affected by the relative concentrations of AMP, ADP and ATP in assay mixtures. 6. The results are discussed in terms of possible mechanisms regulating the rate of adenosine kinase in vivo.

Limulus polyphemus has two electrophoretically distinct arginine kinases, a negative and a neutral (at pH 8.6). The highest concentrations of the neutral are found in muscle, where the total arginine kinase activity is highest. The peptide maps of the two forms are very similar, but the negative has six more acidic peptides than the neutral. Three tryptophan-containing peptides were isolated from tryptic digests of each form. The amino acid compositions of two of the three pairs of peptides were different. This sugests that the isoenzymes are the products of two distinct genes.

Highly purified cAMP-dependent protein phosphokinase from adrenal-cortical tissue contains cAMP-receptor activity. In activating the kinase, cAMP binds to the receptor and causes it to dissociate from its complex with the kinase. The kinase, freed of receptor, is fully activated and no longer stimulable by cAMP. Kinase can be similarly activated by differentially denaturing the receptor with heat. Addition of receptor suppresses kinase activity; this suppression can be overcome by cAMP. After dissociation of receptor, two molecular forms of the activated kinase exist. The cAMP receptor thus functions as a repressor of the protein kinase; binding of cAMP to receptor causes it to dissociate from the kinase, which is then fully activated.

Rats accustomed to eating during the first 8h of a daily 12h dark period re-established about 80% of intact liver weight, protein and DNA within 4 days following partial hepatectomy; further increases were not observed. Liver thymidine kinase activity and thymidine incorporation into liver DNA exhibited marked daily oscillations during liver regeneration. Maximum values were observed near the end of the dark period both in intact growing rats and in rats partially hepatectomized 2h before the end of the dark period. The time of day of surgery affected thymidine kinase activity and thymidine incorporation into DNA at specific times following partial hepatectomy. This seriously affects the interpretation of reports of experiments where the time of day of killing has been held constant and time of surgery varied. Highly significant correlation coefficients were observed for thymidine incorporation before killing versus thymidine kinase activity at time of killing and for thymidine versus orotic acid incorporation into DNA of livers from rats partially hepatectomized 2h before the end of the dark period and killed at 12h intervals. Thymidylate phosphatase activity returned to the normal amount at a rate similar to that for liver protein. Thymidylate phosphatase did not affect the validity of the thymidine kinase assay. The relationship of [14C]orotic acid to [3H]thymidine incorporation into liver DNA varied with the time of day, with the age of the rat and during the regeneration of the liver.

beta-Adrenergic stimulation with isoproterenol inhibits contractility, increases cyclic adenosine monophosphate (AMP) concentration, decreases the concentration of unsaturated cyclic AMP receptor sites, and increases cyclic AMP-independent kinase in the uterus of ovariectomized rats. The total soluble kinase activity is reduced. The protein kinase activity lost from the cytosol was translocated to the microsomal fraction mostly in a cyclic AMP-independent form, suggesting a particulate substrate for the activated enzyme.

Three protein kinase activities have been identified in ribosome-free supernatant from rabbit reticulocytes by DEAE-chromatography. Two of the protein kinase activities have similar substrate specificities, but differ in respect to dependency on cAMP for activation. These kinases preferentially phosphorylate histone, and also phosphorylate identical ribosomal proteins. One protein band in the 40S ribosomal subunit and six protein bands in the 60S subunit are phosphorylated, as demonstrated by Na dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The third kinase activity preferentially phosphorylates casein, instead of histone, and can use both [γ- 32 P]ATP and [γ- 32 P]GTP as phosphate donors. This protein kinase activity phosphorylates one protein band in the 40S subunit different from that phosphorylated by the other two kinases and four bands in the 60S subunit, only one of which is coincident with the proteins phosphorylated by the histone-specific activities.