Wai-Choi Leung

Properties of mitochondrial thymidine kinases of parental and enzyme-deficient HeLa cells

Abstract HeLa(BU25), a mutant subline of HeLa S3 cells, contains mitochondrial thymidine (dT) kinase, despite a marked deficiency in the dT kinase activity of the “cytosol” (high-speed supernatant) cell fraction. The HeLa(BU25) mitochondrial dT kinase differs from the “cytosol” enzyme of parental HeLa S3 cells in sedimentation coefficient, ability to utilize ribonucleoside 5′-triphosphates other than ATP as phosphate donors, sensitivity to inhibition by dCTP, and in disc polyacrylamide gel electrophoretic (disc PAGE) patterns. Two dT kinase activities [relative mobilities (Rm) of 0.4 and 0.6–0.7] were detected after disc PAGE of HeLa(BU25) mitochondrial extracts and both activities migrated more rapidly than the typical cytosol enzyme (Rm = 0.2) of dT kinase-positive human cells. The 0.6 to 0.7-Rm dT kinase of HeLa(BU25) mitochondria, but not the 0.4-Rm activity, utilized GTP and UTP, as well as ATP, as phosphate donors. HeLa S3 mitochondrial fractions contained the 0.6–0.7 Rm and the 0.4-Rm activities, and in addition, a “cytosol-like” 0.2-Rm activity. The 0.6 to 0.7-Rm dT kinase of HeLa S3 mitochondria utilized either UTP or ATP as phosphate donors, but the 0.4- and 0.2-Rm dT kinases utilized only ATP. Similarly, the HeLa S3 cytosol dT kinase efficiently utilized ATP, but not UTP, as a phosphate donor.

Submitochondrial localization and characteristics of thymidine kinase molecular forms in parental and kinase-deficient HeLa cells.

Although HeLa (BU25) cells are deficient in cytosol dT kinase activity, they contain two mitochondrial dT kinases with disc PAGE mobilities (Rm) of 0.4 and 0.6 and isoelectric points (pI) of 8.4 and 5.6, respectively. Mitochondrial extracts of parental HeLa S3 contain the two HeLa (BU25) activities, but also a cytosol-like enzyme (0.25 Rm, pI 9.8). The 0.6-Rm (pI 5.6) mitochondrial activity utilizes ribonucleoside 5′-triphosphates other than ATP (dATP) as phosphate donors and is sensitive to dCTP inhibition. The predominant HeLa S3 cytosol (0.25 Rm) enzyme and the 0.4 Rm mitochondrial enzymeefficiently utilize only ATP as a phosphate donor and are relatively insensitive to dCTP inhibition. Submitochondrial fractionation studies have shown that (1) 74–98% of the mitochondrial dT kinase is located in the matrix plus inner membrane fractions; (2) the matrix fraction has the highest specific activity, contains all the 0.6-Rm activity, most of the HeLa S3 0.25-Rm activity, and some 0.4-Rm activity; (3) the inner membrane fraction is the major site of the 0.4-Rm activity but the outer membrane fraction also contains the 0.4 Rm activity; and (4) all HeLa S3 submitochondrial fractions contain the 0.25-Rm dT kinase activity.

Thymidine Kinases Induced by Avian and Human Herpesviruses

Disc PAGE, isoelectric focusing, and glycerol gradient centrifugation experiments were carried out to characterize thymidine (dT) kinase isozymes induced by herpesvirus of turkeys and infectious laryngotracheitis virus in the cytosol fraction of infected chick cells. The avian herpesvirus dT kinases differed from chick cytosol dT kinase in electrophoretic mobility, isoelectric point and phosphate donor specificity. The avian herpesvirus dT kinases resembled chick mitochondrial dT kinase in electrophoretic mobilityy and isoelectric point, but exhibited larger sedimentation coefficients. The avian herpesvirus enzymes closely resembled dT kinases induced by human herpes simplex viruses types 1 and 2.

Mitochondrial thymidine kinase of bromodeoxyuridine-resistant, kinase-deficient HeLa(BU25) cells

Abstract Total cell extracts of HeLa(BU25), a mutant subline of HeLa S3, are deficient in dT kinase activity. In contrast to the parental HeLa S3 cells, the nuclear DNA of HeLa(BU25) does not exhibit a “heavy” density after the HeLa(BU25) cells are grown in medium with dBU. Despite the loss of the principal dU-dT phosphorylating enzyme, HeLa(BU25) cells contain a mitochondrial dU-dT phosphorylating activity with a specific activity about equal to that of the HeLa S3 mitochondrial enzyme. Phosphorylation of 3 H-dU by the mitochondrial enzyme requires ATP, is markedly inhibited by dTTP or nonradioactive dT, but not by Urd, Cyd, or Ado. Hence, it is a dT kinase. A nucleoside phosphotransferase, weakly active at pH 5.5–6.5, may also be present.

Distinctive properties of mitochondrial thymidine(dT)kinase from bromodeoxyuridine(dBU)-resistant mouse lines

Abstract dBU-resistant mouse lines lack detectable dT kinase activity in the high speed supernatant (cytosol) cell fraction. However, they contain a mitochondrial dT kinase, which sediments more slowly in glycerol gradients than the cytosol enzyme of parental mouse lines, exhibits a disc PAGE mobility relative to the tracking dye (Rm) of about 0.7–0.8, and utilizes ATP, UTP, GTP, and CTP as phosphate donors. The mitochondrial fraction of parental cells also contains this 0.7–0.8 Rm activity and, in addition, a minor dT kinase activity which migrates faster than the cytosol enzyme, but utilizes only ATP as phosphate donor. The cytosol dT kinase of parental mouse lines exhibits an Rm of about 0.2–0.3 and utilizes only ATP as phosphate donor.

Characterization of nucleoside phosphotransferase and thymidine kinase activities of chick embryo cells and of chick-mouse somatic cell hybrids

Abstract Experiments were carried out to characterize the thymidine (dT) phosphorylating activities of chick embryo, chick erythrocytes, and of chick mouse somatic cell hybrids derived from fused chick erythrocytes and dT kinase-deficient LM(TK) mouse cells. Disc PAGE, isoelectric focusing, and glycerol gradient centrifugation analyses revealed that chick embryo cells contained four distinctive dT phosphorylating activities, two dT kinases and two nucleoside phosphotransferases. Thymidine kinase F. found principally in the cytosol, was also detected in mitochondrial and nuclear extracts, but was very low or absent from chick erythrocytes. Thymidine kinase A corresponds to the mitochondrial-specific isozyme found in bromodeoxyuridine-resistant mammalian cells. Nucleoside phosphotransferase activities were very active in chick embryo cytosol and were detected in embryo mitochondria! and nuclear extracts and cytosol and nuclear extracts of chick erythrocytes. Most of the chick embryo nucleoside phosphotransferase activity could be removed by purification of cytosol dT kinase F. Chick-mouse somatic cell hybrids exhibited chick dT kinase F, but neither chick dT kinase A. chick nucleoside phosphotransferase, nor mouse cytosol dT kinase activities. The results indicate (1) the genetic determinant for chick cytosol dT kinase F is on a different chromosome from the determinants for the chick nucleoside phosphotransferases and mitochondrial dT kinase A, and/or (2) only the chick cytosol dT kinase F, but neither the chick nucleoside phosphotransferases nor dT kinase A, was reactivated in the hybrids.

Subcellular Localization and Properties of Thymidine Kinase from Adenovirus-infected Cells

Summary The principal cytosol thymidine kinase activity of African green monkey kidney cells increased approximately threefold at 21 to 29 h after infection with adenovirus type 5. Disc polyacrylamide gel electrophoresis (disc PAGE) analyses showed that the electrophoretic mobility relative to the tracking dye (Rm) of the cytosol thymidine kinase from both mock-infected and adenovirus type 5-infected cells was about 0.23. The cytosol thymidine kinase from normal cells also resembled the cytosol enzyme from infected cells with respect to phosphate donor specificity and sedimentation coefficient. Mitochondria from normal and virus-infected cells contained a cytosol-like enzyme and, in addition, a distinctive mitochondrial isozyme exhibiting an Rm of about 0.6 and a smaller sedimentation coefficient than the cytosol enzyme. The activity of the mitochondrial-specific isozyme of thymidine kinase (Rm = 0.6) was not significantly increased by virus infection. The ratio of the two thymidine kinase activities found in mitochondria also was not markedly changed by virus infection. The results suggest that adenovirus type 5 infection reactivates an inactive molecular form of cytosol thymidine kinase or derepresses the synthesis of the cytosol enzyme, but not that of the mitochondrial-specific thymidine kinase. Adenovirus infection does not alter the electrophoretic mobilities of the cytosol and mitochondrial thymidine kinases.

Biochemical and serological properties of the thymidine-phosphorylating enzymes induced by herpes simplex virus mutants temperature-dependent for enzyme formation

Abstract Evidence is presented in support of the hypothesis that: (i) an enzyme induced by wild-type herpes simplex virus type 1 (HSV-1) is a deoxypyrimidine kinase with a single active site for the phosphorylation of both thymidine (TdR) and deoxycytidine (CdR); (ii) enzymes induced by HSV-1 mutants that are temperature dependent for enzyme induction are altered with respect to nucleoside acceptor specificity and antigenicity; and (iii) altered polypeptides are synthesized in mutant virus-infected cells at both the permissive (31°) and restrictive (37.5°) temperatures. HSV-1 mutants B2010 and B2015 induce less TdR-phosphorylating activities at 31° than wild-type HSV-1 and very low but detectable enzyme activities at 37.5°. The TdR-phosphorylating activities induced by the mutant viruses at 31° have the same electrophoretic mobilities as that of the wild-type HSV-1-induced enzyme but, unlike the wild-type enzyme, the mutant enzymes lack CdR-phosphorylating activity. Immunoglobulin G (IgG) prepared from rabbits immunized with cytosol extracts from rabbit kidney cells infected with wild-type HSV-1 inhibit the TdR-phosphorylating activities induced by wild-type and mutant viruses, as well as the CdR-phosphorylating activity induced by wild-type HSV-1. Extracts purified from cells infected at 31° with wild-type and mutant HSV-1 blocked the neutralizing activity of anti-HSV-1 IgG for the CdR- as well as the TdR-phosphorylating activities of wild-type HSV-1 enzyme, despite the lack of CdR-phosphorylating activity of the mutant enzyme. However, the mutant virus-infected cell extracts were less effective than wild-type extracts in serum blocking activity. Extracts from cells infected with mutant B2015 at 37.5° did not exhibit serum blocking power.

Gel Electrophoresis, Isoelectric Focusing, and Localization of Thymidine Kinase in Normal and Simian Virus 40-Infected Monkey Cells

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.

Identification of chick thymidine kinase determinant in somatic cell hybrids of chick erythrocytes and thymidine kinase-deficient mouse cells.

Abstract Disc polyacrylamide gel electrophoresis (disc PAGE) analyses of chick-mouse somatic cell hybrids [LM(TK−)/CRB]isolated from fusion mixtures of chick erythrocytes and thymidine (TdR) kinase-deficient mouse [LM(TK−)]cells have demonstrated that the somatic cell hybrids contain only chick cytosol TdR kinase F and mouse mitochondrial TdR kinase A activities. Karyotypes were analysed by the method which sequentially reveals Q- and C-bands. Four hybrid clones contained the full complement of mouse chromosomes and 1 to 3 chick micro-chromosomes. Counterselection of the LM(TK−)/CRB hybrids in 5-bromodeoxyuridine (BUdR) medium resulted in the loss of chick cytosol TdR kinase F activity and at least one of the chick chromosomes, but mouse mitochondrial TdR kinase A activity was unaffected. Unlike the LM(TK−)/CRB somatic cell hybrids, the BUdR-resistant clones could not grow in HATG (hypoxanthine-aminopte-rin-thymidine-glycine) medium. The results demonstrate that: (1) the chick cytosol TdR kinase F gene is on a member of the micro-chromosomes; and (2) selection in HATG- and BUdR-containing medium involves only cytosol TdR kinase F.