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.     

New affinity adsorbents containing deoxycytidine, deoxyadenosine, or deoxyguanosine and their interactions with deoxynucleoside-metabolizing enzymes

3'-(4-Aminophenyl phosphate) derivatives of deoxycytidine (dCyd), deoxyadenosine (dAdo), and deoxyguanosine ( dGuo ) were synthesized. The inhibitory effects of these compounds on mammalian and bacterial deoxynucleoside kinases and several other deoxynucleoside-metabolizing enzymes were examined. The same derivatives were coupled to carboxyl-terminal Sepharose CL-6B (3-8 mumol of ligand/mL of gel), and each of the resulting affinity adsorbents was tested with various partially purified enzymes. Reasonable correlation between the inhibitory effect of a soluble deoxynucleoside 3'-phosphate diester and affinity of the corresponding Sepharose adsorbent for the enzyme was observed. Among the three dCyd kinases examined, only the bovine mitochondrial enzyme was adsorbed onto the dCyd-Sepharose column and eluted biospecifically by 1 mM dCyd (1400-fold purification). Its Ki toward the dCyd derivative was relatively low (1.1 mM), whereas no measurable inhibition was seen with mammalian cytosol or bacterial enzymes that did not stick to the column. The Ki of the dAdo derivative toward three dAdo kinases was more than 5 mM in each case, and none of these were retained by dAdo-Sepharose. Among the other dAdo-metabolizing enzymes examined, nucleoside phosphotransferase from barley (Ki = 1.2 mM) was adsorbed to dAdo-Sepharose at pH 5.0 and was biospecifically eluted with dAdo or AMP after suppressing ionic binding by adjusting the pH to 6.0 (480-fold purification to homogeneity). Mammalian mitochondrial dGuo kinase (beef liver) showed the lowest Ki (0.16 mM) among the enzymes tested and was biospecifically purified with dGuo -Sepharose (2800-fold purification).(ABSTRACT TRUNCATED AT 250 WORDS)     

Multisubstrate analogs for deoxynucleoside kinases. Use in novel affinity media applied to resolution of Lactobacillus enzymes

New multisubstrate-type inhibitors of the deoxynucleoside kinases have been synthesized, tested for their specificity as soluble inhibitors of enzymes from Lactobacillus acidophilus, and used to construct media for affinity chromatography. Each inhibitor was a deoxynucleoside 5'-adenosine 5"'-P1,P4-tetraphosphate (abbreviated dNp4A, where dN represents a dAdo, dCyd, dGuo, or dThd moiety linked through its 5'-hydroxyl to the terminal phosphate of adenosine tetraphosphate). At micromolar concentrations, each inhibitor strongly and specifically inhibited the corresponding deoxynucleoside kinase. Each of the four Lactobacillus deoxynucleoside kinase activities was selectively retained on its homologous dNp4A-Sepharose affinity medium. The activity was eluted on addition of the respective dNp4A with up to 70% recovery and 300-500-fold purification (relative to an ammonium sulfate fraction). Whereas dThd kinase was retained only by the dTp4A column, a portion of the dAdo kinase activity was retained, along with all the dCyd kinase or dGuo kinase, on dCp4A- or dGp4A-Sepharose, respectively, and coeluted with these activities. Conversely, all three activities were quantitatively retained on dAp4A-Sepharose, without competition from either dCyd or dGuo, and were eluted simultaneously upon addition of dAp4A. These observations further confirm the understanding that this organism employs paired, and presumably bifunctional, kinases, namely dCyd/dAdo kinase and dGuo/dAdo kinase, along with a separate thymidine kinase.     

Deoxynucleoside kinases encoded by the yaaG and yaaF genes of Bacillus subtilis. Substrate specificity and kinetic analysis of deoxyguanosine kinase with UTP as the preferred phosphate donor

The overlapping yaaG and yaaF genes from Bacillus subtilis were cloned and overexpressed in Escherichia coli. Purification of the gene products showed that yaaG encoded a homodimeric deoxyguanosine kinase (dGK) and that yaaF encoded a homodimeric deoxynucleoside kinase capable of phosphorylating both deoxyadenosine and deoxycytidine. The latter was identical to a previously characterized dAdo/dCyd kinase (M?llgaard, H. (1980) J. Biol. Chem. 255, 8216-8220). The purified recombinant dGK was highly specific toward 6-oxopurine 2'-deoxyribonucleosides as phosphate acceptors showing only marginal activities with Guo, dAdo, and 2',3'-dideoxyguanosine. UTP was the preferred phosphate donor with a Km value of 6 microm compared with 36 microm for ATP. In addition, the Km for dGuo was 0.6 microm with UTP but 6.5 microm with ATP as phosphate donor. The combination of these two effects makes UTP over 50 times more efficient than ATP. Initial velocity and product inhibition studies indicated that the reaction with dGuo and UTP as substrates followed an Ordered Bi Bi reaction mechanism with UTP as the leading substrate and UDP the last product to leave. dGTP was a potent competitive inhibitor with respect to UTP. Above 30 microm of dGuo, substrate inhibition was observed, but only with UTP as phosphate donor.     

Selective inactivation of the deoxyadenosine phosphorylating activity of pure human deoxycytidine kinase: stabilization of different forms of the enzyme by substrates and biological detergents

Deoxycytidine kinase, purified from human leukemic spleen to apparent homogeneity, is a multisubstrate enzyme that also phosphorylates purine deoxyribonucleosides [Bohman & Eriksson (1988) Biochemistry 27, 4258-4265]. In the present investigation we show that the stability and temperature dependence of dCyd kinase activity differed appreciably from the dAdo kinase activity of the same pure enzyme. Selective inactivation of dAdo activity was observed upon an incubation of the enzyme at both 4 and 37 degrees C. The half-life of dAdo activity at 4 degrees C increased from 36 to 84 h, when the protein concentration was increased by addition of bovine serum albumin. However, the half-life of dCyd activity increased from 72 h to more than 7 days under the same conditions. dCyd activity was stable for at least 6 h at 37 degrees C while the half-life of dAdo activity was 2 h. The presence of substrates like ATP, dTTP, or dAdo stabilized dAdo activity at both temperatures, and full maintenance of both activities at 37 degrees C was obtained by the addition of the zwitterionic detergent CHAPS. Furthermore, thermal inactivation of the dAdo activity occurred at a lower temperature (48 degrees C) as compared to the dCyd activity (54 degrees C). The presence of protease inhibitors had no effect on enzyme inactivation, nor was there a difference in the subunit structure of the selectively inactivated enzyme as compared to the fully active form, as revealed by size-exclusion chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel deoxynucleoside-phosphorylating enzymes in mycoplasmas: evidence for efficient utilization of deoxynucleosides

Mycoplasmas are unable to synthesize purine and pyrimidine bases de novo. Therefore, salvage of existing nucleosides and bases is essential for their survival. Four mycoplasma species were studied with regard to their ability to phosphorylate deoxynucleosides. High levels of thymidine kinase (TK), deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK) and deoxyadenosine kinase (dAK) activities were detected in extracts from Mycoplasma pneumoniae, Mycoplasma mycoides subsp. mycoides SC (M. mymySC), Acholeplasma laidlawii (A. laidlawii) and Mycoplasma arginini (M. arginini). Nucleoside phosphotransferase activities were found at high levels in A. laidlawii and low levels in M. arginini. Pyrophosphate-dependent deoxynucleoside kinase activities were detected mainly in A. laidlawii and M. mymySC extracts. Two open reading frames were identified in the M. mymySC genome; one showed 25% sequence identity to human dGK and the other one had about 26% sequence identity to human TK1. The M. mymySC dGK-like enzyme was cloned, expressed in Escherichia coli and affinity-purified. This enzyme phosphorylated dAdo, dGuo and dCyd, and the highest catalytic rate was with dAdo as substrate. Therefore, we suggest that this enzyme should be named deoxyadenosine kinase. The physiological role of mycoplasma dAK and TK may be to support the unusually large dATP and dTTP pools required for replication of mycoplasma genomes.     

Human T-lymphoblast deoxycytidine kinase: purification and properties

Previous observations present tremendous variations in the properties of deoxycytidine kinase. To clarify the properties and physiologic role of deoxycytidine kinase, we have undertaken its purification. Deoxycytidine kinase was purified from cultured human T-lymphoblasts (MOLT-4) to 90% purity with an estimated specific activity of 8 mumol min-1 (mg of protein)-1. The purification procedure included ammonium sulfate precipitation, Superose-12 HPLC gel filtration chromatography, DE-52 ion-exchange chromatography, AMP-Sepharose 4B affinity chromatography, and dCTP-Sepharose-4B affinity chromatography. Deoxyguanosine, deoxyadenosine, and cytidine phosphorylating activities copurified with deoxycytidine kinase to final specific activities of 7.2, 13.5, and 4 mumol min-1 (mg of protein)-1, respectively. The enzyme is very unstable at low protein concentration and is stabilized by storage at -85 degrees C with 1 mg/mL bovine serum albumin, 20% glycerol (v/v), 200 mM potassium chloride, and 25 mM dithiothreitol. The molecular weight was 60,000, and the Stokes radius was 32 A by gel filtration chromatography. The subunit molecular weight was 30,500. This enzyme had apparent Km values of 1.5, 430, 500, 450, and 40 microM for deoxycytidine, deoxyguanosine, deoxyadenosine, cytidine, and cytosine arabinoside, respectively. The pH optimum ranged from 6.5 to 9.0. Mg2+ and Mn2+ were the preferred divalent cations. ATP, GTP, dGTP, ITP, dITP, TTP, and XTP were substrates for the enzymes. Our study indicates that deoxycytidine kinase is a dimer with two subunits and has phosphorylating activity for deoxyguanosine, deoxyadenosine, cytidine, and cytosine arabinoside. This highly purified enzyme will facilitate the study of its regulation and phosphorylation of anticancer or antiviral nucleoside analogues.     

Deoxyadenosine/deoxycytidine kinase from Bacillus subtilis. Purification, characterization, and physiological function

Three different deoxyribonucleoside kinases with specificities toward thymidine, deoxyguanosine, and deoxyadenosine/deoxycytidine, respectively, are identified in Bacillus subtilis. The deoxyadenosin/deoxycytidine kinase is purified 950-fold employing blue Sepharose CL-6B column chromatography. The two deoxyribonucleoside kinase activities copurify and are present in the same band after polyacrylamide gel electrophoresis. The molecular weight is determined by gel filtration to be 47,000. Cytidine, adenosine, arabinosylcytosine, and arabinosyladenine are substrates for the enzyme. The activities toward these substrates are less than 20% of the activities obtained with deoxyadenosin and deoxycytidine. The deoxycytidine and deoxyadenosine saturation curves are hyperbolic with Km values for both nucleosides around 5 microM. The maximal velocities for the two deoxyribonucleosides are nearly identical with GTP as phosphate donor. GTP is the best donor showing hyperbolic saturation curves and Km values around 150 microM depending on the deoxyribonucleoside concentration. dATP and dCTP are inhibitors when GTP is the phosphate donor. They may both act as phosphate donors themselves. A divalent metal ion is required, Mg2+ giving the highest activity. A spontaneous mutant, selected as resistant to 5-fluorodeoxycytidine, lacks both deoxycytidine and deoxyadenosine kinase activity, while it retains normal activities toward deoxyguanosine, deoxyuridine, and thymidine.     

Amino-terminal nucleotide-binding sequences of a Lactobacillus deoxynucleoside kinase complex isolated by novel affinity chromatography

A highly efficient new affinity medium for deoxycytidine kinase, deoxycytidine 5'-tetraphosphate-Sepharose (dCp4-Sepharose), has been constructed. A dCp4-Sepharose column effects a one-step, 19,000-fold, purification to homogeneity of dCyd kinase from the ammonium sulfate fraction of Lactobacillus acidophilus R-26 extract, with 60% recovery. dCTP, a potent end-product inhibitor, is used as an eluent, and it also stabilizes the extremely labile purified enzyme. A noncompeting deoxyadenosine kinase activity accompanies the deoxycytidine kinase activity eluted. Native polyacrylamide gel electrophoresis shows a single protein band, which coincides with both deoxycytidine kinase and deoxyadenosine kinase activities at several gel concentrations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals a single polypeptide band of 26,000 daltons. Since the native enzyme is known to have an Mr of 50,000, it appears that the enzyme is composed of two subunits of similar size. Sequence analysis of the intact protein from the N-terminus reveals but a single amino acid species per residue up to the 17th residue; at the 18th, 21st, 26th, and 27th residue positions of the sequence, however, there appear to be two different amino acids in almost equal amounts. This may indicate that the enzyme is composed of two nonidentical subunits having the same amino acid sequence near the N-terminus. Residues 6-13 contain the highly conserved Gly-X-X-Gly-X-Gly-Lys sequence found at the active sites of kinases and other nucleotide-binding proteins.     

Purification and properties of pyridoxal kinase from bovine brain

A 27,000-fold purification of pyridoxal kinase from bovine brain tissue has been achieved by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, hydroxyapatite chromatography, Sephadex G-150 gel filtration, Blue Sepharose CL-6B chromatography, and Phenyl-Superose chromatography. The final chromatography step yields a homogeneous preparation of high specific activity (2105 nmol/min/mg protein). The molecular mass of the native enzyme was estimated to be approximately 80,000 on gel filtration. The subunit molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be approximately 39,500. This indicates that pyridoxal kinase is a dimeric enzyme.     

Purification of modulator-deficient myosin light-chain kinase by modulator protein-Sepharose affinity chromatography.

Modulator-deficient myosin light-chain kinase from rabbit skeletal muscle was purified by modulator protein-Sepharose 4B affinity chromatography. The purified protein showed a single band (MW 80,000) on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and it exists as a monomer in the native state as determined by gel filtration. The modulator-deficient myosin light-chain kinase (MW 80,000), modulator protein (MW 16,500) and Ca2+ were essential for the kinase activity. The half-maximal activity of the kinase in the presence of excess modulator protein with 10 mM MgCl2 was at pCa 5.1, where full activity of actomyosin-ATPase is observed in the presence of the troponin--tropomyosin system. Assuming a rapid equilibrium between myosin light-chain kinase and two substrates, ATP and g2 light-chain, Km values for ATP and g2 light chain were evaluated as 0.28 mM and 0.024 mM, respectively. Vm/e was 5.7 s-1.     

Identification of the ATP.Mg-dependent protein phosphatase activator (FA) as a myelin basic protein kinase in the brain

The activating factor FA of the ATP.Mg-dependent protein phosphatase FcM was purified to near homogeneity from pig brain by a procedure involving chromatography on phosphocellulose, phosvitin-Sepharose 4B, and Blue Sepharose CL-6B. A specific myelin basic protein (MBP) kinase was found to co-purify with FA in a constant ratio throughout purification. It also proved impossible to separate the two activities on nondenaturing gel electrophoresis and 5-20% sucrose density gradient ultracentrifugation. Kinetic study indicated that MBP, presumably a substrate for FA, could compete with FcM for FA and thereby prevent the FA-mediated activation of the FcM activity. All the results taken together demonstrate that MBP kinase and FA are localized on the same protein. This, together with the data that FA, by activating the ATP.Mg-dependent phosphatase, promotes the dephosphorylation of [32P]MBP, phosphorylated by FA itself, suggests the evidence for a protein bearing two opposing activities involved in the regulation of brain functions. Moreover, since FA is tightly associated with the purified brain myelin membrane, the results further support the notion that FA may well be an endogenous protein kinase responsible for the cyclic phosphorylation-dephosphorylation of the central nervous system myelin.     

Human brain creatine kinase binding to immobilized cibacron blue F3GA: characterization and use in purification of the enzyme.

Creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) from adult human brain grey matter was purified by cibacron blue F3GA-Sepharose affinity chromatography. By gel electrophoresis of the purified enzyme under non-denaturing conditions a single protein band was observed. The dye-bound enzyme was eluted using its substrate, ATP. Reversibility of the binding of purified creatine kinase to blue Sepharose by ATP in a concentration-dependent manner indicated that the cibacron blue molecule which structurally mimics nucleotides occupied the substrate binding site of the enzyme. Also the marked dependence of enzyme binding to blue Sepharose on Mg2+ concentration suggested that Mg2+ ion is capable of combining with the dye moiety to form a site-specific binding complex that is similar to the physiological substrate of creatine kinase, namely Mg(2+)-ATP or Mg(2+)-ADP.     

Multisubstrate analogs for deoxynucleoside kinases. Triphosphate end products and synthetic bisubstrate analogs exhibit identical modes of binding and are useful probes for distinguishing kinetic mechanisms

Comparative inhibition kinetics with natural dNTP end products (dNp3) and new synthetic bisubstrate-type analogs, dNp4A (deoxynucleoside 5'-adenosine 5'-P1,P4-tetraphosphate), have been studied with their target deoxynucleoside kinases from Lactobacillus acidophilus. Analysis of inhibition specificity, inhibition patterns, and Ki(app) under various conditions has revealed the following conclusions. Both dNTP and dNp4A bind to the active site of the corresponding kinase through multiple binding determinants. The deoxynucleoside moiety of dNTP fits optimally at the deoxynucleoside binding site and provides the basis for its inhibition specificity, whereas the triphosphate group interacts with the ATP binding site, reinforcing the affinity of the molecule as a potent end product inhibitor (Ki = 0.4-3 microM). The adenosine moiety of dNp4A does not contribute to the binding of this compound, whereas the tetraphosphate portion is the second binding determinant, just as in the model developed for dNTP. dNTP and dNp4A proved to be useful tools for distinguishing the kinetic mechanisms of kinases which follow sequential pathways, i.e. the rapid equilibrium Random Bi Bi for dCyd and dGuo kinases and the steady state Ordered Bi Bi mechanism for two dAdo kinases associated either with dCyd kinase or with dGuo kinase on different multifunctional proteins.     

Purification and characterization of adenylate kinase isozymes from rat muscle and liver

Isozymes of adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) were purified from skeletal muscle and liver of rats to essentially homogeneous states by acrylamide gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. The isozyme from muscle was purified by acidification to pH 5.0, and column chromatography on phosphocellulose, Sephadex G-75 and Blue Sepharose CL-6B, while that from liver was purified by column chromatography on Blue Sepharose CL-6B, Sephadex G-75 and carboxymethyl cellulose. By these procedures the muscle isozyme was purified about 530-fold in 29% yield, and the liver isozyme about 3600-fold in 27% yield from the respective tissue extracts. The molecular weights of the muscle and liver isozymes were estimated as about 23 500 and 30 500, respectively, by both sodium dodecyl sulfate gel electrophoresis and molecular sieve chromatography, and no subunit of either isozyme was detected. The isoelectric points of the muscle and liver isozymes were 7.0 and 8.1, respectively. The Km values of the respective enzymes for ATP and ADP were similar, but the Km(AMP) of the liver isozyme was about one-fifth of that of the muscle isozyme. Immunological studies with rabbit antiserum against the rat muscle isozyme showed that the muscle isozyme was abundant in muscle, heart and brain, while the liver isozyme was abundant in liver and kidney.     

Striking ability of adenosine-2'(3')-deoxy-3'(2')-triphosphates and related analogues to replace ATP as phosphate donor for all four human,and the Drosophila melanogaster,deoxyribonucleoside kinases

In extension of an earlier report, six non-conventional analogues of ATP, three adenosine-2'-triphosphates (3'-deoxy, 3'-deoxy-3'-fluoro- and 3'-deoxy-3'-fluoroxylo-), and three adenosine-3'-triphosphates (2'-deoxy-, 2'-deoxy-2'-fluoro- and 2'-deoxy-2'-fluoroara-), were compared with ATP as potential phosphate donors for human deoxycytidine kinase (dCK), cytosolic thymidine kinase (TK1), mitochondrial TK2, deoxyguanosine kinase (dGK), and the deoxyribonucleoside kinase (dNK) from Drosophila melanogaster. With one group of enzymes, comprising TK1, TK2, dNK and dCK (with dAdo as acceptor), only 3'-deoxyadenosine-2'-triphosphate was an effective donor (5-60% that for ATP), and the other five analogues much less so, or inactive. With a second set, including dCK (dCyd, but not dAdo, as acceptor) and dGK (dGuo as acceptor), known to share high sequence similarity (approximately 45% sequence identity), all six analogues were good to excellent donors (13-119% that for ATP). With dCK and ATP1, products were shown to be 5'-phosphates. With dCK, donor properties of the analogues were dependent on the nature of the acceptor, as with natural 5'-triphosphate donors. With dCK (dCyd as acceptor), Km and Vmax for the two 2'(3')-deoxyadenosine-3'(2')-triphosphates are similar to those for ATP. With dGK, Km values are higher than for ATP, while Vmax values are comparable. Kinetic studies further demonstrated Michaelis-Menten (non-cooperative) or cooperative kinetics, dependent on the enzyme employed and the nature of the donor. The physiological significance, if any, of the foregoing remains to be elucidated. The overall results are, on the other hand, highly relevant to studies on the modes of interaction of nucleoside kinases with donors and acceptors; and, in particular, to interpretations of the recently reported crystal structures of dGK with bound ATP, of dNK with bound dCyd, and associated modeling studies.     

Snake venom phosphodiesterase: simple purification with Blue Sepharose and its application to poly(ADP-ribose) study.

A rapid method of purifying snake venom phosphodiesterase has been developed using Blue Sepharose or blue dextran/Sepharose as an affinity adsorbent. A sixty-fold purification of the enzyme from commercial preparations is achieved in a single step with a yield of 60%. The purified enzyme preparation is essentially free from phosphatase activities and exhibits a major protein band on SDS-polyacrylamide gel electrophoresis. Chain length analysis of poly(ADP-ribose) exemplifies the usefulness of this technique.     

Purification of the chloroplastic valyl-tRNA synthetase from Euglena gracilis.

Euglena gracilis chloroplast valyl-tRNA synthetase was purified 990 fold to a specific activity of about 1100 units/mg protein, by a series of steps including ammonium sulfate precipitation and chromatography on hydroxyapatite, DEAE-cellulose, Blue Dextran — Sepharose and Sephadex G200. The enzyme gives a single band upon polyacrylamide gel electrophoresis, appears to be a monomer with a molecular weight of 126,000 daltons and has Km values of 1.5 × 10^?5 M for L-valine, 5 × 10^?5 M for ATP, and 6 × 10^?8 for tRNA^Val.     

Identification of apolipoprotein A1 and immunoglobulin as components of a serum complex that mediates activation of human sperm motility

Serum is superior to other body fluids in activating the progressive motility of human spermatozoa and is used in connection with sperm separation for fertilization in vitro. The major activating capacity is localized to a macromolecular fraction, purified to homogeneity by a four-step protocol with ion-exchange chromatography, chromatofocusing, exclusion FPLC (elution corresponding to a molecular mass of about 250 kDa), and Blue Sepharose chromatography (no binding but elimination of albumin). The pure protein, at a concentration of 20-70 nmol/L, activated the motility to the same extent as serum. SDS-polyacrylamide gel electrophoresis under nonreducing conditions showed one band corresponding to a molecular mass of about 180 kDa. In the presence of mercaptoethanol, two bands are obtained corresponding to 50 kDa and about 25 kDa, respectively. Without the Blue Sepharose step, the sample after reduction revealed an additional band at about 67 kDa, suggesting that the fraction is then in complex also with albumin. Amino acid sequence analysis of the Blue Sepharose eluate identified three protein chains--those of apolipoprotein A1 and immunoglobulin heavy and light chains--suggesting that the preparation is an apolipoprotein A1-immunoglobulin complex. Antiserum raised toward the pure preparation in a rabbit inhibited human sperm motility, when added directly to spermatozoa. Pretreatment of human serum with rabbit antiserum significantly reduced its ability to activate sperm motility. The sperm activating capacity of the protein complex was destroyed by heating at 100 degrees C for 5 min, suggesting that the activity was dependent on intact protein conformations. Albumin, apolipoprotein A1, and immunoglobulins by themselves had only minor effects on sperm motility.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ability of adenosine-2'(3')-deoxy-3'(2')-triphosphates and related analogues to replace ATP as phosphate donor for all human and Drosphila melanogaster deoxyribonucleoside kinases

Six non-conventional adenosine-2'- and 3'-triphosphate analogues of ATP were tested as potential phosphate donors for all four human, and D. melanogaster, deoxyribonucleoside kinases. With dCK (only dAdo as acceptor), TK1, TK2 and dNK only 3'-deoxyadenosine-2'-triphosphate was an effective donor (5-60% that for ATP). With dCK (dCyd as acceptor) and dGK (dGuo as acceptor), sharing 45% sequence identity, donor activities ranged from 13 to 119% that for ATP. Products were 5'-phosphates. In some instances, kinetics are dependent on the nature of the acceptor, and donor and acceptors properties are mutually interdependent. Results are highly relevant to studies on the modes of interaction with the enzymes, and to interpretations of reported crystal structures of dCK and dNK with bound ligands.