Involvement of adenosine kinase in the phosphorylation of formycin B in CHO cells

In Chinese hamster ovary cells, [3H]formycin B is metabolized into formycin B-5'-monophosphate, formycin A-5'-monophosphate and higher phosphorylated derivatives of formycin A which are incorporated into RNA. Mutants of CHO cells independently selected for resistance to various adenosine analogs viz. toyocamycin, tubercidin, 6-methylmercaptopurine riboside, which contain no detectable activity of adenosine kinase (AK) in cell extracts, all exhibited between 2- to 3-fold increased resistance to formycin B. Formycin B-resistant mutants of CHO cells are also affected in AK, as indicated by the absence of AK activity in cell extracts. Both types of AK- mutants showed reduced uptake and phosphorylation of [3H]formycin B in comparison to the parental (AK+) cells. In addition, toxicity of formycin B towards CHO cells was reduced in presence of adenosine in a concentration dependent manner. These observations strongly indicate that in CHO cells, formycin B is phosphorylated via AK and that like other nucleoside analogs its phosphorylation may be essential for the drugs cellular toxicity.     

Biochemical genetic analysis of formycin B action in Leishmania donovani

Formycin B is cytotoxic toward Leishmania and is a potential chemotherapeutic agent for leishmaniasis. In order to determine the mechanism of action of formycin B, we have isolated and characterized clonal populations of formycin B-resistant Leishmania donovani. These formycin B-resistant clones are also cross-resistant to formycin A and allopurinol riboside-mediated growth inhibition. Incubation of the formycin B-resistant cells with [3H]formycin B indicates that, unlike wild type cells, the resistant populations cannot accumulate phosphorylated metabolites of exogenous [3H]formycin B. This is due to a defective transport system for formycin B in the resistant cells. However, wild type and mutant cells incorporate [3H]formycin A equally efficiently into [3H]formycin A-containing nucleotides and into RNA. These data suggest that formycin B cytotoxicity in Leishmania is not mediated by its incorporation as the adenosine analog into RNA. A plausible alternative hypothesis is proposed for the mechanism of action of the pyrazolo (4,3-d)pyrimidine C-nucleosides based upon depletion of an essential intracellular metabolite.     

Leishmania donovani: characteristics of adenosine and inosine transporters in promastigotes of two different strains

The nucleoside transport characteristics of two strains of Leishmania donovani promastigotes were studied. Strain S1, growing in fully defined medium, and strain S2 (MHOM/ET/67/HA3) both transported adenosine and inosine, but only strain S1 transported uridine and thymidine. Competition studies in the presence of 100 microM of unlabeled adenosine, inosine, guanosine, 2'-deoxyadenosine, tubercidin, formycin B, 3'-deoxyinosine as well as uridine, thymidine and cytidine, with either 1 microM [3H]adenosine or [3H]inosine as permeant, were carried out. The inhibition profile with [3H]inosine as permeant was essentially identical in S1 and S2 promastigotes, indicating that the same inosine transporter was present in both strains. However, with [3H] adenosine as permeant, significant differences were noted between the two strains. Thus, only adenosine, 2'-deoxyadenosine, tubercidin, uridine, and thymidine were strongly inhibitory in S1 promastigotes, while essentially all nucleosides tested were effective in S2 promastigotes. This indicates that adenosine transport in S2 promastigotes seems to involve a transporter differing from that described for S1 promastigotes.     

Isolation and characterization of angiotensin converting enzyme from human kidney

Angiotensin converting enzyme [EC 3.4.15.1] was solubilized from the membrane fraction of human kidney cortex using trypsin and purified to homogeneity by DEAE-cellulose, hydroxylapatite and DEAE-Sephadex A-50 column chromatographies, preparative isoelectric focusing, and Sephadex G-200 gel filtration. The final recovery of the enzyme was 13.9%. The molecular weight of the enzyme was estimated to be 199,000 by a sedimentation equilibrium method. A value of 170,000 was obtained for the reduced and denatured enzyme by dodecylsulfate-polyacrylamide gel electrophoresis. The enzyme was a glycoprotein consisting of a single polypeptide chain with an isoelectric point of 5.10. Neutral sugar accounted for 13% per weight of the enzyme. The purified enzyme had a specific activity of 96.9 mumol/min/mg protein for hippurylhistidylleucine. The Km value, Kcat value and hydrolytic coefficient (Kcat/Km) of the enzyme for hippurylhistidylleucine were 2.0 mM, 545 s-1 and 273 mM-1 . s-1, respectively. Rabbit antibody against the human kidney converting enzyme inhibited the activities of the enzymes from human lung and serum as equally as that from human kidney, but not those from sheep, dog, or rat sera. The human kidney and lung converting enzymes were immunologically identical on double immunodiffusion analysis.     

Transition-state analysis of a Vmax mutant of AMP nucleosidase by the application of heavy-atom kinetic isotope effects

The transition state of the Vmax mutant of AMP nucleosidase from Azotobacter vinelandii [Leung, H. B., & Schramm, V. L. (1981) J. Biol. Chem. 256, 12823-12829] has been characterized by heavy-atom kinetic isotope effects in the presence and absence of MgATP, the allosteric activator. The enzyme catalyzes hydrolysis of the N-glycosidic bond of AMP at approximately 2% of the rate of the normal enzyme with only minor changes in the Km for substrate, the activation constant for MgATP, and the Ki for formycin 5'-phosphate, a tight-binding competitive inhibitor. Isotope effects were measured as a function of the allosteric activator concentration that increases the turnover number of the enzyme from 0.006 s-1 to 1.2 s-1. The kinetic isotope effects were measured with the substrates [1'-3H]AMP, [2'-2H]AMP, [2'-2H]AMP, [9-15N]AMP, and [1',9-14C, 15N]AMP. All substrates gave significant kinetic isotope effects in a pattern that establishes that the reaction expresses intrinsic kinetic isotope effects in the presence or absence of MgATP. The kinetic isotope effect with [9-15N]AMP decreased from 1.034 +/- 0.002 to 1.021 +/- 0.002 in response to MgATP. The [1'-3H]AMP isotope effect increased from 1.086 +/- 0.003 to 1.094 +/- 0.002, while the kinetic isotope effect for [1',9-14C, 15N]AMP decreased from 1.085 +/- 0.003 to 1.070 +/- 0.004 in response to allosteric activation with MgATP. Kinetic isotope effects with [1'-14C]AMP and [2'-2H]AMP were 1.041 +/- 0.006 and 1.089 +/- 0.002 and were not changed by addition of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formycin B-resistant mutants of Chinese hamster ovary cells: novel genetic and biochemical phenotype affecting adenosine kinase.

Stable mutants which are approximately three- and eightfold resistant to the pyrazolopyrimidine nucleosides formycin A and formycin B (FomR) have been selected in a single step from mutagenized Chinese hamster ovary cells. In cell extracts, the two FomR mutants which were examined were both found to contain no measurable activity of the enzyme adenosine kinase (AK). However, cross-resistance studies with other adenosine analogs such as toyocamycin and tubercidin show that these mutants are distinct from toyocamycin or tubercidin resistant (Toyr) mutants which also contain no measurable AK activity in cell extracts. Studies on the uptake and incorporation of [3H]adenosine and [3H]tubercidin by various mutants and parental cell lines show that unlike the Toyr mutants, which are severely deficient in the phosphorylation of these compounds, the FomR mutants possess nearly normal capacity to phosphorylate these compounds and incorporate them into cellular macromolecules. These results suggest that the FomR mutants contain normal levels of AK activity in vivo. In cell hybrids formed between FomR X FomS cells and FomR X Toyr cells, the formycin-resistant phenotype of both of the FomR mutants behaved codominantly. However, the extracts from these hybrid cells contained either congruent to 50% (FomR X FomS) or no measurable (FomR X Toyr) AK activity, indicating that the lesion in these mutants neither suppresses the wild-type AK activity nor complements the AK deficiency of the Toyr mutants. The presence of AK activity in the FomR mutants in vivo, but not in their cell extracts, along with the codominant behavior of the mutants in hybrids, indicates that the lesions in the FomR mutant are of a novel nature. It is suggested that the genetic lesion in these mutants affects AK activity indirectly and that it may involve an essential cellular function which exists in a complex form with AK. Some implications of these results regarding the mechanism of action of formycin B are discussed.     

Genetic analysis of nucleoside transport in Leishmania donovani.

Genetic dissection of nucleoside transport in Leishmania donovani indicates that the insect vector form of these parasites possesses two biochemically distinct nucleoside transport systems. The first transports inosine, guanosine, and formycin B, and the second transports pyrimidine nucleosides and the adenosine analogs, formycin A and tubercidin. Adenosine is transported by both systems. A mutant, FBD5, isolated by virtue of its resistance to growth inhibition by 5 microM formycin B, cannot efficiently transport inosine, guanosine, or formycin B. This cell line is also cross-resistant to growth inhibition by a spectrum of cytotoxic analogs of inosine and guanosine. A second parasite mutant, TUBA5, isolated for its resistance to 20 microM tubercidin, cannot take up from the culture medium radiolabeled tubercidin, formycin A, uridine, cytidine, or thymidine. Both the FBD5 and the TUBA5 cell lines have about a 50% reduced capacity to take up adenosine, indicating that adenosine is transported by both systems. A tubercidin-resistant clonal derivative of FBD5, FBD5-TUB, has acquired the combined biochemical phenotype of each single mutant. The wild-type and mutant cell lines transport purine bases and uracil with equal efficiency. Mutational analysis of the relative growth sensitivities to cytotoxic nucleoside analogs and the selective capacities to take up exogenous radiolabeled nucleosides from the culture medium have enabled us to define genetically the multiplicity and substrate specificities of the nucleoside transport systems in L. donovani promastigotes.     

8-Hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum: 2. Kinetic and hydrogen-transfer studies

Steady-state kinetic parameters have been obtained for the pure 8-hydroxy-5-deazaflavin-reducing hydrogenase. With H2 and 8-hydroxy-5-deazariboflavin (F0) as substrates, Km (H2) = 12 microM, Km (F0) = 26 microM, and Kcat = 225 s-1. In the back-direction, F0H2 is reoxidized (anaerobically) at 225 s-1. Initial velocity patterns, product inhibition patterns, dead-end inhibition by carbon monoxide, and transhydrogenation to Procion Red HE-3B suggest a two-site hybrid ping-pong mechanism. A kinetic derivation for the rate equation is provided in the Appendix. Studies with D2 and with D2O reveal that no steps involving D transfer are substantially rate determining. Further, D2 yields F0H2 with no deuterium at C5 while in D2O a 5-monodeuterio F0H2 product is formed, indicating complete exchange of hydrogens from H2 with solvent before final transfer of a hydride ion out from reduced enzyme to C5 of F0.     

Nucleoside hydrolase from Crithidia fasciculata. Metabolic role, purification, specificity, and kinetic mechanism

Crithidia fasciculata cells grown on complex medium with added [8-14C, 5'-3H]inosine or [8-14C,5'-3H]adenosine metabolize greater than 50% of the salvaged nucleosides through a pathway involving N-glycoside bond cleavage. Cell extracts contain a substantial nucleoside hydrolase activity but an insignificant purine nucleoside phosphorylase. The nucleoside hydrolase has been purified 1000-fold to greater than 99% homogeneity from kilogram quantities of C. fasciculata. The enzyme is a tetramer of Mr 34,000 subunits to give an apparent holoenzyme Mr of 143,000 by gel filtration. All of the commonly occurring nucleosides are substrates. The Km values vary from 0.38 to 4.7 mM with purine nucleosides binding more tightly than the pyrimidines. Values of Vmax/Km vary from 3.4 x 10(3) M-1 s-1 to 1.7 x 10(5) M-1 s-1 with the pyrimidine nucleosides giving the larger values. The turnover rate for inosine is 32 s-1 at 30 degrees C. The kinetic mechanism with inosine as substrate is rapid equilibrium with random product release. The hydrolytic reaction can be reversed to give an experimental Keq of 106 M with H2O taken as unity. The product dissociation constants for ribose and hypoxanthine are 0.7 and 6.2 mM, respectively. Deoxynucleosides or 5'-substituted nucleosides are poor substrates or do not react, and are poor inhibitors of the enzyme. The enzyme discriminates against methanol attack from solvent during steady-state catalysis, indicating the participation of an enzyme-directed water nucleophile. The pH profile for inosine hydrolysis gives two apparent pKa values of 6.1 with decreasing Vmax/Km values below the pKa and a plateau at higher pH values. These effects are due to the pH sensitivity of the Vmax values, since Km is independent of pH. The pH profile implicates two negatively charged groups which stabilize a transition state with oxycarbonium character.     

Molecular organization of subunits of electroplax (sodium plus potassium)--activated adenosine triphosphatase.

Antisera against each of the two major subunits of detergent-solubilized electroplax (sodium plus potassium)-activated adenosine triphosphatase from Electrophorus electricus were prepared. Antiserum against the small subunit (a glycoprotein, Mr = 58,000) partially inhibits [3H]ouabain binding to the enzyme, but does not interfere with the phosphorylation of enzyme. Conversely, antiserum against the large subunit (the catalytic subunit Mr = 96,000) partially inhibits phosphorylation of the enzyme, but does not interfere with the binding of [3H]ouabain to the enzyme. Since ouabain only interacts with enzyme from the outer surface of the membrane and phosphorylation of enzyme takes place on the inner surface of the membrane, the results suggest that the small subunits are exposed on the outer surface of the membrane, whereas the large subunits are oriented predominantely facing the cytoplasmic side.     

Adenosine Inhibits Choline Kinase Activity and Decreases the Phosphorylation of Choline in Striatal Synaptosomes

The main objective of these studies was to determine whether adenosine inhibits choline kinase in rat striata, leading to a decreased incorporation of choline into phosphorylcholine, a mechanism that may mediate seizure-induced increases in the levels of free choline in brain. Incubation of particulate and soluble fractions of striatal synaptosomes with adenosine or its metabolically stable analogues significantly inhibited enzyme activity. The inhibition was noncompetitive versus choline and competitive versus MgATP. Inhibitor constants for adenosine, 2-chloroadenosine, and 2',5'-dideoxyadenosine at the MgATP site were 94, 49, and 207 microM, respectively; these values were less than the Michaelis constant for MgATP (340 microM). To determine whether adenosine altered the phosphorylation of choline in an intact preparation, synaptosomes were incubated with [3H]choline in the presence or absence of adenosine or its analogues and the amount of [3H]-phosphorylcholine formed from the [3H]choline taken up was measured. All compounds tested significantly reduced the synthesis of [3H]phosphorylcholine. Results suggest that following seizures or hypoxia, when levels of adenosine increase and the concentration of ATP decreases, inhibition of choline phosphorylation may be manifest, resulting in increased levels of free choline in brain.     

Inosine analogs. Their metabolism in mouse L cells and in Leishmania donovani

The growth of Leishmania donovani promastigotes and mouse L cells is differentially inhibited by several inosine analogs with modifications in the imidazole ring. The protozoal and mammalian cells also demonstrate differential metabolism of these analogs. 7-Deazainosine, 7-thia-7,9-dideazainosine, and formycin B were converted to their respective ATP analogs by both cell types. 8-Azainosine was converted to a GTP analog by mouse L cells; L. donovani did not metabolize this nucleoside. 9-Deazainosine and allopurinol riboside were metabolized only to their respective IMP analogs by L cells. L. donovani metabolized 9-deazainosine and allopurinol riboside to their ATP analogs and also metabolized 9-deazainosine to its GTP analog. All nucleosides studied were resistant to cleavage by either organism. From metabolism studies in the presence of a specific enzyme inhibitor, it was deduced that allopurinol riboside, formycin B, and 9-deazainosine were phosphorylated by at least two different routes in the mouse L cells. The metabolism of formycin B was inhibited 65% by the adenosine kinase inhibitor, 5-iodotubercidin, whereas the metabolism of allopurinol riboside (14% inhibition) and 9-deazainosine (0% inhibition) was only slightly affected by this inhibitor. The metabolism of allopurinol riboside and 9-deazainosine by L. donovani was not affected by 5-iodotubercidin. In contrast to the results of L cells, the metabolism of formycin B by L. donovani was also not affected by 5-iodotubercidin. The abilities of mouse L cells and L. donovani to metabolize these inosine analogs to the corresponding nucleotide analogs of ATP or GTP may be considered to be an activating step and correlates well with the respective cytotoxic effects of these compounds.     

[3H]Ouabain binding and Na+, K+-ATPase in resealed human red cell ghosts

The interaction of the cardiac glycoside [3H]ouabain with the Na+, K+ pump of resealed human erythrocyte ghosts was investigated. Binding of [3H]ouabain to high intracellular Na+ ghosts was studied in high extracellular Na+ media, a condition determined to produce maximal ouabain binding rates. Simultaneous examination of both the number of ouabain molecules bound per ghost and the corresponding inhibition of the Na+, K+-ATPase revealed that one molecule of [3H]ouabain inhibited one Na+, K+-ATPase complex. Intracellular magnesium or magnesium plus inorganic phosphate produced the lowest ouabain binding rate. Support of ouabain binding by adenosine diphosphate (ADP) was negligible, provided synthesis of adenosine triphosphate (ATP) through the residual adenylate kinase activity was prevented by the adenylate kinase inhibitor Ap5A. Uridine 5'-triphosphate (UTP) alone did not support ouabain binding after inhibition of the endogenous nucleoside diphosphokinase by trypan blue and depletion of residual ATP by the incorporation of hexokinase and glucose. ATP acting solely at the high- affinity binding site of the Na+, K+ pump (Km approximately 1 microM) promoted maximal [3H]ouabain binding rates. Failure of 5'-adenylyl-beta- gamma-imidophosphate (AMP-PNP) to stimulate significantly the rate of ouabain binding suggests that phosphorylation of the pump was required to expose the ouabain receptor.     

A cyclic nucleotide-independent protein kinase in Leishmania donovani.

Leishmania donovani promastigotes labelled for 2 h with 32Pi incorporated radioactivity into at least 21 different proteins, as determined by SDS/polyacrylamide-gel electrophoresis. Pulse-chase studies with 32Pi demonstrated that the labelled proteins were in a dynamic state: some radiolabelled proteins rapidly disappeared and others appeared after the chase. The possibility of an ectokinase on the parasite was examined; incubation of intact parasites for 10 min at 25 degrees C in an osmotically buffered medium containing [gamma-32P]ATP, but not [alpha-32P]ATP, resulted in the labelling of 10 different protozoal proteins, presumably localized to the surface of the organism's plasma membrane. Intact promastigotes also catalysed the transfer of 32P from [gamma-32P]ATP to histones. The histone-dependent kinase was solubilized by repeated freezing and thawing, and sonication, and purified 118-fold by chromatographing the high-speed (200,000 g, 1 h) supernatant fraction on QAE-Sephadex, Sephadex G-150 and hydroxyapatite columns. The kinase eluted as a single activity peak from all three columns. The partially purified histone-dependent kinase had the following properties: pH optimum, 7.0; optimum temperature, 37 degrees C; Km for mixed calf thymus histone, 0.15 mM; Km for ATP, 0.8 mM; preferred fractionated histone acceptors, H2b greater than H4 greater than H2a greater than H3 (H1 does not serve as an acceptor); optimum activity required 10-20 mM-Mg2+; inhibited 50-80% by 0.01 mM- and 1 mM-Ca2+; activity was not stimulated by calmodulin, cyclic AMP (1 mM) or cyclic GMP (1 mM) nor inhibited by a cyclic AMP-dependent protein kinase inhibitor (50 micrograms/assay); apparent Mr 75,000, as determined by Sephadex G-150 gel filtration chromatography; phosphorylated exclusively serine residues. Protein kinase activity was low in the early exponential phase of the growth curve and increased 6-fold upon entry into the stationary phase.     

Structural role of serine 127 in the NADH-binding site of human NADH-cytochrome b5 reductase

Serine 127 of human NADH-cytochrome b5 reductase was replaced by proline and alanine by site-directed mutagenesis. The former mutation has been found in the genes of patients with hereditary deficiency of the enzyme. Both the mutant enzymes (Ser-127----Pro mutant and Ser-127----Ala mutant) were overproduced in Escherichia coli and purified to homogeneity. The two purified mutant enzymes showed indistinguishable spectral properties which differed from those of the wild-type enzyme. The mutant enzymes showed higher molecular extinction coefficients at 462 nm than that of the wild-type enzyme. Quenching of FAD fluorescence in these mutant enzymes was significantly less than that in the wild-type enzyme. Furthermore, circular dichroism spectra of the mutant enzymes were different, in both the visible and ultraviolet regions, from that of the wild-type enzyme. The spectra of the mutant enzymes in the visible region were restored to almost the same spectrum as the wild type upon reduction with NADH. Ser-127----Pro mutant and Ser-127----Ala mutant showed very low Kcat/Km (NADH) values (5 x 10(7) and 3.5 x 10(7) s-1 M-1, respectively) with cytochrome b5 as an electron acceptor, than that of the wild-type enzyme (Kcat/Km (NADH) = 179 x 10(7) s-1 M-1), while the Kcat/Km (cytochrome b5) value for each enzyme was similar. The mutant enzymes were less thermostable than the wild-type enzyme. These results indicate that serine 127 plays an important role to maintain the structure of the NADH-binding site in the enzyme.     

Phosphorylation of ribosomal protein S6 at multiple sites by a cyclic AMP-independent protein kinase from lymphoid cells

Ribosomes prepared from murine lymphosarcoma cells were phosphorylated by a cyclic AMP-independent protein kinase designated H4P kinase. H4P kinase was isolated as an inactive enzyme which was activated by Mg2+-ATP and an endogenous converting enzyme. In the absence of preactivation by Mg2+-ATP and an endogenous converting enzyme, H4P kinase catalyzed phosphorylation of 80, 60, and 40 S ribosomal subunits at a low rate. After activation, the H4P kinase selectively catalyzed phosphorylation of the S 6 protein in the 40 S ribosomal subunit. Under the assay conditions selected, at least 90% of the [32P]phosphate transferred to the 40 S ribosomal preparation was incorporated into S 6. The apparent Km for 40 S subunits phosphorylated by H4P kinase was 7.2 microM. The calculated Vmax was 50 nmol of Pi transferred per min/mg. Exhaustive phosphorylation of 40 S subunits resulted in incorporation of 3 mol of phosphate/mol of S 6, in contrast to results reported previously which indicated 0.3 mol of phosphate was transferred by a similar enzyme from reticulocyte (Del Grande, R. W., and Traugh, J. A. (1982) Eur. J. Biochem. 123, 421-428). These data are consistent with a potential role for H4P kinase in the insulin-mediated phosphorylation of S 6 at multiple sites.     

Partial purification and some properties of rat brain inositol 1,4,5-trisphosphate 3-kinase.

An enzyme which catalyses the ATP-dependent phosphorylation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was purified approx. 180-fold from rat brain cytosol by (NH4)2SO4 precipitation, chromatography through hydroxyapatite, anion-exchange fast protein liquid chromatography and gel-filtration chromatography. Gel filtration on Sepharose 4B CL gives an Mr of 200 x 10(3) for the native enzyme. The inositol tetrakisphosphate (InsP4) produced by the enzyme has the chromatographic, chemical and metabolic properties of Ins(1,3,4,5)P4. Ins(1,4,5)P3 3-kinase displays simple Michaelis-Menten kinetics for both its substrates, having Km values of 460 microM and 0.44 microM for ATP and Ins(1,4,5)P3 respectively. When many of the inositol phosphates known to occur in cells were tested, only Ins(1,4,5)P3 was a substrate for the enzyme; the 2,4,5-trisphosphate was not phosphorylated. Inositol 4,5-bisphosphate and glycerophosphoinositol 4,5-bisphosphate were phosphorylated much more slowly than Ins(1,4,5)P3. CTP, GTP and adenosine 5'-[gamma-thio]triphosphate were unable to substitute for ATP. When assayed under conditions of first-order kinetics, Ins(1,4,5)P3 kinase activity decreased by about 40% as the [Ca2+] was increased over the physiologically relevant range. This effect was insensitive to the presence of calmodulin and appeared to be the result of an increase in the Km of the enzyme for Ins(1,4,5)P3. Preincubation with ATP and the purified catalytic subunit of cyclic AMP-dependent protein kinase did not affect the rate of phosphorylation of Ins(1,4,5)P3 when the enzyme was assayed at saturating concentrations of Ins(1,4,5)P3 or at concentrations close to its Km for this substrate.     

The kinetics of hydrolysis of some extended N-aminoacyl-L-arginine methyl esters by porcine pancreatic kallikrein. A comparison with human plasma Kallikrein.

The effects of subsite interactions in the S2-S4 region [Schechter & Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162] of porcine pancreatic kallikrein (EC 3.4.21.8) on its catalytic efficiency have been investigated. Kinetic constants (Kcat, Km) have been determined for a series of seven extended N-aminoacyl-L-arginine methyl esters whose sequence is based on either the C-terminal sequence of kallidin (-Pro-Phe-Arg) or (-Gly-)nArg. With these substrates it has been found that neither acylation nor deacylation of the enzyme is rate-limiting. Values of Kcat. range from 21.5 to 2320s-1, indicating that there are interactions with different residues in the N-aminoacyl chain and enzyme subsites in the S2-S4 region. It is shown that possible hydrogen-bonded interactions with the enzyme in the S3-S4 region have a significant effect on catalysis. The presence of L-phenylalanine at P2 has a very large effect on both Kcat, and Km, giving a greatly enhanced catalytic efficiency. Substrates with L-proline at P3 also have a marked effect, but in this case the overall effect is one of lowered catalytic efficiency. By comparison with the results of a similar study with human plasma kallikrein I (EC 3.4.21.8), it has been possible to demonstrate that there are considerable differences in kinetic behaviour between the two enzymes. These are related to relative differences in the rates of acylation and deacylation with ester substrates and also the roles of subsites S2 and S3 of the two enzymes.     

Reactions of 1-bromo-2-[14C]pinacolone with acetylcholinesterase from Torpedo nobiliana. Effects of 5-trimethylammonio-2-pentanone and diisopropyl fluorophosphate

1-Bromo-2-[14C]pinacolone, (CH3)3C14COCH2Br [( 14C]BrPin), was prepared from [1-14C]acetyl chloride and tert-butylmagnesium chloride with cuprous chloride catalyst, followed by bromination. It was examined as an active-site directed label for acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) (AcChE). AcChE, isolated from Torpedo nobiliana, has k(cat) = (4.00 +/- 0.04).10(3) s-1, Km = 0.055 +/- 0.008 mM in hydrolysis of acetylthiocholine, and k(cat) = (5.6 +/- 0.2).10(3) s-1, Km = 0.051 +/- 0.003 mM in hydrolysis of acetylcholine. BrPin, binding in the trimethyl cavity, acts initially as a reversible competitive inhibitor, Ki = 0.20 +/- 0.09 mM, and, with time, as an irreversible covalently bound inactivator. Introduction of 14C from [14C]BrPin into Torpedo AcChE at pH 7.0 was followed by SDS-PAGE, autoradiography and scintillation counting, in the absence and presence of 5-trimethylammonio-2-pentanone (TAP), a competitive inhibitor (Ki = 0.075 +/- 0.001 mM) isosteric with acetylcholine; 1.8-1.9 14C was incorporated per inactivated enzyme unit at 50% inactivation. TAP retarded inactivation by [14C]BrPin, and prevented introduction of 0.9-1.1 14C per unit of enzyme protected. Prior inactivation of AcChE by BrPin prevents reaction with [3H]diisopropyl fluorophosphate [( 3H]DFP). Prior inactivation by DFP or [3H]DFP does not prevent reaction with [14C]BrPin, and this subsequent reaction with BrPin does not displace the [3H] moiety. [14C]BrPin alkylates a nucleophile in the active site, and this reaction does not alkylate or utilize the serine-hydroxyl.     

Sodium-dependent nucleoside transport in mouse intestinal epithelial cells. Two transport systems with differing substrate specificities

Nucleoside transport was examined in freshly isolated mouse intestinal epithelial cells. The uptake of formycin B, the C nucleoside analog of inosine, was concentrative and required extracellular sodium. The initial rate of sodium-dependent formycin B transport was saturable with a Km of 45 +/- 3 microM. The purine nucleosides adenosine, inosine, guanosine, and deoxyadenosine were all good inhibitors of sodium-dependent formycin B transport with 50% inhibition (IC50) observed at concentrations less than 30 microM. Of the pyrimidine nucleosides examined, only uridine (IC50, 41 +/- 9 microM) was a good inhibitor. Thymidine and cytidine were poor inhibitors with IC50 values greater than 300 microM. Direct measurements of [3H]thymidine transport revealed, however, that the uptake of this nucleoside was also mediated by a sodium-dependent mechanism. Thymidine transport was inhibited by low concentrations of cytidine, uridine, adenosine, and deoxyadenosine (IC50 values less than 25 microM), but not by formycin B, inosine, or guanosine (IC50 values greater than 600 microM). These data indicate that there are two sodium-dependent mechanisms for nucleoside transport in mouse intestinal epithelial cells, and that formycin B and thymidine may serve as model substrates to distinguish between these transporters. Neither of these sodium-dependent transport mechanisms was inhibited by nitrobenzylmercaptopurine riboside (10 microM), a potent inhibitor of one of the equilibrative (facilitated diffusion) nucleoside transporters found in many cells.