The kinetic mechanism of S-adenosyl-L-methionine: glutamylmethyltransferase from Salmonella typhimurium

The kinetic mechanism of the CheR methyltransferase, S-adenosyl-L-methionine (AdoMet): protein-L-glutamate O-methyltransferase (EC 2.1.1.24), from Salmonella typhimurium was investigated. Initial velocity, product inhibition, and binding studies were performed, and from the data obtained, it was determined that the mechanism of the reaction catalyzed by the enzyme is random. Initial velocity rates were measured with varied amounts of both substrates, and double-reciprocal plots gave patterns which converged on or near the abscissa. The products, S-adenosyl-L-homocysteine and methylated receptor, were found to be competitive inhibitors with respect to both AdoMet and receptor. Equilibrium dialysis and immunoprecipitation studies indicated that the two substrates can bind to the enzyme independent of each other. These results are consistent with a random mechanism with no abortive complexes being formed. The Michaelis constants calculated for AdoMet and receptor were 8.62 microM and 2.03 mg/ml total membrane protein (approximately 2.10 microM Tar protein), and the apparent dissociation constants of AdoMet and the receptor were 16.8 microM and 4.07 mg/ml total membrane protein (approximately 4.2 microM Tar protein), respectively. The Kd of AdoMet for the enzyme was 10.9 microM as determined by binding studies.     

Purification, characterization, and kinetic mechanism of S-adenosyl-L-methionine: vitexin 2"-O-rhamnoside 7-O-methyltransferase of Avena sativa L

An O-methyltransferase catalyzing the transfer of the methyl group of S-adenosyl-L-methionine to the A-ring 7-hydroxyl group of vitexin 2"-O-rhamnoside has been isolated from oat primary leaves and purified 180-fold by protein fractionation with (NH4)2SO4 and chromatography on DEAE-cellulose and S-adenosyl-L-homocysteine-sepharose. Km values for S-adenosyl-L-methionine and the flavonoid substrate were 1.6 microM and 15 microM, respectively. The lack of methyltransfer to biosynthetic intermediates suggests that the reaction is the last step in the biosynthetic pathway to the oat flavonoid 7-O-methylvitexin 2"-O-rhamnoside. Based on results obtained from kinetic inhibition studies and affinity chromatography a mono-iso Theorell-Chance mechanism is proposed with the nucleotide substrate binding before the flavonoid.     

DNA-[N4-cytosine]-methyltransferase from Bacillus amyloliquefaciens: mechanism of action derived from steady state kinetics

Kinetic analysis of methyl group transfer from S-adenosyl-L-methionine (SAM) to the 5'-GGATCC recognition site catalyzed by the DNA-[N4-cytosine]-methyltransferase from Bacillus amyloliquefaciens [EC 2.1.1.113] has shown that the dependence of the rate of methylation of the 20-meric substrate duplex on SAM and DNA concentration are normally hyperbolic, and the maximal rate is attained upon enzyme saturation with both substrates. No substrate inhibition is observed even at concentrations many times higher than the Km values (0.107 microM for DNA and 1.45 microM for SAM), which means that no nonreactive enzyme-substrate complexes are formed during the reaction. The overall pattern of product inhibition corresponds to an ordered steady-state mechanism following the sequence SAM decreases DNA decreases metDNA increases SAH increases (S-adenosyl-L-homocysteine). However, more detailed numerical analysis of the aggregate experimental data admits an alternative order of substrate binding, DNA decreases SAM decreases, though this route is an order of magnitude slower.     

Catalytic roles for carbon-oxygen hydrogen bonding in SET domain lysine methyltransferases

SET domain enzymes represent a distinct family of protein lysine methyltransferases in eukaryotes. Recent studies have yielded significant insights into the structural basis of substrate recognition and the product specificities of these enzymes. However, the mechanism by which SET domain methyltransferases catalyze the transfer of the methyl group from S-adenosyl-L-methionine to the lysine epsilon-amine has remained unresolved. To elucidate this mechanism, we have determined the structures of the plant SET domain enzyme, pea ribulose-1,5 bisphosphate carboxylase/oxygenase large subunit methyltransferase, bound to S-adenosyl-L-methionine, and its non-reactive analogs Aza-adenosyl-L-methionine and Sinefungin, and characterized the binding of these ligands to a homolog of the enzyme. The structural and biochemical data collectively reveal that S-adenosyl-L-methionine is selectively recognized through carbon-oxygen hydrogen bonds between the cofactor's methyl group and an array of structurally conserved oxygens that comprise the methyl transfer pore in the active site. Furthermore, the structure of the enzyme co-crystallized with the product epsilon-N-trimethyllysine reveals a trigonal array of carbon-oxygen interactions between the epsilon-ammonium methyl groups and the oxygens in the pore. Taken together, these results establish a central role for carbon-oxygen hydrogen bonding in aligning the cofactor's methyl group for transfer to the lysine epsilon-amine and in coordinating the methyl groups after transfer to facilitate multiple rounds of lysine methylation.     

The dual-specific active site of 7,8-diaminopelargonic acid synthase and the effect of the R391A mutation

7,8-diaminopelargonic acid (DAPA) synthase (EC 2.6.1.62) is a pyridoxal phosphate (PLP)-dependent transaminase that catalyzes the transfer of the alpha-amino group from S-adenosyl-L-methionine (SAM) to 7-keto-8-aminopelargonic acid (KAPA) to form DAPA in the antepenultimate step in the biosynthesis of biotin. The wild-type enzyme has a steady-state kcat value of 0.013 s(-1), and the K(m) values for SAM and KAPA are 150 and <2 microM, respectively. The k(max) and apparent K(m) values for the half-reaction of the PLP form of the enzyme with SAM are 0.016 s(-1) and 300 microM, respectively, while those for the reaction with DAPA are 0.79 s(-1) and 1 microM. The R391A mutant enzyme exhibits near wild-type kinetic parameters in the reaction with SAM, while the apparent K(m) for DAPA is increased 180-fold. The 2.1 A crystal structure of the R391A mutant enzyme shows that the mutation does not significantly alter the structure. These results indicate that the conserved arginine residue is not required for binding the alpha-amino acid SAM, but it is important for recognition of DAPA.     

Purification and characterization of Streptomyces griseus catechol O-methyltransferase

A soluble (100,000 x g supernatant) methyltransferase catalyzing the transfer of the methyl group of S-adenosyl-L-methionine to catechols was present in cell extracts of Streptomyces griseus. A simple, general, and rapid catechol-based assay method was devised for enzyme purification and characterization. The enzyme was purified 141-fold by precipitation with ammonium sulfate and successive chromatography over columns of DEAE-cellulose, DEAE-Sepharose, and Sephacryl S-200. The purified cytoplasmic enzyme required 10 mM magnesium for maximal activity and was catalytically optimal at pH 7. 5 and 35 degrees C. The methyltransferase had an apparent molecular mass of 36 kDa for both the native and denatured protein, with a pI of 4.4. Novel N-terminal and internal amino acid sequences were determined as DFVLDNEGNPLENNGGYXYI and RPDFXLEPPYTGPXKARIIRYFY, respectively. For this enzyme, the K(m) for 6,7-dihydroxycoumarin was 500 +/- 21.5 microM, and that for S-adenosyl-L-methionine was 600 +/- 32.5 microM. Catechol, caffeic acid, and 4-nitrocatechol were methyltransferase substrates. Homocysteine was a competitive inhibitor of S-adenosyl-L-methionine, with a K(i) of 224 +/- 20.6 microM. Sinefungin and S-adenosylhomocysteine inhibited methylation, and the enzyme was inactivated by Hg(2+), p-chloromercuribenzoic acid, and N-ethylmaleimide.     

Purification and properties of S-adenosyl-L-methionine:nicotinic acid-N-methyltransferase from cell suspension cultures of Glycine max L

A soluble enzyme which catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the nitrogen atom of pyridine-3-carboxylic acid (nicotinic acid) could be detected in protein preparations from heterotrophic cell suspension cultures of soybean (Glycine max L.). Enzyme activity was enriched nearly 100-fold by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography to study kinetic properties. S-adenosyl-L-methionine:nicotinic acid-N-methyltransferase (EC 2.1.1.7) showed a pH optimum at pH 8.0 and a temperature optimum between 35 and 40 degrees C. The apparent KM values were determined to be 78 microM for nicotinic acid and 55 microM for the cosubstrate. S-Adenosyl-L-homocysteine was a competitive inhibitor of the methyltransferase with a KI value of 95 microM. The native enzyme had a molecular mass of about 90 kDa. The catalytic activity was inhibited by reagents blocking SH groups, whereas other divalent cations did not significantly influence of the enzyme reaction. The purified methyltransferase revealed a remarkable specificity for nicotinic acid. No other pyridine derivative was a suitable methyl group acceptor. To study a potential methyltransferase activity with nicotinamide as substrate, an additional purification step was necessary to remove nicotinamide amidohydrolase activity from the enzyme preparation. This was achieved by affinity chromatography on S-adenosyl-L-homocysteine-Sepharose thus leading to a 580-fold purified enzyme which showed no methyltransferase activity toward nicotinamide as substrate.     

Partial purification, characterization, and kinetic analysis of isoflavone 5-O-methyltransferase from yellow lupin roots

An isoflavone 5-O-methyltransferase was partially purified from the roots of yellow lupin (Lupinus luteus) by fractional precipitation with ammonium sulfate, followed by gel filtration and ion-exchange chromatography using a fast-protein liquid chromatography system. This enzyme, which was purified 810-fold, catalyzed position-specific methylation of the 5-hydroxyl group of a number of substituted isoflavones. The methyltransferase had a pH optimum of 7 in phosphate buffer, an apparent pI of 5.2, a molecular weight of 55,000, no requirement for Mg2+, and was inhibited by various SH-group reagents. Substrate interaction kinetics of the isoflavonoid substrate and S-adenosyl-L-methionine gave converging lines which were consistent with a sequential bireactant binding mechanism. Furthermore, product inhibition studies showed competitive inhibition between S-adenosyl-L-methionine and S-adenosyl-L-homocysteine and noncompetitive inhibition between the isoflavone and either S-adenosyl-L-homocysteine or the 5-O-methylisoflavone. The kinetic patterns obtained were consistent with an ordered bi bi mechanism, where S-adenosyl-L-methionine is the first substrate to bind to the enzyme and S-adenosyl-L-homocysteine is the final product released. The physiological role of this enzyme is discussed in relation to the biosynthesis of 5-O-methylisoflavones of this tissue.     

Protein methylases in Trypanosoma brucei brucei: activities and response to DL-alpha-difluoromethylornithine

Protein methylases I, II and III were detected in extracts of Trypanosoma brucei brucei, and characterized according to the specific amino substituent methylated. Only protein methylase II activity was elevated by difluoromethylornithine treatment of T. b. brucei, and hence this enzyme was characterized further. Protein methylase II transferred methyl groups from S-adenosyl-L-methionine (S-AdoMet) to the carboxyl residues of several protein substrates, exhibiting highest activity with histone VIII-S (arginine-rich subgroup f3). The crude enzyme had an apparent Km for histone VIII-S of 28 mg ml-1 (11.4 mM-aspartyl and 18.4 mM-glutamyl residues methylated), and an apparent Km for S-AdoMet of 8.4 microM. T. b. brucei protein methylase II was sensitive to inhibition by S-adenosyl-L-homocysteine and its analogue sinefungin with apparent Ki values of 12.9 and 1.6 microM, respectively. Using a partially purified preparation, analysis of kinetic data in the presence and absence of sinefungin indicated that this analogue acts as a competitive inhibitor of the S-AdoMet binding site, and as a non-competitive inhibitor of the (protein) histone VIII-S binding site. The possible role of the enzyme in morphological control and its potential as a chemotherapeutic target are discussed.     

Direct photolabeling of the EcoRII methyltransferase with S-adenosyl-L-methionine

Ultraviolet irradiation of EcoRII methyltransferase in the presence of its substrate, S-adenosyl-L-methionine (AdoMet), results in the formation of a stable enzyme-substrate adduct. This adduct can be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after irradiation of the enzyme in the presence of either [methyl-3H]AdoMet or [35S]AdoMet. The extent of photolabeling is low. Under optimal conditions, 4.5 pmol of [3H]AdoMet is incorporated into 100 pmol of enzyme. Use of the 8-azido derivative of AdoMet as the photolabeling substrate increases the incorporation by approximately 2-fold. However, this adduct, unlike the one formed with AdoMet, is not stable when treated with thiol reagents or precipitated with trichloroacetic acid. A catalytically active conformation of the enzyme is needed for AdoMet photolabeling. Heat-inactivated enzyme or proteins for which AdoMet is not a substrate or cofactor do not undergo adduct formation. Two other methyltransferases, MspI and dam methylases are also shown to form adducts with AdoMet upon UV irradiation. The binding constant of the EcoRII methyltransferase for AdoMet determined with the photolabeling reaction is 11 microM, which is similar to the binding constant of 9 microM previously reported (Friedman, S. (1986) Nucleic Acids Res. 14, 4543-4556). The AdoMet analogs S-adenosyl-L-homocysteine (Ki = 0.83 microM) and sinefungin (Ki = 4.3 microM) are effective inhibitors of photolabeling, whereas S-adenosyl-D-homocysteine (Ki = 46 microM) is a poor inhibitor. These experiments indicate that AdoMet becomes covalently bound at the AdoMet-binding site on the enzyme molecule. The EcoRII methyltransferase-AdoMet adduct is very stable and could be used to identify the AdoMet-binding site on DNA methyltransferases.     

Kinetics and inhibition studies of catechol O-methyltransferase from the yeast Candida tropicalis.

The Kms for esculetin and S-adenosyl-L-methionine for catechol O-methyltransferase from the yeast Candida tropicalis were 6.2 and 40 microM, respectively. S-Adenosyl-L-homocysteine was a very potent competitive inhibitor with respect to S-adenosyl-L-methionine, with a Ki of 6.9 microM. Of the catechol-related inhibitors, purpurogallin, with a Ki of 0.07 microM, showed the greatest inhibitory effect. Sulfhydryl group-blocking reagents, such as thiol-oxidizing 2-iodosobenzoic acid and mercaptide-forming p-chloromercuribenzoic acid, provided evidence for sulfhydryl groups in the active site of the enzyme. Yeast catechol O-methyltransferase is a metal-dependent enzyme and requires Mg2+ for full activity. Zn2+ and Mn2+ but not Ca2+ were able to substitute for Mg2+. Mn2+ showed optimal enzyme activation at concentrations 50- to 100-fold lower than those of Mg2+.     

Substrate specificity and kinetic mechanism of mammalian G9a histone H3 methyltransferase

Lysine-specific murine histone H3 methyltransferase, G9a, was expressed and purified in a baculovirus expression system. The primary structure of the recombinant enzyme is identical to the native enzyme. Enzymatic activity was favorable at alkaline conditions (>pH 8) and low salt concentration and virtually unchanged between 25 and 42 degrees C. Purified G9a was used for substrate specificity and steady-state kinetic analysis with peptides representing un- or dimethylated lysine 9 histone H3 tails with native lysine 4 or with lysine 4 changed to alanine (K4AK9). In vitro methylation of the H3 tail peptide resulted in trimethylation of Lys-9 and the reaction is processive. The turnover number (k(cat)) for methylation was 88 and 32 h(-1) on the wild type and K4AK9 histone H3 tail, respectively. The Michaelis constants for wild type and K4AK9 ((K(m)(pep))) were 0.9 and 1.0 microM and for S-adenosyl-L-methionine (K(m)(AdoMet)) were 1.8 and 0.6 microM, respectively. Comparable kinetic constants were obtained for recombinant histone H3. The conversion of K4AK9 di- to trimethyl-lysine was 7-fold slower than methyl group addition to unmethylated peptide. Preincubation studies showed that G9a-AdoMet and G9a-peptide complexes are catalytically active. Initial velocity data with peptide and S-adenosyl-L-methionine (AdoMet) and product inhibition studies with S-adenosyl-L-homocysteine were performed to assess the kinetic mechanism of the reaction. Double reciprocal plots and preincubation studies revealed S-adenosyl-L-homocysteine as a competitive inhibitor to AdoMet and mixed inhibitor to peptide. Trimethylated peptides acted as a competitive inhibitor to substrate peptide and mixed inhibitor to AdoMet suggesting a random mechanism in a Bi Bi reaction for recombinant G9a where either substrate can bind first to the enzyme, and either product can release first.     

Protein-O-carboxylmethyltransferase from cytosol and membranes of chicken erythrocytes

Cytosolic protein-O-carboxylmethyltransferase was purified more than 4,000-fold in specific activity and membrane-associated protein-O-carboxylmethyltransferase carboxymethylase about 900-fold from chicken erythrocytes by use of a combination of affinity chromatography on immobilized S-adenosyl-L-homocysteine and gel filtration on Sephacryl S-200 (Pharmacia), together with 3-((3-cholamidopropyl)-dimethylammonio)-1-propane-sulfonate as a detergent to solubilize the membrane-associated enzyme. The two enzymes were characterized by examining the dependence of their activity on pH and on concentration of S-adenosyl-L-methionine using fetuin as an exogenous methyl-acceptor substrate, and were found to differ somewhat. The cytosolic enzyme had a pH optimum of 6.0 with an apparent Km value of 2.1 microM for S-adenosyl-L-methionine, whereas corresponding values for the membrane-associated enzyme were 6.5 and 0.71 microM. This report deals with the biochemical differences between purified cytosolic and membrane-associated protein carboxymethylase from the same cell source.     

Mechanistic studies of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase from Escherichia coli.

The anomeric specificity and the steady-state kinetic mechanism of homogeneous 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase were investigated. The open-chain 4-deoxy analogue of arabinose-5-phosphate (Ara5P), which is structurally prohibited from undergoing ring closure, was synthesized and tested as a substrate for the synthase. It was found that the analogue functions as a substrate with a similar kcat value to that of the original substrate. The kcat/Km value for the natural substrate is seven-times greater than that of the 4-deoxy analogue. However, taking into account the 9.5% and approximately 1% concentrations of the aldehyde forms of the 4-deoxy analogue and Ara5P in solution, then the 'true' Km values must be in the range 31.5 microM and 0.26 microM, respectively, requiring about a 3 kcal/mol contribution to the binding energy by the 4-hydroxyl group of Ara5P. The data provides evidence that the enzyme acts upon the acyclic form of the natural substrate. The steady-state kinetic study of KDO8P synthase was analyzed via inhibition using the products KDO8P and inorganic phosphate, and D-ribose-5-phosphate as a dead-end inhibitor. First, intersecting lines in double-reciprocal plots of initial-velocity data at substrate concentrations in the micromolar range suggest a sequential mechanism for the enzyme-catalyzed reaction. The inhibition by D-ribose-5-phosphate is competitive for Ara5P and uncompetitive for phosphoenolpyruvate (P-pyruvate). These inhibition patterns are consistent with the model wherein P-pyruvate binding precedes that of Ara5P binding. Furthermore, this order of substrate binding was supported by the observations that KDO8P is a competitive inhibitor for P-pyruvate binding, supporting the concept that KDO8P and P-pyruvate bind to the same enzyme form, and noncompetitively with respect to Ara5P. In addition, the inhibition by inorganic phosphate is noncompetitive with respect to both P-pyruvate and Ara5P, suggesting an apparent ordered release of products such that Pi first, followed by KDO8P. In conclusion, these data suggest a steady-state kinetic mechanism for KDO8P synthase where P-pyruvate binding precedes that of Ara5P, followed by the ordered release of inorganic phosphate and KDO8P.     

Plasmodium falciparum: S-adenosyl-L-methionine decarboxylase

Putrescine-dependent S-adenosyl-L-methionine decarboxylase has been detected in the malaria parasite Plasmodium falciparum. Mg2+ did not affect the enzyme activity. The apparent Km value of the plasmodial enzyme for adenosyl-methionine was found to be 33 microM. Methylglyoxal bis(guanylhydrazone) competitively inhibited the enzyme activity with respect to adenosylmethionine. The inhibition constant for methylglyoxal bis(guanylhydrazone) was determined to be 0.46 microM. Spermidine was the main polyamine detected in the parasite. There was significant decrease in the S-adenosyl-L-methionine decarboxylase activity when the infected erythrocytes were incubated with chloroquine and mefloquine for 2 hr at 1 and 10 microM, respectively. Since at similar concentrations these drugs did not directly affect the plasmodial enzyme activity, the interaction of these drugs with the polyamine biosynthesis remains unclear.     

Restriction and modification in Bacillus subtilis: two DNA methyltransferases with BsuRI specificity. II. Catalytic properties, substrate specificity, and mode of action

The properties of two DNA methyltransferases, termed M. BsuRIa and M. BsuRIb, whose isolation was described in the preceding paper (Günthert, U., Freund, M., and Trautner, T. A. (1981) J. Biol. Chem. 256, 9340-9345) were compared. Both enzymes recognize the same target sequence in double-stranded DNA, leading to methylation of the internal cytosine: 5'GGCC. The enzymes have identical reaction constants with their substrates, DNA (km = 2.7 nM for the 5' GGCC sequence), and S-adenosyl-L-methionine (km = 0.7 microM). Initial rates of methyl group transfer were proportional to enzyme concentration over a range of 50-fold, indicating absence of aggregation. The enzymes are different in their ionic strength requirements using Tris-HCl, pH 8.4. M. BsuRIa is most active at 100 mM, M. BsuRIb at 440 mM. As measured by incorporation kinetics and heat inactivation, M. BsuRIa is the more stable enzyme of the two. Equilibrium dialysis was used to study the mode of methyl group transfer to the DNA with either enzyme. The data indicate that initially S-adenosyl-L-methionine binds to methyltransferase. This complex attaches to either modified or nonmodified DNA. The methyl group will then be transferred to a nonmodified target sequence, leading to the dissociation of enzyme and S-adenosyl-L-homocysteine from the DNA.     

Rat kidney histamine N-methyltransferase: purification and partial characterization

Histamine N-methyltransferase (HMT, EC 2.1.1.8) was purified 8,420-fold in 44% yield from rat kidney. The basic steps in the purification included differential centrifugation, calcium phosphate adsorption, DEAE cellulose chromatography, and affinity chromatography on an S-adenosylhomocysteine-agarose matrix. The resulting protein was homogeneous as determined by gel electrophoresis and was stable for at least five months at -80 degrees C. The apparent molecular weight of the enzyme was found to be 31,500 as determined by gel filtration through Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was determined to be 5.4. The Km's for histamine and S-adenosyl-L-methionine were 12.4 +/- 1.3 microM and 10.2 +/- 0.5 microM, respectively. When S-adenosyl-L-methionine was the variable substrate, the Ki's for S-adenosyl-L-homocysteine and S-adenosyl-D-homocysteine were 31.9 +/- 3.4 microM and 32.0 +/- 3.5 microM, respectively. When histamine was the variable substrate, the Ki for S-adenosyl-L-homocysteine was 11.8 +/- 0.6 microM. Comparison of physico-chemical and catalytic properties of the rat kidney and the guinea pig enzymes suggest that these proteins have similar structural and catalytic characteristics.     

Photoaffinity labeling of terminal deoxynucleotidyl transferase. 1. Active site directed interactions with 8-azido-2'-deoxyadenosine 5'-triphosphate

A photoaffinity analogue of dATP, 8-azido-2'-deoxyadenosine 5'-triphosphate (8-azido-dATP), was used to probe the nucleotide binding site of the non-template-directed DNA polymerase terminal deoxynucleotidyl transferase (EC 2.7.7.31). The Mg2+ form of 8-azido-dATP was shown to be an efficient enzyme substrate with a Km of 53 microM. Loss of enzyme activity occurred during UV photolysis only in the presence of 8-azido-dATP. At saturation (120 microM 8-azido-dATP), 54% of the protein molecules were modified as determined by inhibition of enzyme activity. Kinetic analysis of enzyme inhibition induced by photoincorporation of 8-azido-dATP indicated an apparent Kd of approximately 38 microM. Addition of 2 mM dATP to 120 microM 8-azido-dATP resulted in greater than 90% protection from photoinduced loss of enzyme activity. In contrast, no protection was observed with the addition of 2 mM dAMP. Enzyme inactivation was directly correlated with incorporation of radiolabeled 8-azido-dATP into the protein and UV-induced destruction of the azido group. Photoincorporation of 8-azido-dATP into terminal transferase was reduced by all purine and pyrimidine deoxynucleoside triphosphates of which dGTP was the most effective. The alpha and beta polypeptides of calf terminal transferase were specifically photolabeled by [gamma-32P]-8-azido-dATP, and both polypeptides were equally protected by all four deoxynucleoside triphosphates. This suggests that the nucleotide binding domain involves components from both polypeptides.     

Mechanism of inhibition of Ca2+-transport activity of sarcoplasmic reticulum Ca2+-ATPase by anisodamine

The mechanism of inhibition of Ca2+-transport activity of rabbit sarcoplasmic reticulum Ca 2+-ATPase (SERCA) by anisodamine (a drug isolated from a medicinal herb Hyoscyamuns niger L) was investigated by using ANS (1-anilino-8-naphthalenesulfonate) fluorescence probe, intrinsic fluorescence quenching and Ca 2+-transport activity assays. The number of ANS binding sites for apo Ca2+-ATPase was determined as 8, using a multiple-identical binding site model. Both anisodamine and Ca2+ at millimolar level enhanced the ANS binding fluorescence intensities. Only anisodamine increased the number of ANS molecules bound by SERCA from 8 to 14. The dissociation constants of ANS to the enzyme without any ligand, with 30 mM anisodamine and with 15 mM Ca 2 were found to be 53.0 microM, 85.0 microM and 50.1 microM, respectively. Both anisodamine and Ca2+ enhanced the ANS binding fluorescenc with apparent dissociation constants of 7.6 mM and 2.3 mM, respectively, at a constant concentration of the enzyme. Binding of anisodamine significantly decreased the binding capacity of Ca2+ with the dissociation constant of 9.5 mM, but binding of Ca2+ had no obvious effect on binding of anisodamine. Intrinsic fluorescence quenching and Ca2+-transport activity assays gave the dissociation constants of anisodamine to SERCA as 9.7 and 5.4 mM, respectively, which were consistent with those obtained from ANS-binding fluorescence changes during titration of SERCA with anisodamine and anisodamine + 15 mM Ca2+, respectively. The results suggest that anisodamine regulates Ca2+-transport activity of the enzyme, by stabilizing the trans-membrane domain in an expanded, inactive conformation, at least at its annular ring region.