Fluorescent and spin label probes of the environments of the sulfhydryl groups of porcine muscle adenylate kinase.

The environments of the two sulfhydryl groups of procine muscle adenylate kinase have been investigated by chemical modification reactions. The results indicate that the environments of the two-SH groups of procine muscle adenylate kinase are markedly different and that substrates induce conformational changes in the enzyme in the region of the sulfhydryl groups. The fluorogenic reagent 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-chloride) reacts specifically with the -SH groups of the enzyme at pH 7.9. One thiol group reacts with NBD-chloride approximately 40-fold faster than the other one, and the fast reacting group has been identified as Cys-25 in the amino acid sequence. The similarity of the rate of the more slowly reacting Cys-187 with NBD-chloride to that of glutathione with the same reagent is consistent with its location on the surface of the enzyme as determined by x-ray crystallography structure. The fast reacting Cys-25 in the interior of the structure can be approached by compounds such as NBD-chloride via a cleft. Reaction of Cys-25, presumably located close to the catalytic center, leads to complete inactivation of the enzyme. Substrates such as ATP, MgATP, and ADP which bind to the triphosphate subsite of the enzyme decrease the rate of reaction of Cys-25 by factors up to 3.5 but have only a small effect (approximately equal to 10%) on the reactivity of Cys-187. AMP, however, has a pronounced effect on the reactivity of Cys-187, the slowly reacting group. The multisubstrate analogue P-1, P-5-di-(adenosine-5)pentaphosphate (Ap-5A) decreases the rate of reaction of the fast reacting thiol group by a factor of 300. The behavior of Cys-25 toward NBD-chloride, i.e. super-reactivity in the absense of Ap-5A and slow reactivity in the presence of the multisubstrate inhibitor, was characteristic for both porcin and carp adenylate kinase. In the presence of Ap-5A adenylate kinase can be selectively modified at Cys-187; the introduction of the fluorescent NBD group at this position has no effect on enzymatic activity. A slow transfer of the NBD group occurs from the third groups to the epsilon-amino group of Lys-31. This transfer reaction is further evidence that the structure of adenylate kinase in dilute solution is similar to that of the crystalline enzyme since the x-ray data have shown that the sulfur of Cys-187 and the epsilon-nitrogen of Lys-31 are less than 4 A apart. The strongly fluorescent NBD-NH-enzyme possesses full activity and binds substrates as. cont'd     

Modification of human prostatic acid phosphatase by potassium ferrate

Prostatic acid phosphatase (orthophosphoric-monoester phosphohydrolase, acid optimum, EC 3.1.3.2) reacts with potassium ferrate, K₂FeO₄ a potent oxidizing agent and an analogue of orthophosphate. Treatment of the enzyme with 10^?6m ferrate at pH 7.5 0 C leads to the immediate loss of 95% of the activity. Molybdate, the competitive inhibitor of prostatic phosphatase, partially protects the enzyme from inactivation. Ferrate inactivation at pH 7.5 is accompanied by the modification of 2 histidine, 4 lysine and 4 methionine residues. Histidine is protected by molybdate, whereas methionine is not and lysine is partly protected. Partial inactivation with ferrate leads to the retardation of the modified enzyme on Sephadex G-200 column, which is eluted in the position of the active monomeric unit.     

A cysteine residue (cysteine-116) in the histidinol binding site of histidinol dehydrogenase

Salmonella typhimurium L-histidinol dehydrogenase (EC 1.1.1.23), a four-electron dehydrogenase, was inactivated by an active-site-directed modification reagent, 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl). The inactivation followed pseudo-first-order kinetics and was prevented by low concentrations of the substrate L-histidinol or by the competitive inhibitors histamine and imidazole. The observed rate saturation kinetics for inactivation suggest that NBD-Cl binds to the enzyme noncovalently before covalent inactivation occurs. The UV spectrum of the inactivated enzyme showed a peak at 420 nm, indicative of sulfhydryl modification. Stoichiometry experiments indicated that full inactivation was correlated with modification of 1.5 sulfhydryl groups per subunit of enzyme. By use of a substrate protection scheme, it was shown that 0.5 sulfhydryl per enzyme subunit was neither protected against NBD-Cl modification by L-histidinol nor essential for activity. Modification of the additional 1.0 sulfhydryl caused complete loss of enzyme activity and was prevented by L-histidinol. Pepsin digestion of NBD-modified enzyme was used to prepare labeled peptides under conditions that prevented migration of the NBD group. HPLC purification of the peptides was monitored at 420 nm, which is highly selective for NBD-labeled cysteine residues. By amino acid sequencing of the major peptides, it was shown that the reagent modified primarily Cys-116 and Cys-377 and that the presence of L-histidinol gave significant protection of Cys-116. The presence of a cysteine residue in the histidinol binding site is consistent with models in which formation and subsequent oxidation of a thiohemiacetal occurs as an intermediate step in the overall reaction.     

Catalytic and immunochemical properties of arylsulphatase A from urine, modified by potassium ferrate

Arylsulphatase A (EC 3.1.6.1.) from urine was inactivated with potassium ferrate, a strong oxidizing agent. The inhibition could be prevented by competitive inhibitors, tetraborate and orthophosphate. Tetraborate which was shown to be a powerful competitive inhibitor (determined Ki = 4 X 10(-5) M) gave more efficient protection. The partially inactivated enzyme exhibited a Km value similar to that of the unmodified arylsulphatase A, and its Vmax decreased in proportion to the loss of enzymatic activity. The partially modified enzyme did not lose its ability to catalyse hydrolysis of p-nitrocatechol sulphate according to the "anomalous kinetics" exhibited towards this substrate and characteristic for arylsulphatase A. The immunochemical properties of arylsulphatase A either fully or partially inactivated were similar to those of the native enzyme. The results allow to conclude that ferrate reacts with arylsulphatase A in its active site. Thus ferrate seems to be a very sensitive probe for amino acid residues essential for catalytic activity of arylsulphatase A.     

Ferrate oxidation of Escherichia coli DNA polymerase-I. Identification of a methionine residue that is essential for DNA binding

Treatment of Escherichia coli DNA polymerase-I with potassium ferrate (K2FeO4), a site-specific oxidizing agent for the phosphate group-binding sites of proteins, results in the irreversible inactivation of enzyme activity as judged by the loss of polymerization as well as 3'-5' exonuclease activity. A significant protection from ferrate-mediated inactivation is observed in the presence of DNA but not by substrate deoxynucleoside triphosphates. Furthermore, ferrate-treated enzyme also exhibits loss of template-primer binding activity, whereas its ability to bind substrate triphosphates is unaffected. In addition, comparative high pressure liquid chromatography tryptic peptide maps obtained before and after ferrate oxidation demonstrated that only five peptides of the more than 60 peptide peaks present in the tryptic digest underwent a major change in either peak position or intensity as a result of ferrate treatment. Amino acid analyses and/or sequencing identified four of these affected peaks as corresponding to peptides that span residues 324-340, 437-455, 456-464, and 512-518, respectively. However, only the last peptide, which has the sequence: Met-Trp-Pro-Asp-Leu-Gln-Lys, was significantly protected in the presence of DNA. This latter peptide was also the only peptide whose degree of oxidation correlated directly with the extent of inactivation of the enzyme. Amino acid analysis indicated that methionine 512 is the target site in this peptide for ferrate oxidation. Methionine 512, therefore, appears to be essential for the DNA-binding function of DNA polymerase-I from E. coli.     

Kinetics of irreversible inhibition of yeast alcohol dehydrogenase during modification by 4,4′-dithiodipyridine

The course of inactivation of yeast alcohol dehydrogenase (YADH) using 4,4′-dithiodipyridine (DSDP) has been studied in this paper. The results show that the reaction mechanism between DSDP and YADH is a competitive, complexing inhibition. The microscopic constants for the inactivation of the free enzyme and the enzyme-substrate complex were determined. The presence of the substrate NAD⁺ offers strong protection for this enzyme against inactivation by DSDP. The above results suggest that two Cys residues are essential for activity and are situated at the active site. These essential Cys residues should be Cys-46 and Cys-174 which are ligands to the catalytic zinc ion. Another Cys residue, which can be modified by DSDP, is non-essential for activity of the enzyme.     

Evidence for the existence of a sulfhydryl group in the adenylate cyclase active site

6-Cloro-9-beta-d-ribofuranosylpurine 5'-triphosphate (CIRTP) and 6-mercapto-9-beta-d-ribofuranosylpurine 5'-triphosphate (SRTP) irreversibly inhibit adenylate cyclase from rat brain. Adenosine 5'-[beta, gamma -imido] triphosphate protects the enzyme against inactivation by CIRTP and SRTP and acts as a competitive inhibitor with respect to ATP with the Ki value 2 X 10(-4) M. Study of the pH-dependence of the rate of the enzyme inactivation by CIRTP showed that pK for the group modified by this compound is equal to 7.45. Inactivation is first order with respect to the enzyme; the saturation effect is observed at the increased concentration of CIRTP. The k2 and KI values for irreversible inhibition of brain adenylate cyclase by CIRTP were 0.25 min-1 and 1.9 X 10(-4) M, respectively. Adenylate cyclase inhibition by SRTP is also time-dependent. Partial protection against the enzyme inactivation was observed. Dithiothreitol restores the activity of SRTP-inactivated adenylate cyclase. The results obtained indicate the presence of an -SH group in the purine amino group binding area of the enzyme active site.     

Crystal structure of red sea bream transglutaminase

The crystal structure of the tissue-type transglutaminase from red sea bream liver (fish-derived transglutaminase, FTG) has been determined at 2.5-A resolution using the molecular replacement method, based on the crystal structure of human blood coagulation factor XIII, which is a transglutaminase zymogen. The model contains 666 residues of a total of 695 residues, 382 water molecules, and 1 sulfate ion. FTG consists of four domains, and its overall and active site structures are similar to those of human factor XIII. However, significant structural differences are observed in both the acyl donor and acyl acceptor binding sites, which account for the difference in substrate preferences. The active site of the enzyme is inaccessible to the solvent, because the catalytic Cys-272 hydrogen-bonds to Tyr-515, which is thought to be displaced upon acyl donor binding to FTG. It is postulated that the binding of an inappropriate substrate to FTG would lead to inactivation of the enzyme because of the formation of a new disulfide bridge between Cys-272 and the adjacent Cys-333 immediately after the displacement of Tyr-515. Considering the mutational studies previously reported on the tissue-type transglutaminases, we propose that Cys-333 and Tyr-515 are important in strictly controlling the enzymatic activity of FTG.     

The levels of creatine kinase and adenylate kinase in the plasma of dystrophic chickens reflect the rates of loss of these enzymes from the circulation

The rates of loss of adenylate kinase and creatine kinase from the circulation after intravenous injection of homogenous chicken skeletal muscle enzymes were examined to determine the role of plasma clearance rates in determining the plasma levels of these enzymes in normal and dystrophic chickens. The rapid clearance of adenylate kinase activity (average half-life of 5 min) and the slower biphasic clearance of creatine kinase activity (average half-lives of 0.95 and 11 hr) are consistent with the elevation of creatine kinase but not adenylate kinase in the blood plasma of dystrophic chickens compared to normal chickens. The rates of clearance of these enzymes were similar in normal chickens compared to dystrophic chickens. Radioiodinated enzymes were cleared at similar, but slightly more rapid rates than the loss of enzyme activity. The loss of adenylate kinase activity from the circulation may be due in part to inactivation since adenylate kinase activity is rapidly inactivated in serum in vitro, and because no increase in adenylate kinase activity is observed in the most specific sites of clearance of the radioiodinated enzyme, the liver and spleen. The comparison of enzyme activities in press juices to the activities in high-ionic-strength homogenates of muscle tissue from normal and dystrophic muscle, indicates that adenylate kinase activity is not associated with intracellular structures to the extent that would prohibit release from dystrophic muscle tissue. These results, and those presented previously with regard to plasma levels and clearance rates of AMP aminohydrolase and pyruvate kinase in normal and dystrophic chickens (11) support our hypothesis that the rates of loss of muscle enzyme activities from the circulation are important in determining the circulating levels of muscle enzymes in dystrophic chickens. Furthermore, from the measurement of plasma levels and clearance rates of creatine kinase, it was estimated that the efflux rate of creatine kinase from dystrophic muscle tissue is 2.0% of the total breast muscle creatine kinase per day.     

Inactivation and proteolysis of heat-sensitive adenylate kinase of Escherichia coli CR341 T28

Adenylate kinase from Escherichia coli K12 (strains CR341 and CR341 T28, a temperature-sensitive mutant) was purified by a two-step chromatographic procedure. Denaturation by heat above 60 degrees C of pure or crude preparations of adenylate kinase from both strains of bacteria was shown to be "reversible" if the enzyme was converted to the random coiled state by guanidinium chloride after heat treatment. Like other small monomeric proteins, adenylate kinase refolded rapidly to the native active state by dilution of guanidinium chloride. Adenylate kinase from the mutant strain was irreversibly inactivated by exposure of crude extracts at 40 degrees C. This inactivation is due to proteolysis which follows thermal denaturation (or transconformation) of mutant adenylate kinase at 40 degrees C. ATP, P1, P5-di(adenosine 5')-pentaphosphate, and anti-adenylate kinase antibodies protected the thermosensitive adenylate kinase in crude extracts against denaturation and proteolysis at 40 degrees C.     

Recombinant rat liver guanidinoacetate methyltransferase: reactivity and function of sulfhydryl groups

Rat liver guanidinoacetate methyltransferase, produced in Escherichia coli by recombinant DNA technique, possesses five cysteine residues per molecule. No disulfide bond is present. Analysis of the chymotryptic peptides derived from the iodo[14C]acetate-modified enzyme shows that Cys-90, Cys-15, Cys-219, and Cys-207 are alkylated by the reagent in order of decreasing reactivity. Incubation of the enzyme with excess 5,5'-dithiobis(2-nitrobenzoate) (DTNB) in the absence and presence of cystamine [2,2'-dithiobis(ethylamine)] causes the appearance of 4 and 5 mol of 2-nitro-5-mercaptobenzoate/mol of enzyme, respectively. Reaction of the methyltransferase with an equimolar amount of DTNB results in an almost quantitative disulfide cross-linking of Cys-15 and Cys-90 with loss of a large portion of the activity. The methyltransferase is completely inactivated by iodoacetate following nonlinear kinetics. Comparison of the extent of inactivation with that of modification of cysteine residues and the experiment with the enzyme whose Cys-15 and Cys-90 are cross-linked suggest that alkylation of Cys-15 and Cys-90 results in a partially active enzyme and that carboxymethylation of Cys-219 completely eliminates enzyme activity. The inactivation of guanidinoacetate methyltransferase by iodoacetate or DTNB is not protected by substrates. Furthermore, disulfide cross-linking of Cys-15 and Cys-90 or carboxymethylation of Cys-219 does not impair the enzyme's capacity to bind S-adenosylmethionine. Thus, these cysteine residues appear to occur outside the active-site region, but their integrity is crucial for the expression of enzyme activity.     

Circular dichroism investigation of Escherichia coli adenylate kinase

We examined by circular dichroism (CD) spectroscopy in far- and near-ultraviolet three different molecular forms of Escherichia coli adenylate kinase: the wild type protein, the enzyme carboxymethylated at a single cysteine residue (Cys-77), and the thermosensitive adenylate kinase. The thermosensitive enzyme differs from the wild type protein in that a serine is substituted for a proline residue at position 87 (Gilles, A.-M., Saint Girons, I., Monnot, M., Fermandjian, S., Michelson, S., and Barzu, O. (1986) Proc. Natl. Acad. Sci. U. S. A., 83, 5798-5802). We also examined the CD spectra of isolated peptides resulting from chemical cleavage of adenylate kinase at Cys-77 (C1, residues 1-76; C2, residues 77-214). The secondary structure composition of wild type bacterial adenylate kinase (50% alpha-helix and 15% beta-sheet) was close to that derived from x-ray analysis of pig muscle enzyme (Schulz, G.E., Elzinga, M., Marx, F., and Schirmer, R. H. (1974) Nature 250, 120-123). Carboxymethylation of wild type protein did not greatly affect the CD spectrum. The secondary structure of the thermosensitive adenylate kinase was observed to be significantly different from that of the wild type enzyme (reduction in alpha-helix content to 39%). Changes in ellipticities at 222 nm as a function of temperature indicated that the melting temperature for thermosensitive adenylate kinase was 38 degrees C and that for the wild type enzyme was 54 degrees C. Isolated C1 and C2 peptides had a large proportion of unordered structures. When mixed, C1 and C2 fragments reassociated into structures resembling native, uncleaved adenylate kinase. The recovery of ordered structures, indicated by CD spectroscopy, paralleled the recovery of catalytic activity.     

Tyrosine residues near the FAD binding site are critical for FAD binding and for the maintenance of the stable and active conformation of rat monoamine oxidase A.

Monoamine oxidase is a flavin-containing enzyme located at the mitochondrial outer membrane that catalyzes the oxidative deamination of amines. To investigate the role of tyrosine residues near the FAD-binding site, Cys-406, of monoamine oxidase A, the tyrosine residues at posiyions 402, 407, and 410 were indurdually replaced with alanine or phenylalanine and the effects of the mutations on catalytic activity, FAD binding, and enzyme structure were examined. Half or fewer of the mutant proteins incorporated FAD. The mutation of Tyr-407 to alanine led to an almost completely loss of catalytic activity for serotonin, PEA, tyramine, and tryptamine. A substantial decrease in the catalytic activity was also observed with the enzymes mutated at Tyr-402 and Tyr-410 to alanine, although the effect of the latter mutation was much less. All these mutants were sensitive to trypsin treatment of the purified enzyme, while the wild type enzyme was resistant to treatment. On the other hand, substitution of Tyr-402 or Tyr-407 with phenylalanine had little effect on these properties. Taken together, we conclude that tyrosine residues near Cys-406 may be form a pocket to facilitates FAD incorporation, the catalytic center, and a stable conformation, probably through interactions among the aromatic rings of the tyrosine residues and FAD.     

Amino acid composition and NH2-terminal amino acid sequence of human phospholipase A2 purified from rheumatoid synovial fluid

The amino acid composition and partial NH2-terminal amino acid sequence of an extracellular phospholipase A2 in human rheumatoid synovial fluid were determined. The predominant amino acids in the phospholipase A2 were cysteine, glycine, arginine, and lysine, suggesting that it is a basic one. The NH2-terminal 34 amino acids were found to be as follows: Asn-Leu-Val-Asn-Phe-His-Arg-Met-Ile-Lys-Leu-Thr-Thr-Gly-Lys-Glu-Ala-Ala-Leu- Ser-Tyr-Gly-Phe-Tyr-Gly-Cys-X-Cys-Gly-Val-Gly-Gly-Arg-Gly The enzyme contains Phe-5, Met-8, Ile-9, Tyr-24, Gly-25, Cys-26, Cys-28, Gly-29, Gly-31, Gly-32, and Gly-34 residues, all of which are conserved in most of the sequenced phospholipase A2. The remarkable feature of this enzyme was the absence of Cys-11, which is conserved in the "Group I" enzyme family. This is the first report concerning partial amino acid sequences of human non-pancreatic phospholipase A2.     

Function and reactivity of sulfhydryl groups of rat liver glycine methyltransferase

Rat liver glycine methyltransferase is completely inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Treatment of the inactivated enzyme with KCN results in a reactivated enzyme having values of Vmax and S0.5 for S-adenosyl-L-methionine comparable to those of the native enzyme and about a 4-fold greater Km value for glycine. Kinetics of inactivation and reactivation show that one cysteine residue is involved in this process. Reaction of the methyltransferase with iodoacetate leads to partial inactivation of the enzyme; about 22% of the initial activity is retained in the modified enzyme. The relationship between the loss of enzyme activity and the number of iodoacetate molecules incorporated and the sequence analysis of peptides containing the modified residues indicate that carboxymethylation of Cys-282 is responsible for loss of activity. The observations that the activity of the cyanylated glycine methyltransferase shows no decrease upon incubation with iodoacetate and, conversely, the residual activity associated with the iodoacetate-modified enzyme is not abolished by DTNB suggest that Cys-282 is also involved in the inactivation by DTNB. Besides this residue, Cys-185, Cys-246, and Cys-262 are modified upon prolonged incubation with iodoacetate. 5'-[p-(Fluorosulfonyl)benzoyl]adenosine (FSBA) inactivates glycine methyltransferase by forming 1 disulfide/subunit [Fujioka, M., & Ishiguro, Y. (1986) J. Biol. Chem. 261, 6346-6351]. Despite this stoichiometry, treatment of the FSBA-inactivated enzyme with unlabeled iodoacetate and then with iodo[14C]acetate after reduction with 2-mercaptoethanol and subsequent peptide analysis show that the incorporated radioactivity is distributed equally among Cys-185, Cys-246, Cys-262, and Cys-282.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlations between kinetic and X-ray analyses of engineered enzymes: crystal structures of mutants Cys----Gly-35 and Tyr----Phe-34 of tyrosyl-tRNA synthetase

The crystal structures of two mutant tyrosyl-tRNA synthetases (TyrTS) are reported to test predictions from kinetic data about structural perturbations and also to aid in the interpretation of apparent strengths of hydrogen bonds measured by protein engineering. The enzyme-tyrosine and enzyme-tyrosyl adenylate complexes of the mutant, TyrTS(Cys----Gly-35), have been determined at 2.5- and 2.7-A resolution, respectively. Residue Cys-35 is in the ribose binding site. Small rearrangements in structure are seen in the enzyme-tyrosine complex that are localized around the cavity created by the mutation. The side chain of Thr-51 moves to occupy the cavity, and Ile-52 adopts two significantly populated conformations, one as in the native enzyme and a second unique to the mutant. On binding tyrosyl adenylate, Ile-52 in the mutant crystal structure preferentially occupies the conformation observed in the native structure. The side chain at Thr-51 becomes disordered. The double-mutant test, which was designed to detect interactions between residues, had previously shown a discrepancy of some 0.4 kcal/mol on mutating Cys-35 and Thr-51 separately and together. A crystal structure of a second mutant, delta TyrTS(Tyr----Phe-34), complexed with tyrosine has been determined at 2.7-A resolution. Tyr-34 in wild-type enzyme makes a hydrogen bond with the phenolic oxygen of the bound tyrosine substrate. The mutant crystal structure was solved to discover whether or not a water molecule binds to the substrate instead of the hydroxyl of Tyr-34 as the interpretation of apparent binding energies from site-directed mutagenesis experiments hinges crucially on whether there is access of water to the mutated region.     

Role of strategic cysteine residues in oxidative damage to the yeast plasma membrane H<Superscript>+</Superscript>-ATPase caused by Fe- and Cu-containing fenton reagents

Damage caused to Saccharomyces cerevisiae SY4 plasma membrane H(+)-ATPase by Fe- and Cu-Fenton reagents was determined in secretory vesicles containing enzyme in which Cys residues were replaced singly or in pairs by Ala. Cys-221 situated in a beta-sheet domain between M2 and M3 segments, phosphorylation domain-located Cys-409 and Cys-532 situated at the ATP-binding site play a role in the inactivation. In the presence of all three residues the enzyme exhibited a certain basic inactivation, which did not change when Cys-532 was replaced with Ala. In mutants having intact Cys-532 but lacking one or both other cysteines, replacement of Cys-221 with Ala led to lower inactivation, suggesting that Cys-221 may serve as a target for metal-catalyzed oxidation and intact Cys-532 promotes this target role of Cys-221. In contrast, the absence of Cys-409 caused higher inactivation by Fe-Fenton. Cys-532 thus seems to serve as a target for Fe-Fenton, intact Cys-409 causing a conformational change that makes Cys-532 less accessible to oxidation. The mutant lacking both Cys-221 and Cys-409 is more sensitive to Fe-Fenton than to Cu-Fenton and the absence of both Cys residues thus seems to expose presumable extra Fe-binding sites. These data and those on protection by ATP, ADP, 1,4-dithiothreitol and deferrioxamine B point to complex interactions between individual parts of the enzyme molecule that determine its sensitivity towards Fenton reagents. ATPase fragmentation caused by the two reagents differed in that the Fe-Fenton reagent produced in Western blot "smears" whereas the Cu-Fenton reagent produced defined fragments.     

An affinity label for the regulatory dithiol of ribulose-5-phosphate kinase from maize (Zea mays).

Ribulose-5-phosphate kinase from maize (Zea mays) can exist in either a reduced, active form or an oxidized, inactive form. Reduced ribulose-5-phosphate kinase is rapidly and irreversibly inactivated by the dichlorotriazine dye Reactive Red 1 (Procion Red MX-2B), but the irreversible inactivation of the oxidized form of ribulose-5-phosphate kinase occurs at only 0.05% of this rate. The rate of inactivation of the reduced enzyme by Reactive Red 1 (apparent bimolecular rate constant 10(4)M-1 X s-1 at pH 7.4 and 25 degrees C) is several orders of magnitude greater than previous estimates of the rates of dye-mediated inactivation of other enzymes. The dye-dependent inactivation of the reduced enzyme is inhibited by Hg2+ or p-mercuribenzoate (thiol reagents that reversibly inhibit ribulose-5-phosphate kinase activity), or by ATP and ADP, the nucleotide substrates of the enzyme. Hydrolysed Reactive Red 1, which does not inactivate the enzyme, is a reversible inhibitor of ribulose-5-phosphate kinase. This inhibition is competitive with respect to ATP (Ki approximately 0.5 mM). The dye appears to act as an affinity label for the ATP/ADP-binding site by preferentially arylating a thiol residue generated during the reductive activation of the enzyme that is achieved by dithiothreitol or thioredoxin in vitro or during illumination of leaves.     

Rat Brain Synaptosomal ATP:AMP-Phosphotransferase Activity

Adenylate kinase activity (ATP:AMP-phosphotransferase; EC 2.7.4.3) was studied in various subcellular fractions of rat brain tissues. Because of the presence of other adenosine nucleotide-utilizing enzymes, adenylate kinase activity was assayed in both the forward and reverse directions by using coupled enzyme systems and by using a specific adenylate kinase inhibitor, P1,P5-di(adenosine-5') pentaphosphate. As expected, the highest specific adenylate kinase activity (2.89 mumol/min/mg of protein) was detected in the cytosolic brain fraction. However, substantial enzyme activity (0.68 mumol/min/mg) was also found in the intact synaptosomal fraction isolated on Percoll/sucrose gradients. The increased specific enzyme activity of purified synaptosomes and the differences found between the kinetic parameters of the membrane-bound and cytosolic enzyme forms suggest that the synaptosomal adenylate kinase activity cannot be attributed to the small amount of contaminating cytosol present in our preparations. The adenylate kinase enzyme adhered to purified synaptic plasma membranes and was not released by washings with isoosmotic sucrose medium. The facts that the adenylate kinase enzyme activity could be measured in intact synaptosomal preparations and that both its substrates and its inhibitors do not cross intact plasma membranes support the possibility that the synaptosomal adenylate kinase is an ecto-enzyme.