Isolation of bacterial luciferases by affinity chromatography on 2,2-diphenylpropylamine-Sepharose: phosphate-mediated binding to an immobilized substrate analogue

A covalently immobilized form of an inhibitor of bacterial luciferase, 2,2-diphenylpropylamine (D phi PA), was an effective affinity resin for purifying this enzyme from several distinct bacterial species. The inhibitor is competitive with the luciferase aldehyde substrate but enhances binding of the flavin substrate FMNH2 (reduced riboflavin 5'-phosphate); comparable binding interactions occur with luciferase, the immobilized inhibitor D phi PA-Sepharose, and the substrates [Holzman, T. F., & Baldwin, T. O. (1981) Biochemistry 20, 5524-5528]. The effect of FMNH2 on the binding of luciferase to D phi PA-Sepharose was mimicked by inorganic phosphate; the luciferase-phosphate complex had a greater affinity for D phi PA-Sepharose than did luciferase. This observation led to the development of a method using D phi PA-Sepharose to purify bacterial luciferase. When crude enzyme in a high-phosphate buffer was applied to a column of the affinity matrix, the luciferase activity was removed from solution. After the column was washed with the same buffer to remove unbound protein, the luciferase was eluted with a non-phosphate cationic buffer. The affinity column has proven useful for rapid purification of luciferase in much greater yield than has been previously possible with standard ion-exchange techniques. This approach has allowed one-step purification of luciferases from ammonium sulfate precipitates of Vibrio harveyi, Vibrio fischeri, and Photobacterium phosphoreum. The dissociation constants in 0.10 M phosphate for the affinity ligand: luciferase complexes were 0.49 micro M, 0.28 micro M, and 0.15 micro M, respectively, for the three species. The dissociation constant for the V. harveyi mutant AK-6, which has normal aldehyde binding but greatly reduced affinity for FMNH2, was 0.30 micro M, while that for the V. harveyi mutant AK-20, which has greatly reduced affinity for aldehyde but a slightly increased affinity for FMNH2, was 1.2 microM. Preliminary experiments indicated that the yellow fluorescence protein (YFP) that participates, through energy transfer, in bioluminescent emission in V. fischeri strain Y-1 could be separated from the luciferase in this strain by chromatography on the affinity matrix, whereas other methods of separating luciferase and YFP have had limited success because of the binding of YFP to luciferase.     

Bacterial luciferase: demonstration of a catalytically competent altered conformational state following a single turnover

Ziegler-Nicoli et al. [Ziegler-Nicoli, M., Meighen, E. A., & Hastings, J. W. (1974) J. Biol. Chem. 249, 2385-2392] reported that a highly reactive cysteinyl residue on the alpha subunit of bacterial luciferase resides in or near the flavin binding site such that the enzyme-flavin complex is protected from inactivation by alkylating reagents. These authors also observed that injection of reduced flavin mononucleotide (FMNH2) into an air-equilibrated solution of enzyme protected the enzyme from alkylation for much longer than the lifetime of the 4a-peroxydihydroflavin intermediate resulting from reaction of enzyme-bound FMNH2 with O2. Two related explanations were offered: either the product flavin mononucleotide dissociated from the enzyme much more slowly following a catalytic cycle than would be predicted from the Kd measured by equilibrium binding or the enzyme itself, without bound flavin, was in an altered conformational state in which the thiol was less reactive following a catalytic cycle. Either explanation involves a slow return of the enzyme to its initial state following a catalytic cycle. We have investigated this phenomenon in more detail and found that rapid removal of the flavin from the enzyme by chromatography following catalytic turnover did not return the enzyme to its original state of susceptibility to either alkylating reagents or proteolytic enzymes. The flavin-free enzyme returned to the susceptible conformation with a half-time of ca. 25 min at 0 degree C. Inactivation of the enzyme at intermediate times of relaxation by either a proteolytic enzyme or an alkylating reagent showed biphasic kinetics, indicative of a mixture of the protected and susceptible forms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalytic importance of the substrate binding order for the FMNH2-dependent alkanesulfonate monooxygenase enzyme

The two-component alkanesulfonate monooxygenase system from Escherichia coli includes an FMN reductase (SsuE) and an FMNH2-dependent alkanesulfonate monooxygenase (SsuD) involved in the acquisition of sulfur from alkanesulfonates during sulfur starvation. The SsuD enzyme directly catalyzes the oxidation of alkanesulfonate to aldehyde and sulfite in the presence of O2 and FMNH2. The goal of these studies was to investigate the kinetic mechanism of SsuD through rapid reaction kinetics and substrate binding studies. The SsuD enzyme shows a clear preference for FMNH2 (Kd, 0.32 +/- 0.15 microM) compared to FMN (Kd, 10.2 +/- 0.4 microM) with a 1:1 binding stoichiometry for each form of the flavin. The kinetic trace of premixed SsuD and FMNH2 mixed with oxygenated buffer was best fit to a double exponential with no observed formation of the C4a-(hydro)peroxyflavin. However, when FMNH2 was mixed with SsuD and oxygenated buffer an initial fast phase (kobs, 12.9 s-1) was observed, suggesting that the mixing order is critical for the accumulation of the C4a-(hydro)peroxyflavin. Results from fluorimetric titrations with octanesulfonate imply that reduced flavin must bind first to promote octanesulfonate binding. When octanesulfonate was included in the kinetic studies the C4a-(hydro)peroxyflavin was observed at 370 nm when FMNH2 was not premixed with SsuD, which correlated with an increase in octanal product. There was a clear hyperbolic dependence on octanesulfonate binding, indicating that octanesulfonate binds in rapid equilibrium, and further results indicated there was a second isomerization step following binding. These results suggest that an ordered substrate binding mechanism is important in the desulfonation reaction by SsuD with reduced flavin binding first followed by either O2 or octanesulfonate.     

The devapamil-binding site of the purified skeletal muscle receptor for organic-calcium channel blockers is modulated by micromolar and millimolar concentrations of Ca2+.

The interaction of 2,7-dimethyl-3-(3,4-dimethoxyphenyl)-3-cyan-7-aza-9-(3- methoxyphenyl) nonahydrochloride (devapamil), a stereospecific analog of (3-[2-(3,4-dimethoxyphenyl)ethyl]- methylaminopropyl-3,4-dimethoxy-(1-methylethyl)benzeneacetonitr ile (verapamil), with the purified skeletal muscle receptor for calcium channel blockers (CaCB) was studied at 4 degrees C and 30 degrees C in the absence and presence of calcium. The purified CaCB receptor bound 0.9 mol devapamil/mol calcium-channel alpha 1 subunit, with an apparent Kd of 13 +/- 2.6 nM at 4 degrees C in the presence of 0.4 microM Ca2+. The affinity, and not the density, of the devapamil-binding site was decreased by lowering the pH from 8.5-6.5, or by increasing the Ca2+ concentration from 0.4 microM to 100 mM. The same results were obtained at 30 degrees C, although the ligand-receptor complex was not stable at Ca2+ concentrations below 10 microM. These binding data were confirmed by kinetic experiments. The rate constants calculated for a pseudo-first-order and a second-order reactions were identical and yielded fourfold lower k-1/k+1 (KD) values than the equilibrium experiments performed using 1 nM and 0.4 microM Ca2+, but the same values using 1 mM Ca2+. 1 mM Ca2+ increased the k-1/k+1 (KD) by decreasing 10-fold the association rate at 4 degrees C. The dissociation rate was increased about 10-fold by 5 microM devapamil or 100 microM D-cis-diltiazem, suggesting that the high affinity site is negatively regulated allosterically by millimolar Ca2+ concentrations and by the occupation of a second low-affinity site. Incubation of the CaCB receptors in the absence of Ca2+ and devapamil at 30 degrees C, but not at 4 degrees C, resulted in an apparent loss of devapamil-binding sites. The decrease in binding sites was caused by a reduced affinity. This apparent loss of binding sites was prevented by the addition of Ca2+ with an apparent median effective concentration of 0.4 microM. The apparent half-maximal inactivation times of the devapamil-binding site were 90 s and 12 min in the presence of 1 nM and 0.4 microM Ca2+, respectively. These results show that micromolar Ca2+ concentrations stabilize the CaCB receptor in a conformation which allows high-affinity binding of phenylalkylamines. Millimolar Ca2+ concentrations induce a low-affinity state of the devapamil-binding site on a stable CaCB receptor.     

Transient States of Adenylate Cyclase in Brain Membranes

Basal activity of adenylate cyclase from the amygdala of sheep brain and the neostriatum of turkey brain decays in two phases at 37 degrees C. The first phase is rapid (t1/2 = 2.3 +/- 0.3 min) and results in the loss of 60-70% of basal activity. The second phase is slow (t1/2 approximately 100 min) during which time the catalytic units denature irreversibly. The GTP analogue guanosine-5' (beta-gamma imino) triphosphate (p[NH]ppG) prevents the rapid decay by stabilizing the enzyme at its initial level of activity and also reactivates the enzyme to initial levels during or immediately following the early phase, indicating that denaturation of neither the guanylnucleotide units nor the catalytic units causes the rapid decline in basal activity. Activation by p[NH]ppG is rapid at 37 degrees C, but the binding of p[NH]ppG to the guanylnucleotide subunit also occurs at nonactivatory temperatures. This is determined by the protection of catalytic units from thermal or N-ethylmaleimide inactivation after extensive washing. Thus, at 25 degrees C all of the catalytic units can be stabilized by saturating p[NH]ppG concentrations. At 0 degree C, 35% of the catalytic units can be stabilized by saturating p[NH]ppG concentrations within 30 s. The half-saturation constant for the binding of p[NH]ppG at 0 degree C is identical to that derived in an assay at 37 degrees C, or after an incubation of the membranes for 10 min at 45 degrees C, when the process of thermal denaturation is 80% complete (K1/2 approximately 3 +/- 2 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the flavoprotein moieties of NADPH-sulfite reductase from Salmonella typhimurium and Escherichia coli. Physicochemical and catalytic properties, amino acid sequence deduced from DNA sequence of cysJ, and comparison with NADPH-cytochrome P-450 reductase

NADPH-sulfite reductase flavoprotein (SiR-FP) was purified from a Salmonella typhimurium cysG strain that does not synthesize the hemoprotein component of the sulfite reductase holoenzyme. cysJ, which codes for SiR-FP, was cloned from S. typhimurium LT7 and Escherichia coli B, and both genes were sequenced. Physicochemical analyses and deduced amino acid sequences indicate that SiR-FP is an octamer of identical 66-kDa peptides and contains 4 FAD and 4 FMN per octamer. Potentiometric titrations of SiR holoenzyme, SiR-FP, and FMN-depleted SiR-FP yielded the following redox potentials for the prosthetic groups at pH 7.7: E'1 (FMNH./FMN) = -152 mV; E'2 (FMNH2/FMNH.) = -327 mV; E'3 (FADH./FAD) = -382 mV; E'4 (FADH2/FADH.) = -322 mV. Microcoulometric titration of SiR-FP at 25 degrees C yielded data which were in full agreement with these potentials. Spectroscopic and catalytic studies of native SiR-FP and of SiR-FP depleted of FMN support the following electron flow sequence: NADPH----FAD----FMN. FMN can then contribute electrons to the hemoprotein component of sulfite reductase, as well as to cytochrome c and various diaphorase acceptors. The FMN is postulated to cycle between the FMNH2 and FMNH. oxidation states during catalysis; in this sense SiR-FP shares a catalytic mechanism with NADPH-cytochrome P-450 oxidoreductase. SiR-FP domains involved in binding FMN, FAD, and NADPH are proposed from amino acid sequence homologies with Desulfovibrio vulgaris flavodoxin (Dubourdieu, M., and Fox, J.L. (1977) J. Biol. Chem. 252, 1453-1463) and spinach ferredoxin-NADP+ oxidoreductase (Karplus, P.A., Walsh, K.A., and Herriott, J. R. (1984) Biochemistry 23, 6576-6583). Comparison of the deduced amino acid sequences of SiR-FP and NADPH-cytochrome P-450 oxidoreductase (Porter, T. D., and Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U. S.A. 82, 973-977) also showed identities that suggest these two proteins are descended from a common precursor, which contained binding regions for both FMN and FAD.     

Activity coupling and complex formation between bacterial luciferase and flavin reductases.

Luminous bacteria contain several species of flavin reductases, which catalyze the reduction of FMN using NADH and/or NADPH as a reductant. The reduced FMN (i.e. FMNH(2)) so generated is utilized along with a long-chain aliphatic aldehyde and molecular oxygen by luciferase as substrates for the bioluminescence reaction. In this report, the general properties of luciferases and reductases from luminous bacteria are briefly summarized. Earlier and more recent studies demonstrating the direct transfer of FMNH(2) from reductases to luciferase are surveyed. Using reductases and luciferases from Vibrio harveyi and Vibrio fischeri, two mechanisms were uncovered for the direct transfer of reduced flavin cofactor and reduced flavin product of reductase to luciferase. A complex of an NADPH-specific reductase (FRP(Vh)) and luciferase from V. harveyi has been detected in vitro and in vivo. Both constituent enzymes in such a complex are catalytically active. The reduction of FRP(Vh)-bound FMN cofactor by NADPH is reversible, allowing the cellular contents of NADP(+) and NADPH as a factor for the regulation of the production of FMNH(2) by FRP(Vh) for luciferase bioluminescence. Other regulations of the activity coupling between reductase and luciferase are also discussed.     

Cloning, expression, purification and characterization of fructose-1,6-bisphosphate aldolase from Anoxybacillus gonensis G2

The fructose-1,6-bisphosphate aldolase gene from the thermophilic bacterium, Anoxybacillus gonensis G2, was cloned and sequenced. Nucleotide sequence analysis revealed an open reading frame coding for a 30.9 kDa protein of 286 amino acids. The amino acid sequence shared approximately 80-90% similarity to the Bacillus sp. class II aldolases. The motifs that are responsible for the binding of a divalent metal ion and catalytic activity completely conserved. The gene encoding aldolase was overexpressed under T7 promoter control in Escherichia coli and the recombinant protein purified by nickel affinity chromatography. Kinetic characterization of the enzyme was performed at 60 degrees C, and K(m) and V(max) were found to be 576 microM and 2.4 microM min(-1) mg protein(-1), respectively. Enzyme exhibits maximal activity at pH 8.5. The activity of enzyme was completely inhibited by EDTA.     

Structural and functional relationships of enzyme activities induced by nitrate in barley

1. Nitrate induces the development of NADH-nitrate reductase (EC 1.6.6.1), FMNH(2)-nitrate reductase and NADH-cytochrome c reductase activities in barley shoots. 2. Sucrose-density-gradient analysis shows one band of NADH-nitrate reductase (8S), one band of FMNH(2)-nitrate reductase activity (8S) and three bands of NADH-cytochrome c reductase activity (bottom layer, 8S and 3.7S). Both 8S and 3.7S NADH-cytochrome c reductase activities are inducible by nitrate, but the induction of the 8S band is much more marked. 3. The 8S NADH-cytochrome c reductase band co-sediments with both NADH-nitrate reductase activity and FMNH(2)-nitrate reductase activity. Nitrite reductase activity (4.6S) did not coincide with the activity of either the 8S or the 3.7S NADH-cytochrome c reductase. 4. FMNH(2)-nitrate reductase activity is more stable (t((1/2)) 12.5min) than either NADH-nitrate reductase activity (t((1/2)) 0.5min) or total NADH-cytochrome c reductase activity (t((1/2)) 1.5min) at 45 degrees C. 5. NADH-cytochrome c reductase and NADH-nitrate reductase activities are more sensitive to p-chloromercuribenzoate than is FMNH(2)-nitrate reductase activity. 6. Tungstate prevents the formation of NADH-nitrate reductase and FMNH(2)-nitrate reductase activities, but it causes superinduction of NADH-cytochrome c reductase activity. Molybdate overcomes the effects of tungstate. 7. The same three bands (bottom layer, 8S and 3.7S) of NADH-cytochrome c reductase activity are observed irrespective of whether induction is carried out in the presence or absence of tungstate, but only the activities in the 8S and 3.7S bands are increased. 8. The results support the idea that NADH-nitrate reductase, FMNH(2)-nitrate reductase and NADH-cytochrome c reductase are activities of the same enzyme complex, and that in the presence of tungstate the 8S enzyme complex is formed but is functional only with respect to NADH-cytochrome c reductase activity.     

Complex formation between Vibrio harveyi luciferase and monomeric NADPH:FMN oxidoreductase

A direct transfer of the reduced flavin mononucleotide (FMNH(2)) cofactor of Vibrio harveyi NADPH:FMN oxidoreductase (FRP) to luciferase for the coupled bioluminescence reaction has been indicated by recent kinetic studies [Lei, B., and Tu, S.-C. (1998) Biochemistry 37, 14623-14629; Jeffers, C., and Tu, S.-C. (2001) Biochemistry 40, 1749-1754]. For such a mechanism, a complex formation of luciferase with FRP is essential, but until now, no evidence for such a complex has been reported. In this work, FRP was labeled at 1:1 molar ratio with the fluorophore eosin. The labeled enzyme was about 30% active in either the reductase single-enzyme or the luciferase-coupled assay. The labeled FRP in either the holo- or apoenzyme form was similar to the native FRP in undergoing a monomer-dimer equilibrium. By measuring the steady-state fluorescence anisotropy of eosin-labeled FRP, it was shown that luciferase formed a complex at 1:1 molar ratio with the monomer of either the apoenzyme or the holoenzyme form of FRP with K(d) values of 7 and 11 microM, respectively. Neither the holo- nor the apoenzyme of the labeled FRP in the dimeric form was effective in complexing with luciferase. At maximal in vivo bioluminescence, the V. harveyi cellular contents of luciferase and FRP were estimated to be 172 and 3 microM, respectively. The vast majority of FRP would be trapped in the luciferase/FRP complex. Plausible physiological significance of such a finding is discussed.     

N-acetylcolchinol O-methyl ether and thiocolchicine, potent analogs of colchicine modified in the C ring. Evaluation of the mechanistic basis for their enhanced biological properties

Two colchicine analogs with modifications only in the C ring are better inhibitors than colchicine of cell growth and tubulin polymerization. Radiolabeled thiocolchicine (with a thiomethyl instead of a methoxy group at position C-10) and N-acetylcolchinol O-methyl ether (NCME) (with a methoxy-substituted benzenoid instead of the methoxy-substituted tropone C ring) were prepared for comparison with colchicine. Scatchard analysis indicated a single binding site with KD values of 1.0-2.3 microM. Thiocolchicine was bound 2-4 times as rapidly as colchicine, but the activation energies of the reactions were nearly identical (18 kcal/mol for colchicine, 20 kcal/mol for thiocolchicine). NCME bound to tubulin in a biphasic reaction. The faster phase was 60 times as fast as colchicine binding at 37 degrees C, and a substantial reaction occurred at 0 degrees C. The rate of the faster phase of NCME binding changed relatively little as a function of temperature, so the activation energy was only 7.0 kcal/mol. Dissociation reactions were also evaluated, and at 37 degrees C the half-lives of the tubulin-drug complexes were 11 min for NCME, 24 h for thiocolchicine, and 27 h for colchicine. Relative dissociation rates as a function of temperature varied little among the drug complexes. Activation energies for the dissociation reactions were 30 kcal/mol for thiocolchicine, 27 kcal/mol for NCME, and 24 kcal/mol for colchicine. Comparison of the activation energies of association and dissociation yielded free energies for the binding reactions of -20 kcal/mol for NCME, -10 kcal/mol for thiocolchicine, and -6 kcal/mol for colchicine. The greater effectiveness of NCME and thiocolchicine as compared with colchicine in biological assays probably derives from their more rapid binding to tubulin and the lower free energies of their binding reactions.     

Rapid agonist-induced decrease of 125I-pindolol binding to beta-adrenergic receptors. Relationship to desensitization of cyclic AMP accumulation in intact heart cells

Cyclic AMP accumulation in embryonic chick heart cells and binding of the beta-adrenergic antagonist 125I-pindolol to intact cells has been examined during the first 30 min of (-)-isoproterenol-induced desensitization. Myocardial beta-adrenergic receptors exist in two states which bind agonists with high (KD congruent to 10 nM) and low (KD congruent to 10 microM) affinities. Both activation and desensitization of cyclic AMP accumulation were mediated by (-)-isoproterenol binding to high affinity receptors. (-)-Isoproterenol-induced desensitization of cyclic AMP accumulation occurred with a t1/2 of 3.8 min. Desensitization was accompanied by a decrease in the number of 125I-pindolol binding sites assessed by equilibrium radioligand binding assays conducted at 4 degrees C or short (80 s) binding assays conducted at 37 degrees C. There was an excellent temporal correlation between loss of binding and loss of (-)-isoproterenol-stimulated cyclic AMP accumulation. After (-)-isoproterenol-induced desensitization, most of the remaining receptors assayed at 4 degrees C bound (-)-isoproterenol with high affinity. A rapid (-)-isoproterenol-induced decrease in the number of 125I-pindolol binding sites also occurred in adult canine heart cells and rat adipocytes. The data suggest that agonists do not cause uncoupling of surface receptors. Receptors may be uncoupled as a consequence of rapid sequestration into a hydrophobic compartment.     

Induction of the fibrinogen receptor on human platelets by epinephrine and the combination of epinephrine and ADP

The capacity of epinephrine alone and the combination of low dose epinephrine and ADP to support the binding of fibrinogen to washed human platelets has been examined, 125I-Fibrinogen was bound to epinephrine-stimulated platelets, but 90 min were required to achieve maximal binding at 22 degrees C in contrast to 20 to 30 min with ADP. The overall rate of interaction appeared to reflect the slow binding of fibrinogen to epinephrine-stimulated platelets as opposed to the rate of stimulation of the cell. Divalent ions were required for binding of fibrinogen to epinephrine-stimulated platelets, and both calcium and magnesium supported binding with a prolonged time course. Fibrinogen binding was maximally supported by 20 to 30 microM epinephrine. The combination of low dose epinephrine (5 microM) and low dose ADP (0.5 microM), which acted synergistically to induce platelet aggregation, supported the rapid (10 min) binding of fibrinogen to platelets. With 4 microM epinephrine, more fibrinogen bound per platelet at all ADP doses than with ADP alone. With all the stimuli, saturable binding of fibrinogen to the platelet was observed, and Scatchard plots were linear, yielding very similar apparent association constants. The number of molecules bound per cell was stimulus-dependent, with 30 microM epinephrine inducing the binding of fewer fibrinogen molecules per cell (mean = 20,400) than 10 microM ADP (mean = 35,900) or the combination of 5 microM epinephrine + 0.5 microM ADP (mean = 43,600). The participation of endogenous ADP in fibrinogen binding to epinephrine-stimulated platelets was suggested since enzymes which remove ADP, apyrase, and creatine phosphate/creatine phosphokinase, and the ADP analogue, 2-chloroadenosine, completely inhibited the binding of fibrinogen to the platelet.     

Insulin Binding to Human Astrocytoma Cells and Its Effect on Uridine Incorporation into Nucleic Acid

Binding of [125I]monoiodoinsulin to human astrocytoma cells (U-373 MG) was time dependent, reaching equilibrium after 1 h at 22 degrees C with equilibrium binding corresponding to 2.2 fmol/mg protein: this represents approximately 2,000 occupied binding sites per cell. The t1/2 of 125I-insulin dissociation at 22 degrees C was 10 min; the dissociation rate constant of 1.1 X 10(-2) s-1 was unaffected by a high concentration of unlabeled insulin (16.7 microM). Porcine insulin competed for specific 125I-insulin binding in a dose-dependent manner and Scatchard analysis suggested multiple affinity binding sites (higher affinity Ka = 4.4 X 10(8) M-1 and lower affinity Ka = 7.4 X 10(6) M-1). Glucagon and somatostatin did not compete for specific insulin binding. Incubation of cells with insulin (0.5 microM) for 2 h at 37 degrees C increased [2-14C]uridine incorporation into nucleic acid by 62 +/- 2% (n = 3) above basal. Cyclic AMP, in the absence of insulin, also stimulated nucleoside incorporation into nucleic acid [65 +/- 1% (n = 3)] above basal. Preincubation with cyclic AMP followed by insulin had an additive effect on nucleoside incorporation [160 +/- 4% (n = 3) above basal]. Dipyridamole (50 microM), a nucleoside transport inhibitor, blocked both basal and stimulated uridine incorporation. These studies confirm that human astrocytoma cells possess specific insulin receptors with a demonstrable effect of ligand binding on uridine incorporation into nucleic acid.     

Long-chain acyl-coenzyme A synthetase from rat brain microsomes. Kinetic studies using [1-14C]docosahexaenoic acid substrate

The activation of docosahexaenoic acid by rat brain microsomes was studied using an assay method based on the extraction of unreacted [1-14C]docosahexaenoic acid and the insolubility of [1-14C]docosahexaenoyl-CoA in heptane. This reaction showed a requirement for ATP, CoA, and MgCl2 and exhibited optimal activity at pH 8.0 in the presence of dithiothreitol and when incubated at 45 degrees C. The apparent Km values for ATP (185 microM), CoA (4.88 microM), MgCl2 (555 microM) and [1-14C]docosahexaenoic acid (26 microM) were determined. The presence of bovine serum albumin or Triton X-100 in the incubation medium caused a significant decrease in the Km and Vm values for [1-14C]docosahexaenoic acid. The enzyme was labile at 45 degrees C (t1/2:3.3 min) and 37 degrees C (t1/2:26.5 min) and lost 36% of its activity after freezing and thawing. The transition temperature (Tc) obtained from Arrhenius plot was 27 degrees C with the activation energies of 74 kJ/mol between 0 degrees C and 27 degrees C and 30 kJ/mol between 27 degrees C and 45 degrees C. [1-14C]Palmitic acid activation in rat brain and liver microsomes showed apparent Km values of 25 microM and 29 microM respectively, with V values of 13 and 46 nmol X min-1 X mg protein-1. The presence of Triton X-100 (0.05%) in the incubation medium enhanced the V value of the liver enzyme fourfold without affecting the Km value. Brain palmitoyl-CoA synthetase, on the other hand, showed a decreased Km value in the presence of Triton X-100 with unchanged V. The Tc obtained were 25 degrees C and 28 degrees C for brain and liver enzyme with an apparent activation energy of 109 and 24 kJ/mol below and above Tc for brain enzyme and 86 and 3.3 kJ/mol for liver enzyme. The similar results obtained for the activation of docosahexaenoate and palmitate in brain microsomes suggest the possible existence of a single long-chain acyl-CoA synthetase. The differences observed in the activation of palmitate between brain and liver microsomes may be due to organ differences. Fatty acid competition studies showed a greater inhibition of labeled docosahexaenoic and palmitic acid activation in the presence of unlabeled unsaturated fatty acids. The Ki values for unlabeled docosahexaenoate and arachidonate were 38 microM and 19 microM respectively for the activation of [1-14C]docosahexaenoate. In contrast, the competition of unlabeled saturated fatty acids for activation of labeled docosahexaenoate is much less than that for activation of labeled palmitate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Random and site-directed mutagenesis of bacterial luciferase: investigation of the aldehyde binding site

Numerous luciferase structural gene mutants of Vibrio harveyi have been generated by random mutagenesis and phenotypically characterized [Cline, T.W., & Hastings, J.W. (1972) Biochemistry 11, 3359-3370]. All mutants selected by Cline and Hastings for altered kinetics in the bioluminescence reaction had lesions in the alpha subunit. One of these mutants, AK-20, has normal or slightly enhanced thermal stability and enhanced FMNH2 binding affinity but a much-reduced quantum yield of bioluminescence and dramatically altered stability of the aldehyde-C4a-peroxydihydroflavin-luciferase intermediate (IIA), with a different aldehyde chain length dependence from that of the wild-type luciferase. To better understand the structural aspects of the aldehyde binding site in bacterial luciferase, we have cloned the luxAB genes from the V. harveyi mutant AK-20, determined the nucleotide sequence of the entire luxA gene, and determined the mutation to be TCT----TTT, resulting in a change of serine----phenylalanine at position 227 of the alpha subunit. To confirm that this alteration caused the altered kinetic properties of AK-20, we reverted the AK-20 luxA gene by oligonucleotide-directed site-specific mutagenesis to the wild-type sequence and found that the resulting enzyme is indistinguishable from the wild-type luciferase with respect to quantum yield, FMNH2 binding affinity, and intermediate IIA decay rates with 1-octanal, 1-decanal, and 1-dodecanal. To investigate the cause of the AK-20 phenotype, i.e., whether the phenotype is due to loss of the seryl residue or to the properties of the phenylalanyl residue, we have constructed mutants with alanine, tyrosine, and tryptophan at alpha 227.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uptake and destruction of 125I-CSF-1 by peritoneal exudate macrophages

The binding and uptake of the colony-stimulating factor CSF-1 by peritoneal exudate macrophages (PEM) from lipopolysaccharide insensitive C3H/HeJ mice was examined at 2 degrees C, and at 37 degrees C. At 2 degrees C, 125I-CSF-1 was bound irreversibly to the cell surface. At 37 degrees C, 90% of the cell surface associated 125I-CSF-1 was rapidly internalized and subsequently degraded and the remaining 10% dissociated as intact 125I-CSF-1. Thus classical equilibrium or steady state methods could not be used to quantitatively analyze ligand-cell interactions at either temperature, and alternative approaches were developed. At 2 degrees C, the equilibrium constant (Kd less than or equal to 10(-13) M) was derived from estimates of the rate constants for the binding (kon congruent to 8 x 10(5) M-1 s-1) and dissociation (koff less than or equal to 2 x 10(-7) s-1) reactions. At 37 degrees C, the processes of dissociation and internalization of bound ligand were kinetically competitive, and the data was formally treated as a system of competing first order reactions, yielding first order rate constants for dissociation, koff = 0.7 min-1 (t1/2 = 10 min) and internalization, kin = 0.07 min-1 (t 1/2 = 1 min). Approximately 15 min after internalization, low-molecular weight 125I-labeled degradation products began to appear in the medium. Release of this degraded 125I-CSF-1 was kinetically first order over three half-lives (Kd = 4.3 x 10(-2) min-1, t1/2 = 16 min). Thus CSF-1 binds to a single class of receptors on PEM, is internalized with a single rate limiting step, and is rapidly destroyed without segregation into more slowly degrading intracellular compartments.     

Inhibition of mitochondrial carbonic anhydrase and ureagenesis: a discrepancy examined

The amount of urea synthesized in intact guinea pig hepatocytes in 60 min ([urea]t=60), was determined at 37 degrees C in Krebs-Henseleit buffer plus (in mM) 10 NH4Cl, 5 lactate, and 10 ornithine in 5% CO2-95% O2. The concentrations of sulfonamide carbonic anhydrase (CA) inhibitors required to reduce the rate of urea synthesis by 50% (I50) were (in mM): 0.07 ethoxzolamide, 0.5 methazolamide, 0.7 acetazolamide, and 5.0 p-aminomethylbenzenesulfonamide. At 37 degrees C acetazolamide and ethoxzolamide reduced citrulline synthesis by intact mitochondria in medium containing (in mM) 50 3-(N-morpholino)propanesulfonic acid, 35 KCl, 5 KH2PO4, 2 adenosine triphosphate, 10 ornithine, 10 NH4Cl, 1 [ethylene-bis(oxyethylenenitrile)]tetraacetic acid, 1 MgCl2, 20 pyruvate, and 25 KHCO3 (pH 7.4) in 5% CO2-95% O2; the inhibition by ethoxzolamide was not decreased greater than 50%; 25% inhibition was achieved by 0.65 microM ethoxzolamide. Inhibition constant (Ki) values for CA activity of disrupted mitochondria at 37 degrees C were 0.03 microM ethoxzolamide and 0.16 microM acetazolamide, and for disrupted hepatocytes were 150 microM ethoxzolamide and 50 microM acetazolamide. p-Aminomethylaminosulfonamide-affinity column purification yields one band of 29,000 mol wt for CA V purified from disrupted mitochondria; homogenized whole-liver supernatant yields an additional band of 20,000 mol wt (at greater than 100 times the concentration of CA V), which has some glutathione S-transferase activity. It is concluded that this 20,000-mol wt protein modifies the potency of ethoxzolamide in the liver cytosol.     

Synthesis and biochemical characterization of the new sulfhydryl-reactive ATP analogue 8-thiocyano-ATP. Its interaction with Na,K-ATPase and kinases.

The synthesis of 8-thiocyano-ATP (CNS8-ATP) is described. At 37 degrees C the ATP analogue inactivates Na,K-ATPase, hexokinase, and pyruvate kinase. In all three cases, inactivation can be prevented by the addition of ATP, thus indicating that CNS8-ATP is recognized within the ATP binding site of the above enzymes. Incubation of the inactivated enzymes with dithiothreitol restores the catalytic activities. Therefore, it is likely that in these enzymes a mixed disulfide (E-S-S8-ATP) is formed between a sulfhydryl in the ATP binding site (E-SH) and the ATP analogue: [formula: see text] From the pseudo-first-order inactivation kinetics, a KD = 2.7 microM with k2 = 0.142 min-1 is calculated for the hexokinase and a KD = 40 microM with k2 = 0.347 min-1 is calculated for the pyruvate kinase interactions with the ATP analogue. At 4 degrees C, Na,K-ATPase recognizes CNS8-ATP with a KD = 8.3 microM. At 37 degrees C, the enzyme becomes inactivated by the ATP analogue in a biphasic manner. Inactivation results in the incorporation of [alpha-32P]8-CNS8-ATP into the catalytic alpha-subunit of the enzyme. Limited tryptic digestion in the presence of 150 mM KCl results in the formation of a radioactive peptide of Mr = 56,000, known to bear the purine binding domain of Na,K-ATPase. The results described in this article verify CNS8-ATP as a sulfhydryl-reactive ATP analogue and characterize this new ATP analogue as a useful tool for structure/function studies on ATP-recognizing enzymes.