Bovine Adrenal Tyrosine Hydroxylase: Comparative Study of Native and Proteolyzed Enzyme, and Their Interaction with Anions

Abstract: The pH optimum of native adrenal medulla tyrosine hydroxylase activity is shifted from 5.8 to 6.4 by polyanions (heparin, dextran sulphate), salts (NaCl, Na₂SO₄) and the anionic buffer 2-( N -morpholino)ethanesulphonic acid (MES). Simultaneously, the activity at the optimal pH is increased. Kinetic studies have shown that this activation is associated with a decrease of the apparent K _m of the enzyme for the cofactor 6,7-dimethyltetrahydropterin (DMPH₄) and an increase in the V _max for tyrosine and DMPH₄. The K _m for the tyrosine remained unchanged. These data have been interpreted in terms of the polyelectrolyte theory. The adsorption of tyrosine hydroxylase on various affinity gels containing heparin, dextran sulphate or unsulphated polymer dextran as ligands indicate that the activation of the enzyme is mediated by electrostatic interactions with the anionic species. The site of electrostatic interaction possesses some specificity since the binding constants are higher for heparin or dextran sulphate than for NaCl or MES buffer. Moreover, 3-( N -morpholino)propanesulphonic acid (MOPS) a slightly structurally different buffer inhibits the enzyme activity whereas N -(2-acetamido)-2-amino-ethanesulphonic acid (ACES) has no effect. A limited proteolytic digestion which preserves the enzymatic activity, destroys the effects of the anions. The isoelectric point and the molecular parameters of tyrosine hydroxylase are markedly altered after limited digestion. It is therefore suggested that the interaction between the hydroxylase and anionic compounds occurs on a part of the protein which is different from the active site and which is lost by proteolysis. This portion of the protein might be involved in regulation of native tyrosine hydroxylase.     

Enzyme behaviour and molecular environment. The effects of ionic strength, detergents, linear polyanions and phospholipids on the pH profile of soluble cytochrome oxidase.

The activity vs. pH profile for the oxidation of ferrocytochrome c by purified cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) was investigated as a function of ionic strength (from 10 to 200 mM) in the absence and in the presence of various perturbants: Tween 20, linear polyanions (RNA, heparin, polyglutamic acid) and phospholipids (asolectin, phosphatidylcholine, phosphatidic acid and cardiolipin). The activation induced by Tween 20 and "zero net charge" phospholipid liposomes was not pH dependent. On the other hand, linear polyanions and polyanionic liposomes strongly perturbed the pH profile, mostly at low ionic strength, by shifting the pH optimum about 1.7 pH units towards alkaline pH values. This effect was reversed by increasing ionic strength. These observations are interpreted in the light of polyelectrolyte theory. Since these results show striking with membrane-bound enzyme, it is concluded that in vivo cytochrome oxidase is located within polyanionic sites of the micochondrial membrane. The activation broght about by phospholipids may result from two posible processes: creation of a hydrophobic environment by the non-polar tails, preventing autoaggregation; and creation of a suitable polyelectrolytic environment by the polar heads (of non zero net charge), increasing the intrinsic reaction rate.     

Effect of free and conjugated bile salts on alpha-amylase activity.

The effect of individual bile salts on alpha-amylase hydrolysis of Cibachron Blue starch was studied at pH 6.0. With sodium cholate, taurocholate and taurodeoxycholate, enzyme activity was increased to 150-160 percent of the control value, at a concentration of similar to 1 mmol/l bile salt. The increased activity extended up to 4 mmol/l. The bile salts sodium deoxycholate and taurochenodeoxycholate exerted activation and inhibition depending on the concentration. With deoxycholate (0.75 mmol/l), activation (150 percent) was evident, while inhibition was apparent above 2.5 mmol/l. With taurochenodeoxycholate maximum activity (135 percent) was observed at 0.25 mmol/l, while inhibition was evident above 1.5 mmol/l. Chenodeoxycholate and lithocholate exerted marked inhibition at concentrations as low as 0.5 mmol/l. Inhibition of alpha-amylase by chenodeoxycholate was competitive with both soluble and insoluble starch substrates. Since the pH of the jejunum is in the region of 6.0 the phenomenon of activation and inhibition of alpha-amylase by bile salts at this pH could be of physiological significance.     

L1210 dihydrofolate reductase. Kinetics and mechanism of activation by various agents

Dihydrofolate reductase from a methotrexate-resistant subline (R6) of L1210 mouse leukemia cells is activated (i.e. has its catalytic activity increased severalfold) by treatment with (a) sulfhydryl-modifying agents (p-chloromercuribenzoate (pCMB) or 5,5'-dithiobis(2-nitrobenzoic acid], (b) salts (KCl or NaCl), or (c) chaotropes (urea or guanidinium hydrochloride). With b or c activation is rapid (less than 10 s), but with a the process is much slower; at 25 degrees C, pseudo first-order rate constants for activation by excess pCMB or 5,5'-dithiobis(2-nitrobenzoic acid) are 0.45 and 0.08 min-1, respectively. Activation can also be monitored by conformational changes in the protein as indicated by enhanced fluorescence of 2-p-toluidinylnaphthalene-6-sulfonate or by increased intrinsic fluorescence of tryptophan residues in the enzyme. Pseudo first-order rate constants for the pCMB-induced conformational change, measured by these fluorimetric procedures (0.45 min-1 and about 0.4 min-1, respectively), are in good agreement with the value obtained from the increase in catalytic activity. The rate of modification of the single cysteine residue in the enzyme by excess 14C-labeled pCMB, however, is faster than the rate of activation, indicating that the conformational change follows derivatization and is the rate-limiting step in the overall process. Activated forms of the enzyme are more labile to thermal denaturation or proteolysis than the untreated enzyme; the former process, however, is retarded by the presence of bovine serum albumin. Activation by the various agents is considered to involve a common mechanism in which interaction of the enzyme with the agents is followed by conformational changes in the enzyme, producing a series of forms that differ in microstructure, catalytic activity, and lability.     

Cysteine 904 Is Required for Maximal Insulin Degrading Enzyme Activity and Polyanion Activation

Cysteine residues in insulin degrading enzyme have been reported as non-critical for its activity. We found that converting the twelve cysteine residues in rat insulin degrading enzyme (IDE) to serines resulted in a cysteine-free form of the enzyme with reduced activity and decreased activation by polyanions. Mutation of each cysteine residue individually revealed cysteine 904 as the key residue required for maximal activity and polyanion activation, although other cysteines affect polyanion binding to a lesser extent. Based on the structure of IDE, Asn 575 was identified as a potential hydrogen bond partner for Cys904 and mutation of this residue also reduced activity and decreased polyanion activation. The oligomerization state of IDE did not correlate with its activity, with the dimer being the predominant form in all the samples examined. These data suggest that there are several conformational states of the dimer that affect activity and polyanion activation.     

Gramicidin S-synthetase. Preparation of the multienzymic complex with a high specific activity.

A new purification procedure for the multienzyme of gramicidin S-synthetase has been developed. In vitro proteolysis with partial inactivation is suppressed by protease inhibitors EDTA, phenylmethylsulfonylfluoride, and fast preparation methods during initial separation steps. Activity has only been assayed by the total reaction of gramicidin S-synthetase, not by partial reactions of amino acid activation. The assay has been improved by evaluation of inhibitory concentrations of buffers, salts, and the product gramicidin S. It has been demonstrated that the rate of peptide synthesis in extracts containing both enzymes of gramicidin S-synthetase depends on protein concentration in a second order function. The multienzyme or heavy enzyme has been purified about 1400-fold to a specific activity of 24 nM/min per mg of protein, and the relation of this activity to the calculated in vivo activity is discussed.     

Yeast hexokinase A. Succinylation and properties of the active subunit.

Yeast hexokinase A (ATP:D-hexose 6-phosphotransferase, EC2.7.1.1) dissociates into its subunits upon reaction with succinic anhydride. The chemically modified subunits could be isolated in a catalytically active form. The Km values found for ATP and for glucose were of the some order as those found for the native enzyme. Of the 37 amino groups present per enzyme subunit, 2-3 of these groups might be located in the proximity of the region of subunit interactions. The 50% loss of the initial activity, which follows the succinylation of these more reactive amino groups, does not seem to be due to the modification of a residue on the enzyme active site or to a change of the tertiary structure of the protein. This 50%loss of the enzyme activity may be related to the dissociation of the dimer into monomers. Both native enzyme and the succinylated subunits have the same H-dependent denaturation rate profiles in response to 2 M urea. Moreover, the apparent pK of the group involved in the transition from a more stable conformation of the protein in the acid range to a less stable one at alkaline pH seems to be similar to the pK of the group implicated in the transition between the protonated inactive form of the enzyme and an active deprotonated form. The succinylated subunit presents 'negative co-operativity' with respect to ATP at slightly acid pH; however, the burst-type slow transient in the reaction progress curve and the activation effect induced by physiological polyanions, effects observed for the native enzyme, were not detected in the standard experimental conditions with the succinylated subunit.     

Identification by affinity labeling of potential sites in 23-S rRNA interacting with the 3'' end of tRNA

Tyrosine 3-monooxygenase was purified to homogeneity, as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, from rat adrenal. The specific activity of the final preparation was approximately 1,600 nmol min-1 mg protein-1, which was much higher than the highest yet reported. The enzyme was markedly stabilized in the presence of glycerol, Tween 80 and EDTA. As judged by gel filtration on Ultrogel AcA 34, sodium dodecyl sulfate/polyacrylamide gel electrophoresis and cross-linking studies, the enzyme appeared to be composed of four identical subunits, each possessing a molecular weight of 59,000. The isoelectric point of the enzyme was estimated to be 6.7 in the presence of 8 M urea and 6.6 in its absence. Amino acid analysis of the enzyme revealed a fairly high content of serine residues in this protein. Purification of the enzyme caused changes in the kinetic properties of the enzyme. The Km for 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine decreased from 220 microM to 58 microM. The pH profile for the enzyme activity became more broad and the pH optimum was changed from an acid pH to a neutral pH. Although polyanions, such as heparin and dextran sulfate, markedly stimulated the activity of crude enzyme by increasing the V, they were much less effective in the activation of purified enzyme. A marked stimulation of the enzyme activity by phospholipids, such as phosphatidylserine, phosphatidylinositol, phosphatidylcholine and phosphatidylethanolamine, were not observed in both pure and crude preparations even at low concentrations of the pterin cofactor.     

Allosteric modification of factor XIa functional activity upon binding to polyanions

The effects of several polyanions on the hydrolysis of the chromogenic substrate L-pyroglutamyl-L-prolyl-L-arginyl-p-nitroaniline (S-2366) and on the activation of factor IX by factor XIa have been investigated. Two forms of dextran sulfate (M(r) approximately 500000 and M(r) approximately 10000, DX10) and two forms of heparin (64 disaccharide units, M(r) approximately 14000, and hypersulfated heparin, S-Hep, M(r) approximately 12000) inhibited both factor XIa amidolytic activity and factor IX activation in a concentration-dependent manner. The inhibitory effect was not due to binding of either substrate by the polyanions since only a decrease in V(max) without any effect on K(m) was observed in kinetic assays. Steric inhibition is unlikely since the concentrations of polyanions required for inhibition of small peptide hydrolysis were lower than those required for macromolecular substrate cleavage. In contrast, an allosteric inhibitory mechanism was supported by an enhancement of the dansyl fluorescence of 5-(dimethylamino)-1-(naphthalenesulfonyl)glutamylglycylarginyl- (DEGR-) factor XIa observed when the fluorophore was in complex with either DX10 or S-Hep. Moreover, in the presence of a polyanion the fluorophore was far more resistant to quenching by acrylamide. These results provide compelling evidence that factor XIa binding to the polyanions, dextran sulfate and heparin, results in inhibition of the enzyme by an allosteric mechanism.     

Short chain fatty acid inhibition of rat brain Na-K adenosine triphosphatase

The possibility was considered that the sleep-like state seen after injection of short chain fatty acids salts into animals is a result of inhibition of the sodium-potassium activated ATPase. Tris salts of short chain fatty acids inhibited brain Na-K ATPase activity in vitro at concentrations similar to intravenous levels causing narcosis in vivo. The inhibition depended on the logarithm of the concentration of a given acid. The concentration of acid anion which caused 50 per cent inhibition of the enzyme system (I50) was determined for straight and branched chain acids with 4-12 carbon atoms per molecule. The log of I50 concentrations plotted against the number of carbon atoms in the molecule gave a straight line; the inhibitory capacity of an acid increased by a factor of 2.3 for each--CH2--added to the carbon chain. It is suggested that both fatty acid narcosis and the enzyme inhibition result from fatty acid molecules forming an ordered array along the membrane in association with membrane lipids.     

Effects of salts on thermolysin: activation of hydrolysis and synthesis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester, and a unique change in the absorption spectrum of thermolysin.

It has been reported that neutral salts such as NaCl activate the thermolysin-catalyzed hydrolysis of substrates containing glycine at the P1 position (carboxylic side of the cleavage bond) [Holmquist, B. & Vallee, B.L. (1976) Biochemistry 15, 101-107]. In this paper, we demonstrate that high concentrations (1-4 M) of neutral salts greatly enhance the thermolysin activity in both hydrolysis and synthesis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (ZAPM), a precursor of a peptide sweetener, aspartame, in which the L-aspartyl residue is the P1 residue. The enzyme activity is enhanced with an increase in salt concentration in a pseudo-exponential fashion. The degree of activation by salts was in the order LiCl > NaCl > KCl. The rate of ZAPM hydrolysis in the presence of 3.8 M NaCl was 6-7 times higher than that in its absence, and 50 times or more activation is expected in saturated NaCl solution. The activation is brought about solely through an increase in the catalytic constant (kcat), and the Michaelis constant (Km) is not affected at all by the presence of NaCl. On mixing thermolysin with NaCl, a unique absorption difference spectrum suggesting a conformational change of the enzyme was observed. The intensity increased in a pseudo-exponential fashion with increase of NaCl concentration up to 3 M, and this dependence is similar to that of the enzyme activity.     

Purification and polyamine activation of a high-affinity 3',5'-cAMP phosphodiesterase from rabbit muscle

A high-affinity phosphodiesterase, termed PDE II, has been purified about 1400-fold from rabbit skeletal muscle. This enzyme is activated by treatment with proteases. It is also activated specifically by polyarginine and arginine-rich histones, but not by other polyanions. The activation is counteracted nonspecifically by polycations, such as heparin and chondroitin sulphate. When the enzyme is fully activated by polyarginine it is no longer susceptible to activation by proteases. A conformational or structural change must thus occur in the enzyme by the binding of the polyanions.     

Calcium activation of brain tryptophan hydroxylase.

Using both radioisotopic and fluorometric techniques to measure the activity of midbrain soluble enzyme, we have demonstrated that calcium activates tryptophan hydroxylase. The observed activation apparently results from an increased affinity of the enzyme for both its substrate, tryptophan, and the cofactor 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropteridine (6-MPH₄). The calcium activation of tryptophan hydroxylase appears to be specific for both enzyme and effector: other brain neurotransmitter biosynthetic enzymes, such as aromatic amino acid decarboxylase(s) and tyrosine hydroxylase, are not affected by calcium (at concentrations ranging from 0.01 mM to 2.0 mM); other divalent cations, such as Ba⁺⁺, Mg⁺⁺, and Mn⁺⁺, have no activating effect on tryptophan hydroxylase. This work suggests that increases in brain serotonin biosynthesis induced by neural activation may be due to influx of Ca⁺⁺ associated with membrane depolarization and resulting activation of nerve ending tryptophan hydroxylase.     

Effect of surface-active agents on peroxidase activity. IV. Effect of lipids and fatty acids from milk

The effect of surfactants, lipids and fatty acid salts isolated from cow milk on the activity of heme-containing horseradish peroxidase in solution was studied. As the surfactant concentration increases, the rate of the enzymic reaction successively decreases, increases, and again decreases, down to zero in the case of the fatty acid salts. The initial deceleration of the reaction rate results from the enzyme inhibition. The subsequent increase is caused by an improved accessibility for the substrate and the enhanced activity of the catalytic site of the enzyme due to its immobilization in the surfactant aggregates. A shielding of the protein by these aggregates can explain the secondary deceleration of the enzymic reaction rate. The general character of the dependence is similar and does not depend on the surfactant structure for a series of fatty acid salts and phospholipids; however, it is quite different in the case of cholesterol and sphingomyelin.     

Nitrilase of Rhodococcus rhodochrous J1. Conversion into the active form by subunit association.

Nitrilase-containing resting cells of Rhodococcus rhodochrous J1 converted acrylonitrile and benzonitrile to the corresponding acids, but the purified nitrilase hydrolyzed only benzonitrile, and not acrylonitrile. The activity of the purified enzyme towards acrylonitrile was recovered by preincubation with 10 mM benzonitrile, but not by preincubation with aliphatic nitriles such as acrylonitrile. It was shown by light-scattering experiments, that preincubation with benzonitrile led to the assembly of the inactive, purified and homodimeric 80-kDa enzyme to its active 410-kDa aggregate, which was proposed to be a decamer. Furthermore, the association concomitant with the activation was reached after dialysis of the enzyme against various salts and organic solvents, with the highest recovery reached at 10% saturated ammonium sulfate and 50% (v/v) glycerol, and by preincubation at increased temperatures or enzyme concentrations.     

Characterization of microsomal choloyl-coenzyme A synthetase.

Choloyl-CoA synthetase (EC 6.2.1.7) was characterized for the first time under appropriated assay conditions. The p/ optimum for the reaction is pH 7.2.-7.3. The reaction has an absolute requirement for bivalent cation. Several different metal ions fulfil this requirement, but Mn2+ and Mg2+ were the most effective. The KAppm (apparent Km) for CoA, extrapolated from kinetic data, is 50 micronM, but in fact the rate of reaction is increased little by concentrations of CoA above 25 micronM. The KAppm for ATP is 600 micronM. High concentrations of ATP appear to cause substrate inhibition. The KAppm for cholate was 6 micronM. The enzyme was inhibited by treating the microsomal fraction with N-ethylmaleimide. The inclusion of various conjugated and unconjugated bile salts in the assay also inhibited the enzyme. Unconjugated bile salts were more potent inhibitors than the conjugated bile salts. High concentrations of oleic acid inhibited the enzyme. The properties of choloyl-CoA synthetase were not modified by alterations of the properties of the lipid phase of the microsomal membrane. Treatment with phospholipase A did not alter activity directly. Triton N-101 and Triton X-100 also were without effect on activity, and the enzyme was insensitive to temperature-induced phase transitions within the lipid portion of the membrane. The enzyme can be solubilized from the microsomal membrane in an active form by treatment with Triton N-101.     

Enzymatic properties of ubiquinol:cytochrome c2 oxidoreductase in situ in Rhodopseudomonas palustris membranes

The presence of ubiquinol:cytochrome c2 oxidoreductase was shown in the membranes from a photosynthetic bacterium, Rhodopseudomonas palustris. Some properties of the enzyme in situ were investigated. The optimal pH of this enzyme activity was 7.0 in the intact membranes. The activity was inhibited by both antimycin and myxothiazol. Maximal activity (Vmax) was 3-4 mol cytochrome c (c2) reduced/mol cytochrome c1.s. Apparent activity of the enzyme with horse heart cytochrome c as the electron acceptor decreased as the concentration of salts in the reaction mixture increased, whereas when R. palustris cytochrome c2 was used as the electron acceptor, the activity increased as the concentration of salts increased. Moreover, the activity of the enzyme did not depend on the species or concentration of anions but on both the concentration and valency of the cations of the salts. These salt effects were thought to be due to the change of effective concentration of cytochrome molecules caused by cations near the membrane surface, which was net negatively charged. Apparent Km for ubiquinol-1 was about 80 microM irrespective of the species of cytochrome and the presence of salts.     

Combined action of Escherichia coli glycogen synthase and branching enzyme in the so-called "unprimed" polyglucoside synthesis.

Mutants of Escherichia coli which are unable to synthesize glycogen were used to study the so-called “unprimed” synthesis of glycogen. The glycogen synthase has been partially purified from these mutants. During the purification, attempts were made to separate the activity which requires the addition of an exogenous primer (primed activity) from the activity which does not require a primer but is highly dependent on the presence of some salts such as citrate and EDTA (unprimed activity). No separation between these two activities could be achieved but the results obtained by chromatography on DEAE-Sephadex indicate that there is a single form of glycogen synthase which is responsible for both unprimed and primed activity. The evidence that a single protein was necessary to catalyze these two reactions was given by the findings that mutants defective in glycogen synthase activity were unable to catalyze glucosyl transfer without added primer. At low concentration, the glycogen synthase purified from a branching enzyme negative mutant catalyzed the unprimed reaction at a slow rate even in presence of salts. A protein activator of this reaction was found in mutants lacking glycogen synthase but not in mutants lacking branching enzyme. The hypothesis that this activator is the branching enzyme itself was supported by the observation that it co-purified with the branching enzyme from a E. coli strain defective in glycogen synthase activity. EDTA or Triton X-100 increased the stimulation of the unprimed synthesis by the branching enzyme. The apparent affinity of the glycogen synthase for glycogen was increased twofold in the presence of EDTA but the branching enzyme further increased the effect of EDTA. The combined action of the glycogen synthase and the branching enzyme on the endogenous glucan associated with the synthase may account for the unprimed activity observed in vitro.     

Mobilization of B and T lymphocytes and haemopoietic stem cells by polymethacrylic acid and dextran sulphate.

The leucocytosis which can be evoked by the polyanions dextran sulphate (DS), polymethacrylic acid (PMAA) and the copolymer of PMAA and styrene (PMAA--STYR) was studied in mice. After intravenous administration of these polyanions peak numbers of leucocytes were found in the peripheral blood 3 hr after injection. All three types of polyanions increased the number of lymphocytes, granulocytes and monocytes. Dose--response studies revealed that the nature of the polyanion determined the degree of leucocyte mobilization. The most potent mobilizer was found to be DS. This polyanion could evoke a six-fold increase of the number of peripheral blood leucocytes. By means of the membrane fluorescence technique it could be demonstrated that optimal doses of DS, PMAA and PMAA--STYR mobilized both B and T lymphocytes. The ratio between the number of B and T cells mobilized was greater for DS than for the other two polyanions. Intravenous injection of DS, PMAA and PMAA--STYR also increased the number of circulating haemopoietic stem cells (CFU-S). The most potent stem cell mobilizer appeared to be PMAA--STYR. This polyanion evoked a twenty-five-fold increase in the number of CFU-S.