Detection and properties of l-histidinol dehydrogenase in wheat germ

The presence and partial characterization of the properties of l-histidinol dehydrogenase (EC 1.1.1.23), the enzyme catalysing the last step in the pathway of histidine biosynthesis, has been described in higher plants for the first time. The activity has been found in cell-free extracts from wheat germ, turnip root, radish root and squash fruit. The enzyme has been partially purified and characterized from extracts of acetone powders of wheat germ. DEAE-cellulose chromatography revealed two peaks of histidinol dehydrogenase activity. In one there was a rapid reduction of NAD⁺ in the absence of histidinol; however, the rate was stimulated by the addition of histidinol. The rate in the absence of substrate became quite low after several min and the histidinol-dependent rate was then easily observed. The second peak of activity did not reduce NAD⁺ unless l-histidinol was present in the assay mixture. The K^ms for l-histidinol and NAD⁺ were determined for this latter enzyme. The values obtained at saturating concentrations of the other substrate were l-histidinol, 8.8 μM and NAD⁺, 0.14 mM. The product of the dehydrogenase reaction was histidine as determined by paper chromatography.     

Kinetic mechanism of histidinol dehydrogenase: histidinol binding and exchange reactions

Salmonella typhimurium histidinol dehydrogenase produces histidine from the amino alcohol histidinol by two sequential NAD-linked oxidations which form and oxidize a stable enzyme-bound histidinaldehyde intermediate. The enzyme was found to catalyze the exchange of 3H between histidinol and [4(R)-3H]NADH and between NAD and [4(S)-3H]NADH. The latter reaction proceeded at rates greater than kcat for the net reaction and was about 3-fold faster than the former. Histidine did not support an NAD/NADH exchange, demonstrating kinetic irreversibility in the second half-reaction. Specific activity measurements on [3H]histidinol produced during the histidinol/NADH exchange reaction showed that only a single hydrogen was exchanged between the two reactants, demonstrating that under the conditions employed this exchange reaction arises only from the reversal of the alcohol dehydrogenase step and not the aldehyde dehydrogenase reaction. The kinetics of the NAD/NADH exchange reaction demonstrated a hyperbolic dependence on the concentration of NAD and NADH when the two were present in a 1:2 molar ratio. The histidinol/NADH exchange showed severe inhibition by high NAD and NADH under the same conditions, indicating that histidinol cannot dissociate directly from the ternary enzyme-NAD-histidinol complex; in other words, the binding of substrate is ordered with histidinol leading. Binding studies indicated that [3H]histidinol bound to 1.7 sites on the dimeric enzyme (0.85 site/monomer) with a KD of 10 microM. No binding of [3H]NAD or [3H]NADH was detected. The nucleotides could, however, displace histidinol dehydrogenase from Cibacron Blue-agarose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiological comparison of L-serine dehydratase and tryptophanase from Bacillus alvei

Tryptophanase from Bacillus alvei also possesses serine dehydratase activity. A comparison of this enzyme with l-serine dehydratase [l-serine hydro-lyase (deaminating), EC 4.2.1.13] in toluene-treated whole cell preparations of the organism was undertaken. Tryptophanase is a constitutive enzyme in B. alvei. The dehydratase undergoes a repression-derepression-repression sequence as the l-serine level in the growth medium is increased from 0 to 0.1 m. Tryptophanase activity is decreased in organisms grown in medium containing glucose. Both enzymes are repressed in organisms grown in glycerol-containing medium. l-Serine dehydratase has a pH optimum of 7.5 in potassium phosphate buffer; tryptophanase functions optimally in this buffer at pH 8.2. Both enzymes lose activity in the presence of tris(hydroxymethyl)aminomethane buffer. Either K(+) or NH(4) (+) is required for full tryptophanase activity, but Na(+) is markedly inhibitory. These three cations are stimulatory to l-serine dehydratase activity. Both enzymes are subject to apparent substrate inhibition at high concentrations of their respective amino acids, but the inhibition of tryptophanase activity can be completely overcome by the removal of indole as it is formed. The dehydratase does not catalyze cleavage of d-serine, l-threonine, or alpha-substituted serine analogues at the concentrations tested. However, activity of the enzyme in cleaving l-serine is competitively inhibited by d-serine, indicating that the d-isomer can occupy an active site on the enzyme. The enzyme catalyzes cleavage of some beta-substituted serine analogues.     

Purification of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans. An iron-sulfur protein

1. The (R)-2-hydroxyglutaryl-CoA dehydratase system from Acidaminococcus fermentans was separated by chromatography of cell-free extracts on Q-Sepharose into two components, an activator and the actual dehydratase. The latter enzyme was further purified to homogeneity by chromatography on blue-Sepharose. It is an iron-sulfur protein (Mr 210,000) consisting of two different polypeptides (alpha, Mr 55,000, and beta, Mr 42,000) in an alpha 2 beta 2 structure with probably two [4Fe-4S] centers. After activation this purified enzyme catalysed the dehydration of (R)-2-hydroxyglutarate only in the presence of acetyl-CoA and glutaconate CoA-transferase, demonstrating that the thiol ester and not the free acid is the substrate of the dehydration. The result led to a modification of the hydroxyglutarate pathway of glutamate fermentation. 2. The activation of the dehydratase by the flow-through from Q-Sepharose concentrated by ultrafiltration required NADH, MgCl2, ATP and strict anaerobic conditions. This fraction was designated as Ao. Later when the concentration was performed by chromatography on phenyl-Sepharose, an NADH-independent form of the activator, designated as A*, was obtained. This enzyme, which required only ATP for activation of the dehydratase, was purified further by affinity chromatography on ATP-agarose. It contains neither iron nor inorganic sulfur. A*, as well as the activated dehydratase, were irreversibly inactivated by exposure to air within less than 15 min. The activated dehydratase but not A* was also inactivated by 1 mM hydroxylamine or by 0.1 mM 2,4-dinitrophenol. 3. The (R)-2-hydroxyglutaryl-CoA dehydratase system is closely related the that of (R)-lactoyl-CoA dehydratase from Clostridium propionicum as described by R. D. Kuchta and R. H. Abeles [(1985) J. Biol. Chem. 260, 13,181-13,189].     

Increase of L-serine dehydratase activity under gluconeogenic conditions in adult-rat hepatocytes cultured on collagen gel/nylon mesh.

Hormonal regulation of L-serine dehydratase [L-serine hydro-lyase (deaminating), EC 4.2.1.13] was studied in primary cultures of adult-rat hepatocytes. The hepatocytes were isolated by collagenase perfusion and maintained in culture on collagen-gel/nylon-mesh substrata. L-Serine dehydratase activity was measured with [14C]threonine as substrate. The enzyme activity in hepatocytes of normal adult rats was low and declined rapidly in culture in L-15 medium containing 0.1 micro M-insulin and even more in the presence of glucose. L-Serine dehydratase activity in hepatocytes of rats with streptozotocin-induced diabetes was initially 20-fold higher than that of normal rats, but fell rapidly to a low value by 4 days in culture. Hormonal regulation of the enzyme activity was manifested by treatment of the cultured hepatocytes with insulin (0.1 micro M), glucagon (0.3 micro M), dexamethasone (10 micro M) and combinations of these hormones. Either glucagon or dexamethasone in the absence of insulin enhanced the activity of L-serine dehydratase, but failed to do so in the presence of insulin. Treatment with both hormones resulted in a 2-3-fold increase in enzyme activity in culture on days 3 and 4. Under conditions in which the enzyme activity was enhanced, glucose production by the cultured hepatocytes was concomitantly increased. Glucose production resulted in part from gluconeogenesis from pyruvate and not entirely from glycogenolysis. The gluconeogenic conditions of culture resulted in a decrease in cellular lipids in the cultured hepatocytes, as evidenced by ultrastructural studies.     

Purification and properties of 5-aminolaevulinate dehydratase from human erythrocytes.

A new procedure for the isolation of homogeneous human 5-aminolaevulinate dehydratase (porphobilinogen synthase, EC 4.2.1.24) is described in which the enzyme is purified 35000-fold and in 65-74% yield. The specific activity of the purified enzyme, 24 units/mg, is the highest yet reported. An efficient stage for the removal of haemoglobin is incorporated in the method, which has general application to the purification of other erythrocyte enzymes. The erythrocyte dehydratase (Mr 285 000) is made up of eight apparently identical subunits of Mr 35 000. The enzyme is sensitive to oxygen, and its activity is maintained by the presence of thiols such as dithioerythritol. Zn2+ is obligatory for enzyme activity, the apoenzyme being essentially inactive (approximately equal to 12% of control) when assayed in buffers devoid of Zn2+. Addition of Zn2+ to the apoenzyme restores activity as long as the sensitive thiol groups are fully reduced; optimal stimulation occurs between 100 and 300 microM-Zn2+. The human enzyme is inhibited by Pb2+ in a non-competitive fashion [KiI (dissociation constant for E X S X Pb2+ complex) = 25.3 +/- 3.0 microM; KiS (dissociation constant for E X Pb2+ complex) = 9.0 +/- 2.0 microM]. Modification of thiol groups, inactivation by oxidation, alkylation or reaction with thiophilic reagents demonstrates the importance of sensitive thiol groups for full enzymic activity.     

Tyrosyl-tRNA synthetase from wheat germ

Tyrosyl-tRNA synthetase (TyrRS) was purified 5,000-fold from wheat germ extract by ultracentrifugation, precipitation with ammonium acetate, and column chromatography. Under denaturing conditions the enzyme ran as a single band on SDS-polyacrylamide electrophoresis with an apparent Mr of 55,000. The native molecular weight determined by gel filtration was 110,000, suggesting a quaternary structure of an alpha 2 type for native TyrRS. Purified enzyme activity, based on the aminoacylation reaction, was studied in terms of Mg2+, ATP, pH, and KCl dependence. Optimum concentrations were 6 mM Mg2+, 4 mM ATP, and 200 mM KCl at pH 8. The Km values for ATP, tyrosine, and tRNA were 40, 3.3, and 1.5 microM, respectively. The instability of the TyrRS activity and the methods used for stabilizing it are discussed. In wheat germ extract we found a second tyrosylating activity that works with Escherichia coli tRNA, but not with wheat germ tRNA. We believe that this enzyme is the mitochondrial tyrosyl-tRNA synthetase of wheat germ.     

Evidence for the phosphorylation of the type II insulin-like growth factor receptor in cultured cells

The ATP pools of monolayer cultures of rat embryo fibroblasts and rat liver cells (BRL-3A2) were labeled with [32P]H3PO4. The type II insulin-like growth factor (IGF) receptor was purified by affinity chromatography on wheat germ lectin-Sepharose and IGF-II-Sepharose columns. A phosphorylated species having the expected size of the type II receptor (Mr = 220,000 without reduction, Mr = 260,000 with reduction) was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. IGF-II stimulated phosphorylation of the type II receptor in BRL-3A2 rat liver cells. Lability of the receptor phosphate bonds to alkaline pH suggests that the bulk of phosphorylation was occurring on serine residues.     

A Novel Class of Herbicides (Specific Inhibitors of Imidazoleglycerol Phosphate Dehydratase)

A new mode of herbicidal action was established by finding specific inhibitors of imidazoleglycerol phosphate dehydratase, an enzyme of histidine (His) biosynthesis. Three triazole phosphonates inhibited the reaction of the enzyme with Ki values of 40 [plus or minus] 6.5, 10 [plus or minus] 1.6, and 8.5 [plus or minus] 1.4 nM, respectively, and were highly cytotoxic to cultured plant cells. This effect was completely reversed by the addition of His, proving that the cytotoxicity was primarily caused by the inhibition of His biosynthesis. These inhibitors showed wide-spectrum, postemergent herbicidal activity at application rates ranging from 0.05 to 2 kg/ha.     

The hepatic glucagon receptor. Solubilization, characterization, and development of an affinity adsorption assay for the soluble receptor

The hepatic glucagon receptor was covalently labeled with [125I-Try10]monoiodoglucagon [( 125I]MIG) by use of the heterobifunctional cross-linker hydroxysuccinimidyl p-azidobenzoate. Labeling of the Mr = 63,000 peptide was sensitive to glucagon and GTP at concentrations at which they affect [125I]MIG binding to the receptor. The labeled receptor was solubilized with Lubrol-PX, and the hydrodynamic characteristics of the receptor were determined. The molecular parameters of the solubilized receptor are: S20,w = 4.3 +/- 0.1, Stokes radius = 6.3 +/- 0.1 nm, frictional coefficient f/f0 = 1.8, and a calculated Mr = 119,000. Incubation of liver membranes at 32 degrees C for 15 min prior to the addition of [125I]MIG permitted us to identify the high molecular weight form (Mr = approximately 113,000) of the receptor by direct sodium dodecyl sulfate-gel electrophoretic analysis. The Mr = 63,000 peptide can be adsorbed to wheat germ lectin-Sepharose. The glycoprotein nature of the receptor has been utilized to develop an assay for the detergent-solubilized receptor that uses wheat germ lectin-Sepharose as a solid matrix to adsorb the [125I] MIG-receptor complex. The free hormone remains in the liquid phase and is removed in the supernatant after low speed centrifugation. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) solubilizes receptors with retention of [125I]MIG binding activity. [125I]MIG binding to the CHAPS-solubilized receptor is specifically affected by unlabeled glucagon. Interaction of [125I]MIG with the soluble receptor is insensitive to the presence of GTP. IC50 for glucagon using the soluble receptor was 33-70 nM, irrespective of the presence or absence of GTP, while when the membrane-bound receptor was used, the IC50 in the absence of GTP was 2-4 nM and in the presence of GTP was 35-80 nM. These data allow us to conclude that the hepatic glucagon receptor in the membrane and in the nondenaturing detergent solution is a dimer of the Mr = 63,000 hormone-binding subunit and a glycoprotein. The soluble receptor does not display any functional interaction with the stimulatory regulator.     

Purification of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase from Acanthamoeba castellanii and identification of a subunit of the enzyme.

UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) from the soil amoeba Acanthamoeba castellanii has been purified over 100,000-fold by means of wheat germ agglutinin-Sepharose affinity chromatography, DEAE-cellulose chromatography, concanavalin A-Sepharose affinity chromatography, orange A-agarose dye chromatography, and gel filtration on Superose 6. The most purified enzyme has an estimated specific activity of at least 5 mumol of GlcNAc-phosphate transferred/min/mg of protein using alpha-methylmannoside as acceptor. The molecular weight of the native enzyme is approximately 250,000, as determined by gel filtration and glycerol gradients in H2O and D2O. A protein with an apparent M(r) of 97,000 in small scale preparations and its putative proteolytic fragment of 43,000 in large scale preparations co-purifies with the enzyme activity. This protein is covalently modified with GlcNAc-[32P]phosphate when the enzyme preparation is incubated with [beta-32P]UDP-GlcNAc in the absence of an acceptor substrate. The labeling of the 97(43)-kDa protein requires active enzyme and is completely inhibited by the addition of the acceptor substrate alpha-methylmannoside. The GlcNAc-[32P]phosphate transferred to the protein is not bound to serine, threonine, tyrosine, or mannose residues. The 97(43)-kDa protein with covalently bound GlcNAc-P does not serve as a kinetically competent enzyme-substrate intermediate. However, preincubation of GlcNAc-phosphotransferase with UDP-GlcNAc does result in a decrease in the Vmax of the enzyme in subsequent assays. Taken together, these data are consistent with the 97(43)-kDa protein being a subunit of GlcNAc-phosphotransferase.     

Improved purification of pyruvate decarboxylase from wheat germ. Its partial characterisation and comparison with the yeast enzyme

An improved procedure was developed for the isolation of pyruvate decarboxylase from wheat germ. Its final step, an electrophoresis of the native apoenzyme in concave pore gradient polyacrylamide gels, followed by superficial activity-staining, produced two bands of different molecular masses and chain compositions. The high-molecular-mass band occurred in low quantity and consisted of, probably eight, apparently identical chains of Mr = 33,000, as judged from sodium dodecyl sulfate electrophoreses. The low-molecular-mass band contained two types of chains with Mr alpha = 63,000-65,000 and Mr beta = 61,000-62,000. The N termini of both chains were threonine, whereas their C-terminal sequences were different: alpha, -(Val)-(Ser)-(Ala)-Leu; beta, -(His)-(Asp)-(Ala)-Ser. Their amino acid composition was too different to be compatible with our original concept of one chain being produced from the other by proteolytic shortening. Limited proteolysis by Staphylococcus aureus V8 proteinase yielded peptides partly identical size and partly quite different. In all properties investigated, the low-molecular-mass enzyme largely resembled yeast pyruvate decarboxylase; the holoenzyme appeared to possess (alpha beta)2 structure, the apoenzyme alpha beta. SH reagents inactivated the enzyme. Binding and fluorescence of 2-p-toluidinonaphthalene-6-sulfonate indicated a similar lipophilicity of the active site as found earlier for the yeast enzyme. 2-Hydroxy-5-nitrobenzyl modification of exposed tryptophan residues left the holoenzyme intact, but in the apoenzyme it destroyed most of the cofactor-binding ability and hence the activity. The strength of cofactor binding and the maximal specific activity were found somewhat lower than in yeast pyruvate decarboxylase.     

The identification and characterization of two separate carboxylesterases in guinea-pig serum.

The esterase activity of guinea-pig serum was investigated. A 3-fold purification was achieved by removing the serum albumin by Blue Sepharose CL-6B affinity chromatography. The partially purified enzyme preparation had carboxylesterase and cholinesterase activities of 1.0 and 0.22 mumol of substrate/min per mg of protein respectively. The esterases were labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated electrophoretically on sodium dodecyl sulphate/polyacrylamide gels. Two main labelled bands were detected: band I had Mr 80 000 and bound 18-19 pmol of [3H]DiPF/mg of protein, and band II had Mr 58 000 and bound 7 pmol of [3H]DiPF/mg of protein. Bis-p-nitrophenyl phosphate (a selective inhibitor of carboxylesterase) inhibited most of the labelling of bands I and II. The residual labelling (8%) of band I but not band II (4%) was removed by preincubation of partially purified enzyme preparation with neostigmine (a selective inhibitor of cholinesterase). Paraoxon totally prevented the [3H]DiPF labelling of the partially purified enzyme preparation. Isoelectrofocusing of [3H]DiPF-labelled and uninhibited partially purified enzyme preparation revealed that there were at least two separate carboxylesterases, which had pI3.9 and pI6.2, a cholinesterase enzyme (pI4.3) and an unidentified protein that reacts with [3H]DiPF and has a pI5.0. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these enzymes showed that the carboxylesterase enzymes at pI3.9 and pI6.2 corresponded to the 80 000-Mr subunit (band I) and 58 000-Mr subunit (band II). The cholinesterase enzyme was also composed of 80 000-Mr subunits (i.e. the residual labelling in band I after bis-p-nitrophenyl phosphate treatment). The unidentified protein at pI5.0 corresponded to the residual labelling in band II (Mr 58 000), which was insensitive to neostigmine and bis-p-nitrophenyl phosphate. These studies show that the carboxylesterase activity of guinea-pig serum is the result of at least two separate and distinct enzymes.     

Isolation and characterization of cDNAs encoding imidazoleglycerolphosphate dehydratase from Arabidopsis thaliana.

cDNA clones encoding imidazoleglycerolphosphate dehydratase (IGPD; EC 4.2.1.19) from Arabidopsis thaliana were isolated by complementation of a bacterial auxotroph. The predicted primary translation product shared significant identity with the corresponding sequences from bacteria and fungi. As in yeast, the plant enzyme is monofunctional, lacking the histidinol phosphatase activity present in the Escherichia coli protein. IGPD mRNA was present in major organs at all developmental stages assayed. The Arabidopsis genome appears to contain two genes encoding this enzyme, based on DNA gel blot and polymerase chain reaction analysis.     

The iron-sulfur cluster in the L-serine dehydratase TdcG from Escherichia coli is required for enzyme activity

The anaerobically inducible L-serine dehydratase, TdcG, from Escherichia coli was characterized. Based on UV-visible spectroscopy, iron and labile sulfide analyses, the homodimeric enzyme is proposed to have two oxygen-labile [4Fe-4S]2+ clusters. Anaerobically isolated dimeric TdcG had a kcat of 544 s(-1) and an apparent KM for L-serine of 4.8 mM. L-threonine did not act as a substrate for the enzyme. Exposure of the active enzyme to air resulted in disappearance of the broad absorption band at 400-420 nm, indicating a loss of the [4Fe-4S]2+ cluster. A concomitant loss of dehydratase activity was demonstrated, indicating that integrity of the [4Fe-4S]2+ cluster is essential for enzyme activity.     

Decrease of Clonidine Binding Affinity to α2-Adrenoceptor by ADP-Ribosylation of 41,000-Dalton Proteins in Rat Cerebral Cortical Membranes by Islet-Activating Protein

The IC50 value for inhibition of specific [3H]yohimbine binding to rat cerebral cortical membranes by clonidine was increased, and the Hill coefficient (nH) approached unity in the presence of 150 microM GTP. Pretreatment of membranes with islet-activating protein (IAP) in the presence of NAD caused an increase in IC50 and nH values for clonidine compared with control membranes in the absence of GTP, the addition of which was without effect. Scatchard analysis showed that the Bmax value of the high-affinity component in [3H]clonidine binding was decreased by pretreatment with IAP/NAD. GTP in a concentration range of 0.1 microM-1 mM caused a significant elevation of [3H]yohimbine binding. In IAP/NAD-pretreated membranes, however, [3H]yohimbine binding was no longer affected by GTP, although IAP/NAD significantly (p less than 0.01) increased [3H]yohimbine binding compared to control. IAP ADP-ribosylated 41,000 dalton proteins of cerebral cortical membranes. From these results, it can be suggested that inhibitory guanine nucleotide regulatory protein with Mr 41,000 couples to alpha 2-adrenoceptors to regulate binding affinity of agonists and antagonists in membranes of the rat cerebral cortex.     

Phosphoglycerate mutase from wheat germ: studies with 18O-labeled substrate, investigations of the phosphatase and phosphoryl transfer activities, and evidence for a phosphoryl-enzyme intermediate.

From studies using unlabeled phospho-D-glycerate in solutions enriched in H2(18)O, and from experiments involving [18O]phospho-D-glycerate, it is shown that the intramolecular isomerization of 2- and 3-phospho-D-glycerate that is catalyzed by the phosphoglycerate mutase from wheat germ does not involve an intermediate 2,3-cyclic phosphate. It is also shown that phosphoglycerate mutase catalyzes the hydrolysis of the substrate analogues 2-phosphoglycolate, 2-phospho-D-lactate, 3-phosphohydroxypropionate, phosphoenolpyruvate, and phosphohydroxypyruvate. The substrates 3- and 2-phospho-D-glycerate are not hydrolyzed, nor are 2,3-bisphospho-D-glycerate, 2-phospho-L-lactate, 3-phospho-L-glycerate, or sn-glycerol 3-phosphate. Although no exchange of D-[14C]glycerate into phospho-D-glycerate can be detected, the enzyme catalyzes the transfer of the phosphoryl group from "unnatural" donors such as 2-phosphoglycolate, to the "natural" acceptor, D-glycerate. It is concluded that the intramolecular phosphoryl transfer catalyzed by the wheat germ phosphoglycerate mutase follows a pathway involving a phosphoryl-enzyme intermediate.     

Covalent cross-linking of prostaglandin E receptor from bovine adrenal medulla with a pertussis toxin-insensitive guanine nucleotide-binding protein

Prostaglandin E2 (PGE2) specifically bound to 100,000 X g pellet prepared from bovine adrenal medulla, and [3H]PGE2-bound proteins were solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The dissociation of bound [3H]PGE2 from the proteins was enhanced by GTP. [3H]PGE2-specifically bound proteins were adsorbed onto a wheat germ agglutinin column and GTP treatment decreased the amount of [3H]PGE2 retained on the column. When [3H]PGE2-bound proteins were cross-linked in the membrane by dithiobis(succinimidyl propionate) and solubilized, bound [3H]PGE2 was no longer dissociated by GTP treatment, suggesting that cross-linking produced a stable and high-affinity complex of PGE receptor with a GTP-binding protein. Covalent cross-linking of the complex was attested by adsorption of dithiobis(succinimidyl propionate)-treated [3H]PGE2-bound proteins to GTP-Sepharose, and co-elution of [35S]guanosine 5'-O-(3-thiotriphosphate) binding activity and immunoreactivities of alpha o and beta subunits of a GTP-binding protein. The cross-linked [3H]PGE2-bound complex was eluted as an apparently single radioactive peak at the position of Mr = 200,000 by gel filtration. These results have demonstrated that PGE receptor is a glycoprotein with an approximate Mr of 110,000, assuming that the Mr of the GTP-binding protein is 90,000. PGE2 neither activated nor inhibited adenylate cyclase activity, and pertussis toxin (islet-activating protein) did not affect PGE2 binding and its GTP sensitivity. These results suggest that the PGE receptor may be functionally associated with a pertussis toxin-insensitive GTP-binding protein and is not coupled to the adenylate cyclase system in bovine adrenal medulla.     

Purification and properties of phosphofructokinase 2/fructose 2,6-bisphosphatase from chicken liver and from pigeon muscle

Phosphofructokinase 2 and fructose 2,6-bisphosphatase extracted from either chicken liver or pigeon muscle co-purified up to homogeneity. The two homogeneous proteins were found to be dimers of relative molecular mass (Mr) close to 110,000 with subunits of Mr 54,000 for the chicken liver enzyme and 53,000 for the pigeon muscle enzyme. The latter also contained a minor constituent of Mr 54,000. Incubation of the chicken liver enzyme with the catalytic subunit of cyclic-AMP-dependent protein kinase in the presence of [gamma-32P]ATP resulted in the incorporation of about 0.8 mol phosphate/mol enzyme. Under similar conditions, the pigeon muscle enzyme was phosphorylated to an extent of only 0.05 mol phosphate/mol enzyme and all the incorporated phosphate was found in the minor 54,000-Mr constituent. The maximal activity of the native avian liver phosphofructokinase 2 was little affected by changes of pH between 6 and 10. Its phosphorylation by cyclic-AMP-dependent protein kinase resulted in a more than 90% inactivation at pH values below 7.5 and in no or little change in activity at pH 10. Intermediary values of inactivation were observed at pH values between 8 and 10. Muscle phosphofructokinase 2 had little activity at pH below 7 and was maximally active at pH 10. Its partial phosphorylation resulted in a further 25% decrease of its already low activity measured at pH 7.1 and in a negligible inactivation at pH 8.5. Phosphoenolpyruvate and citrate inhibited phosphofructokinase 2 from both origins non-competitively. The muscle enzyme and the phosphorylated liver enzyme displayed much more affinity for these inhibitors than the native liver enzyme. Fructose 2,6-bisphosphatase from both sources had about the same specific activity but only the chicken liver enzyme was activated about twofold upon incubation with ATP and cyclic-AMP-dependent protein kinase. All enzyme forms were inhibited by fructose 6-phosphate and this inhibition was released by inorganic phosphate and by glycerol 3-phosphate. Both liver and muscle fructose 2,6-bisphosphatases formed a 32P-labeled enzyme intermediate when incubated in the presence of fructose 2,6-[2-32P]bisphosphate.