Studies of the regulation and reaction mechanism of the carbamyl phosphate synthetase and aspartate transcarbamylase of bakers' yeast.

Kinetic studies of the carbamyl phosphate synthetase activity (CPSase) of bakers' yeast revealed an absolute requirement for K+ ions ; KM values for two of the substrates, glutamine and bicarbonate, were found to be 5 X 10(-4) M and 3 X 10(-3) M respectively. CPSase activity of the purified enzyme aggregate (M.W. 800,000) was extremely sensitive to UTP with a Ki of 2.4 X 10(-4) M. The purine nucleotide intermediate, XMP, was a strong activator of CPSase, acting at a site different from the regulatory site at which UTP binds ; XMP activation diminished at high concentrations of the substrate Mg-ATP. Studies of the reaction mechanism of CPSase revealed that it involved the sequential addition of the substrates bicarbonate and Mg-ATP, liberation of ADP, addition of glutamine, binding of ATP and then release of ADP and the product carbamyl phosphate. Studies of the reaction mechanism of the aspartate transcarbamylase (ATCase) of the aggregate yielded data which were not compatible with any of the usual models ; whichever reaction mechanism is ultivately found to fit the data, it will probably prove applicable both to the ATCase of the aggregate and to the disaggregated ATCase subunit (MW 138,000).     

In vitro effects of lead ions on peripheral benzodiazepine receptors and adenylyl cyclase activity in the mantle of Mytilus galloprovincialis

As an extension of our previous work, where the density of peripheral benzodiazepine receptors (PBR) increased in mantle mitochondria of the marine mollusk Mytilus galloprovincialis Lmk. under chronic exposure to lead, the present study investigates the in vitro effects of an exogenous source of lead ions on PBR and on adenylyl cyclase (AC) complex in mantle membranes of mussels collected from a non-polluted coastal area. PBR binding experiments used the specific isoquinoline carboxamide derivative [3H]PK 11195, and AC activity was measured using a modified procedure adapted to M. galloprovincialis. Lead ions (Pb2+) dose-dependently decreased either the [3H]PK 11195 specific binding in mitochondria or basal AC velocity in plasma membranes of mussel mantle. The IC50 values for lead ions were 10 microM with [3H]PK 11195 binding and 25 microM with AC activity, with maximal inhibition values of 60% and 70%, respectively. Moreover, lead behaved as a non-competitive inhibitor on [3H]PK 11195 binding and as a 'mixed' inhibitor on AC activity. The present results suggest that some of the early effects induced by lead in mussel cell metabolism consist in significant changes of the PBR density and cyclic AMP production in the mantle of M. galloprovincialis.     

Phosphorylation-activated 6-phosphofructo-2-kinase from mantle tissue of marine mussels.

PKF-2 from mussel mantle was phosphorylated by cAMP-dependent protein kinase. The phosphorylation does not change the enzyme activity at neutral pH values, but at acid pH the activity of the phosphorylated form is higher than the native PFK-2. With respect to the native enzyme, the activation consisted of a reduction in the Km for Fru-6-P and a decrease in the inhibitory effect of PEP. These results are in keeping with the stabilized concentration of Fru-2,6-P2 found in the mussel mantle during the physiological hypoxia caused by the closure of the valves.     

Purification and properties of GTP-cyclohydrolase of the yeast Pichia guilliermondii

GTP-cyclohydrolase was isolated from the Fe-deficient cells of Pichia guilliermondii and purified 440-fold by treatment of extracts with streptomycin sulfate as well as by protein fractionation with (NH4)2SO4 at 25-45% saturation, gel filtration through Sephadex G-200 and DEAE-cellulose chromatography. The curves for the dependence of specific activity of GTP-cyclohydrolase on substrate and cofactor concentrations are non-hyperbolic; the values of [S]0.5 for GTP and Mg2+ are 2.2 X 10(-5) and 2 X 10(-4) M, respectively. The enzyme activity is inhibited by pyrophosphate ([I]0.5 = 5.8 X 10(-4) M), orthophosphate ([I]0.5 = 4.5 X 10(-3) M), heavy metal ions and chelating agents. The temperature optimum for the enzyme activity lies at 42-45 degrees C. The enzyme is labile at 4 degrees C but can well be stored at -15 degrees C. The pyrimidine product of the cyclohydrolase reaction, 2.5-diamino-6-oxy-4-ribosyl-aminopyrimidine-5'-phosphate, as well as pyrophosphate were purified from the reaction medium and identified.     

Purification and properties of myo-inositol-1-phosphatase from rat brain

myo-Inositol-1-phosphatase [EC 3.1.3.25] was purified from a cytosolic fraction of rat brain. The purified enzyme appeared homogeneous on SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 29,000. The molecular weight of the native enzyme was 55,000 as determined by molecular sieve chromatography. These values indicated that the native enzyme was composed of two identical subunits. The isoelectric point of the enzyme was 4.6. The enzyme hydrolyzed inositol-1-phosphate, 2'-AMP, 2'-GMP, beta-glycerophosphate, and alpha-glycerophosphate; the ratio of the reaction rates was 100 : 84 : 73 : 64 : 32. The Km values for inositol-1-phosphate, 2'-AMP, and beta-glycerophosphate were 1.2 X 10(-4) M, 1.9 X 10(-4) M, and 7.7 X 10(-4) M, respectively. Mn2+ and Ca2+ were strong competitive inhibitors against Mg2+, with Ki values of 3 microM and 20 microM, respectively. This result suggests that myo-inositol-1-phosphatase might be regulated by intracellular Ca2+ and/or Mn2+. Li+, which is known to show a therapeutic effect on manic-depressive disease and also to prolong the intrinsic periods of circadian rhythms in various organisms, was a potent uncompetitive inhibitor and inhibited 50% of the activity at 1 mM. The possibility that myo-inositol-1-phosphatase and inositol phospholipid metabolism are involved in circadian rhythm oscillation is discussed in terms of Li actions.     

The effect of peptides on the motility of the stomach, intestine and rectum in the skate (Raja)

1. Pentagastrin (10(-8)-2 X 10(-6) M) was found to increase motor activity in the cardiac stomach and spiral intestine but only occasionally in the pyloric stomach and not at all in the rectum. 2. Substance P increased motor activity in both parts of the stomach and the rectum (10(-8)-5 X 10(-7) M) but had only a slight effect on the spiral intestine. 3. No effect on the activity of any part of the gut was seen with VIP (10(-7) M), neurotensin (2 X 10(-6) M) or bradykinin (2 X 10(-5) M). 4. The responses to pentagastrin or substance P were not abolished by TTX (10(-6) M). 5. The implications of these results for the understanding of the control of gut motility in elasmobranchs is discussed.     

Thiamine intestinal transport and phosphorylation : a study in vitro of potential inhibitors of small intestinal thiamine-pyrophosphokinase using a crude enzymatic preparation.

Using as enzymatic source the cytoplasmatic fraction of enterocytes isolated from the rat small intestine, thiamine-pyrophosphokinase activity was studied with a radiometric method using [thiazole-2-(14)C] thiamine. The Km value for thiamine was 2.14 X 10(-6) M and V 0.87 nmol of thiamine pyrophosphate mg-1 protein h-1. Eleven thiamine structural analogs and derivatives were assayed for their inhibitory action on the small intestine thiamine-pyrophosphokinase activity. Their Ki values were : pyrithiamine, 2.25 X 10(-6) M; thiamine monophosphate, 4 X 10(-6) M; 2'-ethylthiamine, 8 X 10(-6) M; 2'-butylthiamine, 6 X 10(-6) M; chloroethylthiamine and dimethalium, 1.5 X 10(-5) M; amprolium, 1.8 X 10(-4) M; L-582571, 1.65 X 10(-4) M; oxythiamine, 4.2 X 10(-3) M. Of the miscellaneous compounds tested (toxopyrimidine, Na-pyrophosphate, choline, L-phenylalanine, ethyl-urethane and 5-fluorouracil), none had any inhibitory action on intestinal thiamine-pyrophosphokinase activity, even if used at concentrations hundred times higher than that of labelled thiamine.     

The influence of the peptide chain length on the activity of peptidyl-tRNA hydrolase from E. coli.

The dependence of the Vmax and Km on the length of the peptide moiety in the peptidyl-tRNA series (Gly)n-Val tRNA, was measured in the system peptidyl-tRNA hydrolase-peptidyl-tRNA. It was found that the Km value decreases from 7.2 X 10-7 M for Gly-Val-tRNA to 4.6 X 10-7 M FOR (Gly)2-Val-tRNA and to 1.7 X 10-7M for (Gly)3-Val-tRNA; further increase of the peptide chain is not followed by decrease of the Km. The Vmax values are 5.7 pmole/min/EU for Gly-Val-tRNA and 42 pmole/min/EU for (Gly)3-Val-tRNA. The enzyme activity is inhibited competitively by uncharged tRNA with a KI value of about 10-5M. The significance of these results described in this paper, in relation to the fact that peptides and peptide esters do not inhibit the enzyme activity, and in relation to the proposed physiological role of the enzyme, is discussed.     

Purification and characterization of a catalase-peroxidase from the photosynthetic bacterium Rhodopseudomonas capsulata

Catalase-peroxidase was isolated from aerobically grown Rhodopseudomonas capsulata. The enzyme resembles typical catalases in some of its physicochemical properties. It has an apparent molecular weight of 236,000 and is composed of four identical subunits. It shows a typical high spin ferric heme spectrum with absorption maxima at 403 and 635 nm and shoulders at 503 and 535 nm. Upon binding of cyanide, the enzyme is converted to the low spin state, as shown by the shift of the Soret maximum to 418 nm and the band at 532 nm. It has an isoelectric point at pH 4.5. The enzyme differs from typical catalases in also having a strong peroxidatic activity with dianisidine, pyrogallol, and diaminobenzidine as electron donors. Both the catalatic and the peroxidatic activities are similarly inactivated by treatment with 1 mM H2O2, heating to 50 degrees C, exposure to ethanol/chloroform, and photooxidative conditions. In contrast to typical catalases, but similarly to peroxidases, the enzyme is reduced by sodium dithionite. The pH optimum of the peroxidatic activity is 5-5.3 (in contrast to 6-6.5 of the catalatic activity). 50% of the apparent maximal activities are reached at 0.3 and 4.2 mM H2O2 for the peroxidatic and catalatic activities, respectively. Both enzymic activities are equally inhibited by cyanide, 50% inhibition being achieved with 2.2 X 10(-5) M KCN. Contrarily, the two activities differ in their response to hydroxylamine and azide. 50% inhibition of the catalatic activity is obtained with 1.5 X 10(-4) M azide or 2.15 X 10(-6) M hydroxylamine; 50% inhibition of the peroxidatic activity requires 7.3 X 10(-4) M azide or 7.8 X 10(-5) M hydroxylamine. The activation energies of the catalatic and the peroxidatic activities are 1.9 and 1.7 kcal/mol, respectively.     

Purification and characterization of two components of acid alpha-glucosidase from pig liver

Acid alpha-glucosidase [EC 3.2.1.3] was purified from pig liver by a procedure including Sephadex G-100 affinity chromatography. Electrophoresis on SDS-polyacrylamide gel of the purified enzyme indicated the presence of two components with molecular weights of 73K and 64K. The two components of the enzyme were completely separated, in reasonable yield, by chromatography on a DEAE-5PW column. Both components catalyzed the hydrolysis of the alpha-1,4 and alpha-1,6 linkages of glycogen, maltose, isomaltose, dextrin, and a synthetic glucoside at acid pH. The pH optima of both components were 4.3 for maltase and glucoamylase, and 4.8 for isomaltase and dextrinase. But as to the activity on 4MU-alpha-Glc, the pH optimum of the larger component was 4.8 and that of the smaller component 5.3. The Km values of both components for 4MU-alpha-Glc, maltose, glycogen, isomaltose, and dextrin were 1.0 X 10(-4) M, 9.1 X 10(-3) M, 16.7 mg/ml, 6.7 X 10(-2) M, and 12.5 mg/ml, respectively. Erythritol, Tris, and turanose inhibited the two components competitively. The Ki values of the larger component were 5.0 X 10(-2) M, 13.3 X 10(-3) M, and 3.2 X 10(-3) M, and those of the smaller component were 2.5 X 10(-2) M, 6.1 X 10(-3) M, and 4.7 X 10(-3) M, for erythritol, Tris, and turanose, respectively.     

Calmodulin-induced activation of ATP-dependent Ca2+ transport in plasma membranes of the myometrium

Calmodulin activates the ATP-dependent transport of Ca2+. The V0 value for this reaction in the absence of calmodulin is 0.82, that in the presence of 10(-7) M calmodulin is 5 times as high, i. e. 4.5 nmol 45Ca2+/mg protein/min. The Vmax value in the absence of calmodulin is 2.07, that with the activator is 4.33 nmol 45Ca2+/mg protein/min. The corresponding Km values are 0.75 X 10(-6) M and 0.66 X 10(-7) M, respectively, i. e., the affinity of the Ca-pump for Ca2+ increases. The half-maximum Ca-binding activity of calmodulin measured with a help of the fluorescent probe, N-phenyl-1-naphthylamine (PNA), is observed at 5 X 10(-7) M Ca2+. Mg2+ (3 mM) decreases 10-fold the Ca-binding affinity. No significant effect of ATP on the Ca-binding properties of calmodulin was found; the Hill coefficient is suggestive of a positive cooperativity of this reaction. A comparison of dependences of the calmodulin-stimulated component of ATP-dependent transport of Ca2+ in myometrium plasma membranes and of the Ca-binding activity of calmodulin measured with a help of PNA suggests that the effect of calmodulin on the affinity of the Ca-pump for Ca2+ can also be realized when some (but not all) Ca-binding sites in the calmodulin molecule are saturated with Ca2+.     

Subunit interaction: A molecular basis of heterosis

Acid phosphatase, a dimeric enzyme, in Drosophila malerkotliana was studied in isogenic flies to explore the molecular basis of heterosis. As the enzyme activity in heterozygotes is 34% more than that in the better parent (S/S), heterosis is indicated. Vmax, Km, and Ki values are 14.60, 3.6 X 10(-4) M, and 0.45 X 10(-4) M, respectively, for the enzyme from F/S flies and 11.80, 4.0 X 10(-4) M, and 0.37 X 10(-4) M, respectively, for the enzyme from S/S flies. Thus heterosis for enzyme activity results from a better enzyme in F/S flies. The higher efficiency and better quality of the enzyme in F/S flies were traced to the heterodimeric allozyme, present only in heterozygotes. Enzyme activity, Vmax, Km, and Ki values are 0.739, 42.1; 3.6 X 10(-4) M, and 0.50 X 10(-4) M, respectively, for the heterodimeric and 0.513, 36.8; 4.1 X 10(-4) M, and 0.37 X 10(-4) M, respectively, for the better parental homodimeric allozyme. On an equimolar basis the enzyme activity of the heterodimer is 44% higher than that of the better homodimer. The better performance of the heterodimer is probably a reflection of superior conformation resulting from interaction between component subunits (F and S polypeptides).     

Lack of effect of somatostatin on the stimulation of hepatic glycogenolysis by epinephrine in isolated canine hepatocytes

The effects of somatostatin on epinephrine's ability to stimulate glucose output have been examined in hepatocytes isolated from dogs fasted overnight. Half-maximal stimulation of phosphorylase a activity and glucose output occurred at an epinephrine concentration of approx. 5 X 10(-9) M. Somatostatin at 10, 100 or 1000 ng/ml had no effect on the ability of a maximal (1 X 10(-7) M) and a submaximal (1 X 10(-8) M) dose of epinephrine to activate phosphorylase at 2 min, or to stimulate glucose output over 20 min. Since the doses of somatostatin used in the present study are up to 50-fold higher than the blood concentrations commonly found when somatostatin is used in vivo to inhibit pancreatic hormone secretion, it seems unlikely that use of somatostatin in this way would affect stimulation of hepatic glycogenolysis by epinephrine in vivo.     

Inhibition of mammalian aspartate transcarbamylase by quinazolinone derivatives

Quinazolinone derivatives have been studied as both in vitro and in vivo inhibitors of aspartate transcarbamylase (ATCase). In vitro treatment of mammalian ATCase with four compounds revealed that they inhibited enzyme activity and that 2-phenyl-1,3-4(H)benzothiazin-4-thione was the most potent one. This compound acts as a noncompetitive inhibitor towards both aspartate and carbamoyl phosphate. The values of the inhibition constant (K_i) indicate that this compound exerts a potent inhibitory effect upon ATCase activity. Moreover, in vivo treatment with different doses of these derivatives showed also an inhibitory effect upon ATCase, the relative activity being decreased by 40%–58% with a 1 mg dose. These data support the inhibition of ATCase by quinazolinone derivatives as a new type of inhibitor for the enzyme.     

Effect of some insecticides on the periodic activity of the fresh-water mussel (Anodonta cygnea L.).

The effect of the insecticides Hungaria L-7, Dimecron-50, Thimet-10 G and Satox 20 WSC containing lindane, phosphamidon, phorate and trichlorfon, respectively, as active ingredient was investigated on the survival and pumping behaviour of the fresh-water mussel Anodonta cygnea L. The shortening of active periods was used as indicator of the sublethal effects. Hungaria L-7, Thimet-10 G and Satox 20 WSC caused significant inhibition of the filtering activity, while Dimecron-50 was less effective. Fifty per cent shortening of active periods required 6 X 10-3 g/l Hungaria L-7, 8 X 10-3 g/l Thimet-10 G and 2 X 10-2 ml/l Satox 20 WSC. Thimet and Satox were lethal at 10-1 g/l and 1 m/l concentrations, respectively. Although the mechanism of the effect can be different with various substances, the results have shown, that monitoring of the activity of mussels can be a suitable test for the biological indications of water pollution.     

Effect of dehydrogenation/hydrogenation on the linear and nonlinear optical properties of Li@porphyrins

In the present work, Li@porphyrins and their derivatives were designed in order to explore the effect of dehydrogenation/hydrogenation on linear and nonlinear optical properties. Their stable structures were obtained by the M06-2X method. Moreover, the M06-2X method showed that dehydrogenation/hydrogenation has greatly influences polarizabilities (α (0) values) and hyperpolarizabilities (β (tot) and γ (tot) values): α (0) values ranged from 331 to 389 au, β (tot) values from 0 to 2465 au, and γ (tot) values from -21.2?×?10(4) to 21.4?×?10(4) au. This new knowledge of the effect of dehydrogenation/hydrogenation on nonlinear optical properties may prove beneficial to the design and development of high-performance porphyrin materials.     

Opiate-prostaglandin interactions in the regulation of insulin secretion from rat islets of Langerhans in vitro

The inadequate insulin secretory response to glucose stimulation in non-insulin dependent diabetes has been attributed to many factors including high PGE2 levels blunting the secretory response, and to the existence of inhibitory opiate activity in vivo. The purpose of the present work was to see if there was a connection between these two independent theories. Radioimmunoassayable PGE2 in islets of Langerhans was found to be proportional to islet number and protein content and was typically 4 to 5pg/micrograms islet protein. Indomethacin (2.8 X 10(-5) M), sodium salicylate (1.25 X 10(-3) M) and chlorpropamide (7.2 X 10(-5) M) all lowered islet PGE2 levels and stimulated insulin release in vitro. Dynorphin (1-13), stimulated insulin release at a concentration of 6 X 10(-9) M, while lowering islet PGE2. Conversely, at a higher concentration, (6 X 10(-7) M), dynorphin had no stimulatory effect on insulin secretion and did not lower PGE2 levels in islets or in the incubation media. The stimulatory effects of dynorphin and sodium salicylate on insulin secretion were blocked by exogenous PGE2 (10(-5) M). PGE2 at a lower concentration (10(-9) M) did not exert any inhibitory effect on dynorphin- or sodium salicylate-induced insulin release. This concentration of exogenous PGE2 stimulated insulin release in the presence of 6mM glucose. Results from these experiments suggest that since an opioid peptide can lower endogenous PGE2 production in islets and since the stimulatory effects of the opioid peptide are reversed by exogenous PGE2 there may be interactions between these two modulators of insulin secretion.     

Pharmacological induction of larval settlement and metamorphosis in the blue mussel Mytilus edulis L

The blue mussel Mytilus edulis L. is an important aquaculture and fouling species in northern seas. Although the general role of chemical cues for settlement of larvae of the blue mussel has been proposed, few studies have focused on induction of settlement and metamorphosis by pharmacological agents. In this study, the induction of larval settlement of the blue mussel by pharmacological compounds was investigated through a series of laboratory experiments with an aim of identifying artificial cues for laboratory bioassay systems in fouling and antifouling research. Gamma-aminobutiric acid (GABA), dihydroxyphenyl L-alanine (DOPA), isobutyl methylxanthine (IBMX) and acetylcholine chloride (ACH) at 10(-7)-10(-2) M as well as KCl at 10-40 mM K+ in excess of the level in normal seawater were tested for their inductive effect on larval settlement. In filtered seawater (FSW) < 9% of the larvae settled after 48 h. Elevated K+ and GABA levels had no effect on larval settlement and metamorphosis. DOPA at 10(-5) M and IBMX at 10(-6)-10(-4) M induced 41-83% larval settlement and ACH at 10(-7)-10(-5) M induced < 40% larval settlement. While the highest settlement rates were observed after 48 h exposure to the chemical, most of the larvae settled within 24 h. Compounds at concentrations of 10(-3)-10(-2) M were either toxic to larvae or retarded the growth of the post-larvae shell. Juveniles resulting from induction by lower concentrations of chemicals had a very high survival rate, completed metamorphosis and grew as well as the juveniles that metamorphosed spontaneously. IBMX at 10(-6)-10(-4) M and L-DOPA at 10(-5) M are effective agents for induction of settlement and metamorphosis for future studies using juvenile M. edulis.     

1-Amino-2-phenylethylphosphonic acid: an inhibitor of L-phenylalanine ammonia-lyase in vitro

L(-)-, and D(+)-enantiomers of 1-amino-2-phenylethylphosphonic acid (PheP), a phosphonic analogue of phenylalanine, inhibit the activity of L-phenylalanine ammonia-lyase (EC 4.3.1.5) of potato tuber tissue in vitro. The apparent type of inhibition depends on concentration of PheP; as the concentration of D-PheP is raised from 10(-5) M to 2.5 X 10(-3) M, the type of inhibition shifts from competitive through mixed and non-competitive to uncompetitive. L-PheP exerts either a competitive or mixed-type inhibition at low (10(-6)-10(-5) M) or moderate (5 X 10(-5)-2 X 10(-4) M) concentration. Ki for the concentration range of competitive inhibition were 6.5 X 10(-6) M, 5.3 X 10(-5)M and 1.6 X 10(-5) M for L-, D-, and D,L-PheP, respectively. These Ki values are valid for a relatively narrow range of L-Phe concentration (0.2-4 mM) as L-phenylalanine ammonia-lyase does not follow the Michaelis-Menten kinetics of the reaction.     

Pyruvate kinase in mussel foot (Mytilus edulis L.). Purification and general properties

The specific activity of pyruvate kinase in mussel foot is markedly higher than that from mantle and digestive gland. The foot enzyme shows maximum pH activity in the range between 7.0 and 7.5 and is stable (15 min, 37 degrees C) at pH values between 7.0 and 9.0. The activation energy value is 23 kJ/mol with a Q10 coefficient of 1.4. All of these experiments were carried out using partially purified extracts with (NH4)2SO4 treatment (30-60%) and posterior dialysis with EDTA 1.2 mM. No isoenzymatic forms could be detected using the column chromatography techniques with Sephadex G-150, DEAE Sephadex A-50 and polyacrylamide gel electrophoresis.