Inhibition of glucagon-induced glycogenolysis in isolated rat hepatocytes by the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate

The ability of the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp cAMPS) to inhibit glucagon-induced glycogenolysis was studied in hepatocytes isolated from fed rats. Preincubation of the cells for 20 min with progressively higher concentrations of Rp cAMPS followed by a 1 X 10(-9) M glucagon challenge resulted in a 50% inhibition of glucose production over a 30-min period at 2-3 X 10(-6) M Rp cAMPS. A maximal inhibition of 50-74% was achieved, the actual value depending upon the length of preincubation with Rp cAMPS. The inhibitory effect did not increase when the concentration of Rp cAMPS was increased from 3 X 10(-6) to 3 X 10(-4) M. Addition of 1 X 10(-5) M Rp cAMPS to the cells followed by 10(-11) to 10(-6) M glucagon shifted the glucagon concentration required for half-maximal glucose production measured at 10 min to 6-fold higher glucagon concentrations and the concentration of glucagon required for apparent maximal glucose production measured at 10 min to greater than 10-fold higher glucagon concentrations. The cAMP-dependent protein kinase activation curve was similarly shifted to higher concentrations of glucagon. These data show that Rp cAMPS acts as a cAMP antagonist capable of opposing the glucagon-induced activation of cAMP-dependent protein kinase and the concomitant activation of the glycogenolytic cascade.     

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.     

N-hydroxy-2-acetylaminofluorene as effective inhibitor of aldehyde oxidase

N-Hydroxy-2-acetylaminofluorene has been found to be an effective inhibitor of aldehyde oxidase. At concentrations of 1 X 10(-6) M and 1 X 10(-5) M, 38% and 88% inhibition was observed on the oxidase activity towards N1-methylnicotinamide. The inhibition was of noncompetitive type and had a Ki value of 4.4 X 10(-6) M. In contrast, little inhibition of the enzyme was observed with 2-aminofluorene, 2-acetylaminofluorene and acetohydroxamic acid even at a concentration of 1 X 10(-4) M.     

Cooperativity in the dissociation of nitric oxide from hemoglobin.

The dissociation of nitric oxide from hemoglobin, from isolated subunits of hemoglobin, and from myoglobin has been studied using dithionite to remove free nitric oxide. The reduction of nitric oxide by dithionite has a rate of 1.4 X 10(3) M-1 S-1 at 20 degrees in 0.05 M phosphate, pH 7.0, which is small compared with the rate of recombination of hemoglobin with nitric oxide (25 X 10(6) M-1 S-1 (Cassoly, R., and Gibson, Q. H. (1975) J. Mol. Biol. 91, 301-313). The rate of NO combination with chains and myoglobin was found to be 24 X 10(6) M-1 S-1 and 17 X 10(6) M-1 S-1, respectively. Hence, the observed progress curve of the dissociation of nitric oxide is dependent upon the dithionite concentration and the total heme concentration. Addition of excess carbon monoxide to the dissociation mixture reduces the free heme yielding a single exponential process for chains and for myoglobin which is dithionite and heme concentration independent over a wide range of concentrations. The rates of dissociation of nitric oxide from alpha chains, from beta chains, and from myoglobin are 4.6 X 10(-5) S-1, 2.2 X 10(-5) S-1, and 1.2 X 10(4) S-1, respectively, both in the presence and in the absence of carbon monoxide at 20 degrees in 0.05 M phosphate, pH 7.0. Analogous heme and dithionite concentration dependence is found for the dissociation of nitric oxide from tetrameric hemoglobin. The reaction is cooperative, the intrinsic rate constants for the dissociation of the 1st and 4th molecules of NO differing about 100-fold. With hemoglobin, replacement of NO by CO at neutral pH is biphasic in phosphate buffers. The rate of the slow phase is 1 X 10(-5) S-1 and is independent of pH. The amplitude of the fast phase increases with lowering of pH. By analogy with the treatment of the HbCO + NO reaction given by Salhany et al. (Salhany, J.M., Ogawa, S., and Shulman, R.G. (1975) Biochemistry 14, 2180-2190), the fast phase is attributed to the dissociation of NO from T state molecules and the slow phase to dissociation from R state molecules. Analysis of the data gives a pH-independent value of 0.01 for the allosteric constant c (c = Kr/Kt where Kr and Kt are the dissociation constants for NO from the R and T states, respectively) and pH-dependent values of L (2.5 X 10(7) at pH 7 in 0.05 M phosphate buffer). The value of c is considerably greater than that for O2 and CO. Studies of the difference spectrum induced in the Soret region by inositol hexaphosphate are also reported. This spectrum does not arise directly from the change of conformation between R and T states. The results show that if the equilibrium binding curve for NO could be determined experimentally, it would show cooperativity with Hill's n at 50% saturation of about 1.6.     

Inhibition of mRNA synthesis in rabbit bone marrow nuclei in vitro by quinone metabolites of benzene

mRNA synthesis by rabbit bone marrow nuclei has been shown to be inhibited by the quinone metabolites of benzene, hydroquinone and p-benzoquinone, in a concentration-dependent manner with 50% inhibitory concentration (IC50[M]) for both compounds of 6 X 10(-6) M. Catechol and 1,2,4-benzenetriol also showed a concentration-dependent inhibition of synthesis, however, 50% inhibition was not reached by 10(-4) M. Phenol did not inhibit mRNA synthesis even at 10(-3) M. It is possible that myelotoxicity from benzene might result from such an inhibition of mRNA synthesis by quinone metabolites in pluripotent and/or committed bone marrow stem cells.     

Decreased interaction of fibronectin, type IV collagen, and heparin due to nonenzymatic glycation. Implications for diabetes mellitus

The nonenzymatic glycation of basement membrane proteins, such as fibronectin and type IV collagen, occurs in diabetes mellitus. These proteins are nonenzymatically glycated in vivo and can also be nonenzymatically glycated in vitro. After 12 days of incubation at 37 degrees C with 500 mM glucose, purified samples of human plasma fibronectin and native type IV collagen showed a 13.0- and 4.2-fold increase, respectively, in glycated amino acid levels in comparison to control samples incubated in the absence of glucose. Gelatin (denatured calfskin collagen) was glycated 22.3-fold under the same conditions. Scatchard analyses were performed on the binding of radiolabeled fibronectin to gelatin or type IV collagen. It was found that there is a 3-fold reduction in the affinity of fibronectin to type IV collagen due to the nonenzymatic glycation of fibronectin. The dissociation constant (KD) for the binding of control fibronectin to type IV collagen was 9.6 X 10(-7) M while the KD for glycated fibronectin and type IV collagen was 2.9 X 10(-6) M. This was similar to the 2.7-fold reduction in the affinity of fibronectin for gelatin found as a result of the nonenzymatic glycation of fibronectin (KD of 4.5 X 10(-7) M for the interaction of control fibronectin with gelatin vs. KD of 1.2 X 10(-6) M for the interaction of nonenzymatically glycated fibronectin with gelatin). The molecular association of control fibronectin or its glycated counterpart with [3H]heparin was also determined. Scatchard analyses of this interaction showed no difference between control fibronectin and glycated fibronectin in [3H]heparin binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of 17 beta-hydroxysteroid dehydrogenase activity in human endometrium by adrenal androgens

The effect of dehydroepiandrosterone sulphate (DHA-S) and its metabolites dehydroepiandrosterone (DHA) and 5-androstene-3 beta, 17 beta-diol (ADIOL) on the activity of 17 beta-hydroxysteroid dehydrogenase in human endometrial tissue was investigated by an isotope ratio technique. The apparent KM for oestradiol was 1.59 X 10(-6) M. All three androgens inhibited the metabolism of oestradiol and the apparent Ki values were: ADIOL, 2.05 X 10(-6) M; DHA-S and DHA, 1.59 X 10(-6) M. However, ADIOL acted by direct competition with oestradiol for the active enzyme site whereas inhibition by DHA and its sulphate was non-competitive. DHA-S and DHA were more potent inhibitors of oestradiol metabolism than was ADIOL. These results support the hypothesis that adrenal androgens could be involved in the development of endometrial hyperplasia and adenocarcinoma. Inhibition of oestradiol metabolism could increase the concentration of oestradiol in endometrial tissue and if unopposed by progesterone, e.g. after the menopause or in subjects with ovulatory defects, could stimulate abnormal endometrial growth.     

Modulation of Ca2+- and voltage-activated K+ channels by internal Mg2+ in salivary acinar cells

High-conductance K+ channels are known to be activated by internal Ca2+ and membrane depolarization. The effects of changes in internal Mg2+ concentration have now been investigated in patch-clamp single-channel current experiments on excised membrane fragments from mouse acinar cells. It is shown that Mg2+ in the concentration range 10(-6)-10(-3) M evokes a dose-dependent K+ channel activation at a constant Ca2+ concentration of 10(-8) M. The demonstration that changes in [Mg2+]i between 2.5 X 10(-4) and 1.13 X 10(-3) M has effects on the channel open-state probability indicates that fluctuations in [Mg2+]i in intact cells may influence the control of channel opening.     

Studies on rat liver argininosuccinate synthetase. Inhibition by various amino acids.

Inhibition studies of crystallized rat liver argininosuccinate synthetase [EC 6.3.4.5] are described. 1. L-Argininosuccinate, L-histidine, and L-tryptophan inhibited the enzyme activity at saturating amounts of the substrates. 2. L-Norvaline, L-argininosuccinate, L-arginine, L-isoleucine, and L-valine competitively inhibited the enzyme activity at a low concentration of L-citrulline, with Ki values of 1.3 x 10(4) M, 2.5 X 10(-4) M, 6.7 X 10(-4) M, 6.3 X 10(-4) M, and 6.0 x 10(-4) M, respectively. 3. L-Argininosuccinate and L-arginine competitively inhibited the enzyme activity at a low concentration of L-aspartate, with Ki values of 9.5 x 10(-4) M and 1.2 x 10(-3) M, respectively. 4. The modes of inhibition by L-histidine were mixed-noncompetitive, uncompetitive, and noncompetitive types with respect to L-citrulline, L-aspartate, and ATP, respectively. 5. When the enzyme was preincubated with L-citrulline, the enzyme activity was slightly increased in the presence of a low concentration of L-histidine in the assay mixture. 6. The conformation of the enzyme was markedly changed by the addition of L-histidine as judged from the CD spectrum. This change was partially reversed by incubation with L-citrulline.     

Reversible inhibition of microtubules and cell growth by the bicyclic colchicine analogue MTC

2-methoxy-5-(2,3,4-trimethoxyphenyl) 2,4,6-cycloheptatrien-1-one (MTC) is a synthetic colchicine analogue, lacking the B ring of the alkaloid (Fitzgerald: Biochem. Pharmacol. 25:1381-1387, 1976). MTC has been shown to bind reversibly to the colchicine binding site of tubulin and to inhibit microtubule assembly in vitro (Andreu et al: Biochemistry 23:1742-1752, 1984; Bane et al: J. Biol. Chem. 259:7391-7398, 1984). Its action on different cultured cell lines (PtK2, Pk15, and SV-3T3) has now been studied. 0.2 X 10(-6) M MTC stopped Pk15 and SV-3T3 cell growth, inducing an accumulation of mitoses in a few hours. Removal of MTC from the culture medium rapidly restored normal mitotic index and growth rates. Partial depolymerization of the cytoplasmic microtubules of PtK2 cells was observed at concentrations ranging from 2 to 5 X 10(-7) M. Maximal microtubule network depolymerization was obtained after 4 h of treatment with 2 to 5 X 10(-6) M MTC or at a higher MTC concentration (2 X 10(-5) M) for less than 2 h. Removal of 2 X 10(-5) M MTC (the highest MTC concentration used) from the culture medium resulted in almost complete microtubule polymerization after 10 min of drug recovery and a normal microtubule network in 20-30 min. MTC constitutes an antimitotic drug directed to the colchicine site. It is water-soluble, shows a fast and reversible action, and may therefore be employed as a convenient tool to study cellular microtubule-dependent functions.     

Characterization of monoclonal antibodies to the beta-adrenergic antagonist alprenolol as models of the receptor binding site

Antibodies to receptor ligands have been valuable in understanding the nature of receptor-ligand interactions. We have developed four monoclonal antibodies to the beta-adrenergic receptor antagonist alprenolol by immunizing A/J mice with (-)-alprenolol coupled to keyhole limpet hemocyanin. The antisera from these mice displayed specific [3H]dihydroalprenolol ([3H]DHA) binding that was inhibited by alprenolol, propranolol, and isoproterenol. Somatic cell fusion of spleen cells from the immunized mice to SP2/0 myeloma cells, followed by limited dilution subcloning, resulted in the isolation of four hybridomas (1B7, 5B7, 5D9, and 2G9) demonstrating three different classes of ligand binding characteristics. 1B7 had the highest binding affinity for antagonists based on Scatchard analysis (Kd [125I]- CYP = 1.4 X 10(-10) M; Kd [3H]DHA = 6.5 X 10(-9) M), and was the only antibody to demonstrate agonist-inhibition of [3H]DHA binding. Ki values computed from competitive inhibition curves of [3H]DHA binding to 1B7 resulted in a rank order of potency similar to that of beta-2-adrenergic receptors: (-)-propranolol greater than acebutolol amine greater than isoproterenol greater than (+)-propranolol greater than epinephrine greater than norepinephrine. 5B7 and 5D9 exemplified a second class of antibody. This pair had lower antagonist binding affinities (Kd [3H]DHA = 2 X 10(-8) M and 2.5 X 10(-7) M, respectively) and was stereoselective in binding receptor antagonists: (-)-propranolol greater than (+)-propranolol greater than acebutolol amine. Agonist inhibition of [3H]DHA binding to these antibodies could only be observed at very high concentrations (greater than 10(-4) M agonist), and was not dose-dependent. Finally, the class of anti-alprenolol monoclonal antibodies represented by 2G9 had the lowest antagonist binding affinity of all (IC50 alprenolol = 1 X 10(-5) M), did not demonstrate ligand stereoselectivity, and did not recognize agonists. We propose that antibodies raised against beta-adrenergic receptor ligands demonstrating stereoselective agonist binding will also demonstrate high affinity antagonist binding, and that they will closely parallel the binding characteristics of the receptor. According to this "agonist best-fit hypothesis," anti-idiotypic antibodies raised against the binding site of these idiotypes might contain true mirror images of the beta-adrenergic receptor binding site.     

Bioproduction of rubratoxin in a glucose-mineral salts broth with nutritional supplements and metabolic inhibitors.

A sterile glucose-salts broth fortified with various metabolic inhibitors and nutritional supplements was inoculated with conidia of Penicillium rubrum P3290, and incubated quiescently at 28 degrees C for 14 days. Potassium sulfite and sodium metabisulfite at all test concentrations caused moderate reduction in rubratoxin formation; at high concentrations (greater than or equal to 2.7 X 10(-2)M) accumulation of fungal tissue was also retarded. Production of rubratoxin and cell mass was inhibited by p-aminobenzoic acid; syntheses of toxin were completely blocked by 7.5 X 10(-2)M of the vitamin. Effects of sodium fluoride on P. rubrum cultures grown on inorganic nitrogen sources varied from inhibition of mold growth and (or) rubratoxin A production to reduction in formation of rubratoxin B. With organic nitrogen sources, fluoride caused a 30 and 60% reduction in synthesis of rubratoxins A and B, respectively. Sodium acetate at all test concentrations enhanced formation of rubratoxin; mold growth was enhanced when acetate concentration was larger than or equal to 6.0 X 10(-2)M. A moderate reduction in mold growth was caused by lower acetate concentrations (1.2 X 10(-2)M or 2.4 X 10(-2)M). Sodium arsenite and iodoacetate at test concentrations blocked mold growth and toxin formation; sodium azide and 2,4-dinitrophenol caused a marked reduction in mold growth but inhibited toxin formation completely. However, sodium azide permitted slight growth and toxin formation when mold cultures were incubated for 28 days.     

Reduction of extracellular dehydroascorbic acid by K562 cells

K562 erythroleukaemic cells produced ascorbate when incubated with dehydroascorbic acid. The reduction depended on the number of cells and on the concentration of dehydroascorbic acid. The observed rate consists of a high affinity (apparent) K_m 7 μM , V_max 3·25 pmol min^?1 (10⁶ cells)^?1 and a low affinity component, which was non-saturable up to 1 mM of DHA (rate increase of 0·1 pmol min^?1 (10⁶ cells)^?1 (1 μM of DHA^?1). The rate was dependent on temperature and was stimulated by glucose and inhibited by phloretin, N-ethylmaleimide, parachloro-mercuribenzoate and thenoyltrifluoroacetone. Although uptake of DHA proceeded at a higher rate than its extracellular reduction, the generation of extracellular ascorbate from DHA cannot be accounted for by intracellular reduction and the release of ascorbate, since the latter was not linear with time and had an initial rate of approximately 3 pmol min^?1 (10⁶ cells^?1). At a concentration of DHA of 100 μM this is 25 per cent of the observed reduction.     

Prostaglandin E1 binding protein on the surface of primary cultured hepatocytes

Prostaglandin E1 (PGE1) was bound to primary cultured rat hepatocytes in a receptor-dependent manner in serum-free medium at 4 degrees C. When added at a concentration of 2 X 10(-9) M, maximal specific binding occurred within 60-90 min. Trypsin treatment of the cells reduced the binding capacity to about 50% of that of untreated cells. Scatchard-analysis of the binding data showed that the cells had an apparent dissociation constant of 1.2 X 10(-8) M and a binding capacity of 580 fmol (approximately 3.5 X 10(11) PGE1 receptors)/mg of protein. In experiments at 37 degrees C, maximal specific binding occurred within 5 min and was 6-7 times that at 4 degrees C, but the amount of bound PGE1 decreased rapidly after 5 min due to metabolism of PGE1 in the hepatocytes. Thin-layer chromatographic analysis showed that the material bound to the cell surface consisted of intact PGE1 and its metabolites at 37 degrees C, but PGE1 only at 4 degrees C.     

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.     

Production of docosahexaenoic acid by Thraustochytrium aureum

Summary Thraustochytrium aureum ATCC 34 304 contained approximately 50% of total fatty acids as docosahexaenoic acid (DHA). Lipid content of the biomass was dramatically influenced by medium composition and ranged from 0.3 to 16% of the biomass weight. Increasing the culture medium glucose concentration from 5 to 20 g/l caused the lipid content of the biomass to increase from 2.7 to 16.5% and the DHA yield to increase from 26 to 270 mg/l of whole culture broth. The proportion of DHA in lipids was independent of glucose concentration. The fatty acid profile observed in the high-yielding DHA culture was: 16:0, 19.2%; 18:1, 9.8%; 18:2, 2.4%; 20:4, 4.9%; 20:5, 3.6%; 22:6, 48.5% and others, 7.1%. For DHA production, fungal cultures were incubated on an orbital shaker under light at 25° C for 6 days. Offprint requests to: O. P. Ward     

The p-nitrophenyl phosphatase activity of ubiquitin from bovine erythrocytes

Ubiquitin, a unique protein with esterase and carbonic anhydrase activity, has been found to have also a p-nitrophenyl phosphatase activity. This phosphomonoesterase activity of ubiquitin has an acidic pH optimum; its true substrate appears to be the phosphomonoanion, with a Km of 1.8 X 10(-3) M. It is competitively inhibited by the typical acid phosphatase inhibitors, arsenate (Ki = 1.3 X 10(-3) M), molybdate (Ki = 1.2 X 10(-6) M), and phosphate (Ki = 1.4 X 10(-3) M). These inhibitors have no effect on the CO2 hydration and p-nitrophenyl acetate esterase activities of the ubiquitin. Acetazolamide slightly inhibited the p-nitrophenyl phosphatase activity.     

Hormonal regulation of prolactin release by human prolactinoma cells cultured in serum-free conditions

Thyrotropin-releasing hormone (TRH) stimulates the prolactin (PRL) release from normal lactotrophs or tumoral cell line GH3. This effect is not observed in many patients with PRL-secreting tumors. We examined in vitro the PRL response to TRH on cultured human PRL-secreting tumor cells (n = 10) maintained on an extracellular matrix in a minimum medium (DME + insulin, transferrin, selenium). Addition of 10(-8) M TRH to 4 X 10(4) cells produced either no stimulation of PRL release (n = 6) or a mild PRL rise of 32 +/- (SE) 11% (n = 4) when measured 1, 2 and 24 h after TRH addition. When tumor cells were preincubated for 24 h with 5 X 10(-11) M bromocriptine, a 47 +/- 4% inhibition of PRL release was obtained. When TRH (10(-8) M) was added, 24 h after bromocriptine, it produced a 85 +/- 25% increase of PRL release (n = 8). This stimulation of PRL release was evident when measured 1 h after TRH addition and persisted for 48 h. The half maximal stimulatory effect of TRH was 2 X 10(-10) M and the maximal effect was achieved at 10(-9) M TRH. When tumor cells were pretreated with various concentrations of triiodothyronine (T3), the PRL release was inhibited by 50% with 5 X 10(-11) M T3 and by 80% with 10(-9) M T3. Successive addition of TRH (10(-8) M) was unable to stimulate PRL release at any concentration of T3. The addition of 10(-8) M estradiol for up to 16 days either stimulated or had no effect upon the PRL basal release according to the cases. In all cases tested (n = 4), preincubation of the tumor cells with estradiol (10(-8) M) modified the inhibition of PRL release induced by bromocriptine with a half-inhibitory concentration displaced from 3 X 10(-11) M (control) to 3 X 10(-10) M (estradiol). These data demonstrate that the absence of TRH effect observed in some human prolactinomas is not linked to the absence of TRH receptor in such tumor cells. TRH responsiveness is always restored in the presence of dopamine (DA) at appropriate concentration. This TRH/DA interaction seems specific while not observed under T3 inhibition of PRL. Furthermore, estrogens, while presenting a variable stimulatory effect upon basal PRL, antagonize the dopaminergic inhibition of PRL release.     

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.     

Fibrinogen binding to human platelet plasma membranes. Identification of two steps requiring divalent cations

Fibrinogen binding to platelet plasma membranes, which is a prerequisite for platelet aggregation, was determined by incubating 125I-labeled fibrinogen with isolated membranes and measuring the amount of radioactivity sedimenting with the membranes through 15% sucrose. Fibrinogen binding was optimal at 10(-3) M Ca2+. Scatchard analyses of the fibrinogen binding showed that the membrane capacity for fibrinogen was 1.6 X 10(-12) mol/mg of membrane protein, with a dissociation constant (Kd) = 1.2 X 10(-8) M. When Ca2+ levels were manipulated by the addition of varying amounts of EGTA at a fixed Mg2+ concentration of 3 X 10(-3) M, specific binding of fibrinogen to platelet membranes occurred only at Ca2+ concentrations greater than or equal to 10(-6) M. Membranes isolated from platelets of an individual with Glanzmann's thrombasthenia bound only 12% as much fibrinogen as control platelets. The data in the present study suggest that there are two divalent cation binding sites that must be occupied for fibrinogen to bind: one site is specific for calcium and is saturated at 10(-6) M Ca2+; the other site is less specific and is saturated at a 10(-3) M concentration of either Ca2+ or Mg2+. Fibrinogen binding to intact platelets and, consequently, platelet aggregation only required 10(-3) M extracellular divalent cation and was not specific for Ca2+. These data indicate that the cytoplasm is a potential source for the requirement of 10(-6) M Ca2+, and that changes in the intracellular concentration of Ca2+ may cause the expression of fibrinogen receptors during ADP-induced platelet activation.