Hill Number Research Articles

Radiometric oil well assay for glucokinase in microscopic structures

Glucokinase (ATP: d-glucose 6-phosphotransferase, EC 2.7.1.1) plays a pivotal role in hepatic glucose metabolism and serves as the glucose sensor in pancreatic islet β-cells. Biochemical studies of this enzyme are complicated by the cellular heterogeneity of the liver and the pancreas and because the presence of hexokinases (ATP: d-hexose 6-phosphotransferases, EC 2.7.1.1) seriously interferes with currently available analytical procedures. A radiometric assay was designed to deal with these problems. It is based on the liberation of 3H 2O from d-[2- 3H(N)]glucose 6-phosphate, the product of the glucokinase reaction, using exogenous phosphoglucose isomerase ( d-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9). Interference by hexokinases was largely eliminated by using glucose 6-phosphate as inhibitor and the sensitivity of the assay was greatly increased by using small volumes with the oil well procedure. The assay was sufficiently sensitive to detect about 1 pg of glucokinase. It thus allowed the application of quantitative histochemical procedures to the study of intralobular hepatic glucokinase profiles and the pancreatic β-cell glucose sensor. The quantitative histochemical procedures were sufficiently sensitive and reliable for measuring important kinetic constants of glucokinase (i.e., the K m and the Hill number) in microscopic samples of tissue.

Use of the fluorescent probe 1-N-phenylnaphthylamine to study the interactions of aminoglycoside antibiotics with the outer membrane of Pseudomonas aeruginosa.

The mode of interaction of the polycationic aminoglycoside antibiotics with the surface of Pseudomonas aeruginosa cells was studied with the hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN). The addition of the aminoglycoside gentamicin to intact cells in the presence of NPN led to a shift in the fluorescence emission maximum from 460 to 420 nm. At the same time the NPN fluorescence intensity increased fourfold. Gentamicin caused no such effects when added to outer membrane vesicles, suggesting that the increased fluorescence resulted from the interaction of gentamicin with intact cells. Gentamicin-promoted NPN uptake was inhibited by the divalent cations Mg2+ and Ca2+, but occurred in the absence of gentamicin transport across the inner membrane. Low concentrations of gentamicin (2 micrograms/ml) caused NPN fluorescence to increase over a period of 4 min in a sigmoidal fashion. At higher concentrations (50 micrograms/ml) the increase occurred within a few seconds. The final fluorescence intensity was almost independent of the gentamicin concentration. A centrifugation technique was used to demonstrate that gentamicin caused actual uptake of NPN from the supernatant. The initial rate of NPN uptake varied according to the gentamicin concentration in a sigmoidal fashion. Similar data were obtained for seven other aminoglycoside antibiotics. The data, when reanalyzed as a Hill plot, gave a series of lines with a mean slope (the Hill number) of 2.26 +/- 0.26, suggesting that the interaction of aminoglycosides with the cell surface to permeabilize it to NPN involved at least three sites and demonstrated positive cooperativity. There was a statistically significant relationship between the pseudoassociation constant K, from the Hill plots and the minimal inhibitory concentrations for the eight antibiotics. These results are consistent with the concept that aminoglycosides interact as a divalent cation binding site on the P. aeruginosa outer membrane and permeabilize it to the hydrophobic prove NPN.

Purification, molecular and kinetic properties of glucosamine-6-phosphate isomerase (deaminase) from Escherichia coli

Glucosamine-6-phosphate isomerase (deaminase), (2-amino-2-deoxy- d-glucose-6-phosphate ketol isomerase (deaminating), EC 5.3.1.10) has been purified to homogeneity from Escherichia coli B as judged by several criteria of purity. The procedure included ammonium sulfate fractionation, anion-exchange chromatography and a biospecific affinity chromatography step with N-ϵ- amino-n- caproyl- d-glucosamine 6-phosphate bound to agarose as the ligand, the elution being performed with GlcNAc6 P. The enzyme appears to be an hexamer of about 178 kDa, composed of six subunits of 29 700 ± 300 Da; the isoelectric point was 6.0–6.1 and the sedimentation constant 9.0 S. The amino-acid composition of the enzyme was determined and a value for E 275 1% of 4.55 was calculated. The molecular activity was 1800 s −1 for the deamination reaction and 455 s −1 for the reaction of GlcN6 P formation. A positive homotropic cooperativity was found for both sugar substrates; it was stronger for GlcN6 P in the deamination reaction (Hill number 2.7 at pH 7.7). Ammonia behaved as a Michaelian substrate. Cooperativity was abolished by 0.1 mM GlcNAc6 P; this allosteric modulator activated the reaction in both directions, with a positive K-effect upon both sugar phosphates, but had no effect on K m for ammonia. The initial velocity patterns for the amination reaction were obtained under conditions of hyperbolic kinetics produced by GlcNAc6 P; the K m values for the allosteric substrates were determined under the same conditions, and their dependence upon pH was studied.

Nature of nicotine binding to rat brain P 2 fraction

(−)-Nicotine may bind to as many as 5 sites in the rat brain P 2 preparation: A very high affinity site (K D∼2.2×10 −11 M); a positive cooperativity site; a high affinity site (K D∼5.2×10 −9 M); a low affinity site (K D∼4.5×10 −5 M) and a very low affinity site. The curvilinear nature of both Scatchard plots and kinetic curves indicates the presence of multiple binding sites. Evidence for a positive cooperativity site includes: (1) The configuration of Scatchard plots (at low concentrations) of saturation as well as inhibition curves for (−)- and (+)-nicotine. (2) The Hill number of 1.37 for the binding of low concentrations of (±)-[ 3 H]nicotine . (3) Selectivity among cholinergic drugs for producing positive cooperativity. (4) Markedly different specificities of drugs for the positive cooperativity site. Thus while only (+)- and (−)-nicotine interacted with the very high affinity site, acetylcholine, atropine, mecamylamine, lobeline, carbachol, (+)-nicotine and (−)-nicotine enhanced the binding of (±)-[ 3 H]nicotine and cytisine, anabasine, cotinine and choline selectively inhibited binding at the high affinity site. Several lines of evidence indicate that there is stereospecificity. (+)-Nicotine was more potent than (−)-nicotine in inducing positive cooperativity whereas (−)-nicotine was 80 times more potent than (+)-nicotine in inhibiting binding at the high affinity site. Further, the specificity of the binding sites can be altered by changing the concentration of the buffer which gives additional evidence for the lability of the nicotine binding site. Although the pharmacologic significance of the different binding sites has not been determined, these data taken together indicate that (±)-[ 3 H]nicotine binds with specificity to multiple sites in the rat brain P 2 preparation with a complexity not addressed heretofore.

Courbe de dissociation de l'hémoglobine et 2,3-diphosphoglycérate intraérythrocytaire dans le choc septique

The oxyhaemoglobin dissociation curve (OHDC) and intraerythrocytic 2,3-diphosphoglycerate (2,3-DPG) play an important role in oxygen delivery, especially during shock. 62 determinations in eight septic shocks were carried out. Hem.O.Scan® drew the OHDC at standard temperature and P co 2; P 50 was modified for pH 7.40 (P 50 std) by algorithm. When the patients had recovered from the shock, their average P 50 std was higher than during the shock (p < 0.02), and much more so than in normal subjects (p < 0.01). There was a positive correlation between P 50 std and 2,3-DPG, arterial blood pH, haemoglobin and cardiac index, as well as between 2,3-DPG and arterial blood pH. On the other hand, there existed no correlation between P 50 std or 2,3-DPG, and Pa o 2, Pa co 2 and phosphoremia. The Hill number was normal.

Purification and properties of NADP‐dependent glutamate dehydrogenase from Sphaerostilbe repens

NADP‐dependent glutamate dehydrogenase (EC 1.4.1.4) extracted from Sphaerostilbe repens was purified to homogeneity by using ammonium sullate fractionation hydroxyapatite and DEAE‐cellulose column chromatography and, finally, preparative polyacrylamide gel electrophoresis. The turnover number of the enzyme for the amination reaction was about 66000 mol substrate transformed min‐1 (molecule of GDH)‐1. Molecular weight of the native enzyme was estimated to be 280000 dalton by polyacrylamide gradient gel electrophoresis. The same technique in the presence of sodium dodecyl sulfatc gave a single protein band that corresponded to the subunit molecular weight of 48000 dalton. Thus, it is concluded that NADP‐GDH is composed of six identical polypeptidic chains.The pH optimums were 6.9 and 8.4 for the forward and reverse reactions respectively. The NADP‐GDH lost practically none of its activity for ten days at 4°C and for 15 h at room temperature, but was inactivated by higher temperatures. Thiol compounds such as 2‐mercaptoethanol and dithiolhrcitol protected the enzyme from rapid inactivation. The Michaelis constants for GDH were 0.64, 0.049. 0.043 and 5.5 mM for α‐ketoglutaratc. NADPH, NADP and glutamate, respectively. The enzyme had a negative cooperativity for ammonium (Hill number of 0.66), and its Km value increased from 2.6 to 21.2 mM when the ammonium concentration exceeded 16 mM. The deamination reaction was highly sensitive to inhibition by ammonium, while the amination reaction was only slightly inhibited by glutamate. These results, considered together with the Km values, indicate that the NADP‐GDH in Sphaerostilbe repens is primarily concerned with glutamate biosynthesis.

Differential inhibition of brain specific [3H]flunitrazepam binding by several types of dyes.

Several dyes, representing different structural classes, inhibit [3H]flunitrazepam binding to brain specific receptors in the rat with 50% inhibition in the 1 to 100 microM range. Crystal Violet and Methyl Violet 2B inhibited more potently in the forebrain than in the cerebellum. Congo Red yielded a Hill number near 2.3, probably reflecting positive cooperativity between interacting binding sites in benzodiazepine receptor complexes. Toluidine Blue 0 was the most potent of the dyes tested (IC50 = 1 microM in cerebellum) and inhibited more potently in cerebellum than in forebrain.

Special effects of UDP-sugar binding to bovine liver uridine diphosphoglucose dehydrogenase

The binding of NADH to uridine diphosphate glucose dehydrogenase has been examined by equilibrium dialysis. There is an absolute requirement for the presence of UDP-glucose for the binding of NADH. Other analogs such as UDPxylose, UDPgalactose and UDPglucuronic acid cannot replace UDPglucose as an effector of NADH binding. UDPxylose competes with UDPglucose for the UDP-sugar-binding site, and in so doing releases the bound NADH. The binding of NADH to UDPglucose dehydrogenase in the presence of UDPglucose reaches a saturation limit of 3 mol NADH bound per enzyme hexamer, and displays positive cooperativity, Hill number = 1.34. The effects of UDP-sugars on the fluorescence of UDPglucose dehydrogenase derivatized at the catalytic sites with a fluorophore have also been studied. Two classes of UDPxylose-binding site have been detected. One class has high affinity ( K diss = 3 μM, determined by equilibrium dialysis) but does not affect fluorophore fluorescence, and the other has lower affinity ( K diss = 120 μM) and leads to red-shifted fluorescence quenching, presumably by effecting exposure of the fluorephore to solvent. The high-affinity sites are identified as the UDP-sugar subsites of the undreivatized catalytic sites, and the low-affinity sites os UDP-sugar subsites of the fluorophore-labeled catalytic sites.

Isolation and Characterization of Inner Membrane-Associated and Matrix NAD-Specific Isocitrate Dehydrogenase in Potato Mitochondria

The isotherm for isocitrate oxidation by potato (Solanum tuberosum L. var. Russet Burbank) mitochondria in the presence of exogenous NAD is characterized by a hyperbolic phase at isocitrate concentrations below 3 millimolar, and a sigmoid, or positively cooperative phase from approximately 3 to 30 millimolar. The two forms of isocitrate dehydrogenase were separated and characterized following the sonication of mitochondria in 15% glycerol in the absence of buffer, followed by fractionation in a density step gradient to yield inner membrane and matrix components. The membrane-associated isocitrate dehydrogenase was found to have a Hill, or cooperativity, number of 1, while the Hill number of the matrix enzyme was 2.5. Upon digitonin extraction the cooperativity number of the membrane enzyme rose to 3.5. The isocitrate K(m) for the membrane enzyme was calculated to be approximately 5.9 x 10(-4) molar, while the S(0.5) for the matrix was 6.9 x 10(-4) molar. The NAD K(m) for both enzymes was 150 micromolar. Whereas the membrane enzyme proved indifferent to adenine nucleotides, the matrix enzyme was arguably inhibited by AMP and ADP, and inhibited some 25% by 5 millimolar ATP. Both enzymes were negatively responsive to the mole fraction of NADH, the membrane enzyme being 50% inhibited at a mole fraction of 0.26, and the matrix enzyme by a mole fraction of 0.32. The suggestion is offered that the enzymes in question constitute two forms of a single enzyme, one peripherally associated with the inner membrane, and one soluble in the matrix. It is proposed that a degree of regulation may be achieved by the apportionment of the enzyme between the bound and free forms.

A high-affinity [formula omitted] in plasma membranes of rat ascites hepatoma AH109A cells

The activity of calcium-stimulated and magnesium-dependent adenosinetriphosphatase which possesses a high affinity for free calcium (high-affinity (Ca 2+ + Mg 2+)-ATPase , EC 3.6.1.3) has been detected in rat ascites hepatoma AH109A cell plasma membranes. The high-affinity (Ca 2+ + Mg 2+)-ATPase had an apparent half saturation constant of 77 ± 31 nM for free calcium, a maximum reaction velocity of 9.9 ± 3.5 nmol ATP hydrolyzed/mg protein per min, and a Hill number of 0.8. Maximum activity was obtained at 0.2 μM free calcium. The high-affinity (Ca 2+ + Mg 2+)-ATPase was absolutely dependent on 3–10 mM magnesium and the pH optimum was within physiological range (pH 7.2–7.5). Among the nucleoside trisphosphates tested, ATP was the best substrate, with an apparent K m of 30 μM. The distribution pattern of this enzyme in the subcellular fractions of the ascites hepatoma cell homogenate (as shown by the linear sucrose density gradient ultracentrifugation method) was similar to that of the known plasma membrane marker enzyme alkaline phosphatase (EC 3.1.3.1), indicating that the ATPase was located in the plasma membrane. Various agents, such as K +, Na +, ouabain, KCN, dicyclohexylcarbodiimide and NaN 3, had no significant effect on the activity of high-affinity (Ca 2+ + Mg 2+)-ATPase . Orthovanadate inhibited this enzyme activity with an apparent half-maximal inhibition constant of 40 μM. The high-affinity (Ca 2+ + Mg 2+)-ATPase was neither inhibited by trifluoperazine, a calmodulin-antagonist, nor stimulated by bovine brain calmodulin, whether the plasma membranes were prepared with or without ethylene glycol bis(β- aminoethyl ether)-N,N,N′,N′- tetraacetic acid . Since the kinetic properties of the high-affinity (Ca 2+ + Mg 2+)-ATPase showed a close resemblance to those of erythrocyte plasma membrane (Ca 2+ + Mg 2+)-ATPase , the high-affinity (Ca 2+ + Mg 2+)-ATPase of rat ascites hepatoma cell plasma membrane is proposed to be a calcium-pumping ATPase of these cells.

Properties of (Ca2+ + Mg2+)-ATPase in the plasma membranes of liver malignant tumor.

The activity of (Ca2++ Mg2+)-ATPase has been detected in the plasma membranes of rat ascites hepatoma AH10 9A cells. The (Ca2++ Mg2+)-ATPase had a Ka of 77 nM for free calcium, a Vmax of 9.9 nmol ATP hydrolyzed/mg/min, and a Hill number of 0.8. This enzyme was dependent on 3-10 mM magnesium, its optimal pH was within the physiological range(pH 7.2-7.5). ATP was the major substrate with a Km of 30 μM. Various agents such as K+, Na+, ouabain, mitochondrial ATPase blockers, calmodulin, and trifluoperazine had no significant effect on the enzyme activity. Orthovanadate inhibited the enzyme activity with a Ki of 40 μM. The localization of the enzyme in the cell was similar to that of alkaline phosphatase, a plasma membrane marker enzyme, using the sucrose density gradient centrifugation method. Since kinetic properties of the enzyme showed close resemblance to those of erythrocyte plasma membrane (Ca2++ Mg2+)-ATPase, this (Ca2++ Mg2+)-ATPase may be a calcium pumping ATPase in the plasma membranes of rat ascites hepatoma cell. In the plasma membrane fraction from normal liver of rat, however, similar (Ca2++ Mg2+)-ATPase was not detected, and the properties of the high affinity Ca2+-ATPase of the membrane reported (Y. Iwasa et al.; BBRC, 105, 488, 1982) were different from those of the (Ca2++ Mg2+)-ATPase in the present study.

Effect of seed tuber and seed piece size on growth and incidence of hollow heart in Norgold Russet potatoes

Small seed pieces decreased yield and number of tubers. Both seed tuber and seed piece size affected the percentage of tubers with hollow heart. Hollow heart increased from 14 to 22% as the seed tuber size increased from 57 to 228 g and decreased from 27 to 19% as the seed piece size increased from 28 to 57 g. The least hollow heart (11%) occurred in tubers of plants grown from 57 g whole seed pieces and the most (26%) in tubers of plants grown from 228 g seed tubers cut into 28 g seed pieces. Hollow heart was positively correlated with the mean tuber size and negatively correlated with the number of mainstems per hill, total yield, and total number of tubers.

Glutamate Synthase from Rice Leaves

Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) from rice leaves (Oryza sativa L. cv Delta) was purified 206-fold with a final specific activity of 35.9 mumoles glutamate formed per min per milligram protein by a procedure including ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephacryl S-300 gel filtration, and ferredoxin-Sepharose affinity chromatography. The purified enzyme yielded a single protein band on polyacrylamide gel electrophoresis. Molecular weight of the native enzyme was estimated to be 224,000 daltons by Sepharose 6B gel filtration. Electrophoresis of the dissociated enzyme in sodium dodecyl sulfate-polyacrylamide gel gave a single protein band which corresponds to the subunit molecular weight of 115,000 daltons. Thus, it is concluded that the glutamate synthase is composed of two polypeptidic chains exhibiting the same molecular weight. Spectrophotometric analysis indicated that the enzyme is free of iron-sulfide and flavin. The pH optimum was 7.3. The enzyme had a negative cooperativity (Hill number of 0.70) for glutamine, and its K(m) value increased from 270 to 570 mum at a glutamine concentration higher than 800 mum. K(m) values for alpha-ketoglutarate and ferredoxin were 330 and 5.5 mum, respectively. Asparagine and oxaloacetate could not be substituted for glutamine and alpha-ketoglutarate, respectively. Enzyme activity was not detected with pyridine nucleotides as electron donors. Azaserine and several divalent cations were potent inhibitors. The purified enzyme was stabilized by dithiothreitol.

Characteristics of β-adrenergic-agonist binding to rat adipocyte membranes: Evidence that [formula omitted]hydroxybenzylisoproterenol interacts selectively with the adipocyte β-adrenergic receptors

The binding characteristics of the β-adrenergic agonist (±)-[ 3 H]hydroxybenzylisoproterenol to rat adipocyte membranes were studied. Binding was rapid, reaching equilibrium within 10 min at 37°C (second order rate constant k 1=1.37·10 7·M −1·min −1). Dissociation of specific binding by 0.5 mM (−)-isoproterenol suggested dissociation from two different sites with respective dissociation rate constants k 2 of 0.106·min −1 and 0.011·min −1.[ 3H]Hydroxybenzylisoproterenol binding was saturable ( B max=690±107 fmol/mg protein), yielding curvilinear Scatchard plots. Computer modeling of these data were consistent with the existence of two classes of [ 3H]hydroxybenzylisoproterenol binding sites, one having high affinity ( K D=3.5±0.7 nM) but low binding capacity (10% of the total sites) and one haveing low affinity ( K D=101±20 nM) but high binding capacity (90% of the sites). Adrenergic ligands competed with [ 3H]hydroxybenzylisoproterenol binding with the following order of potency=(−)-propranolol>(−)-isoproterenol>(−)-norepinephrine≈ (−)-epinephrine>>(+)-isoproterenol=(+)-propranolo, which is consistent with binding to β 1-adrenergic receptors. Competition curves of [ 3H]hydroxybenzylisoproterenol binding by the β-agonist (−)-isoproterenol were shallow and modeled to two affinity states of binding, whereas, competition curves by β-antagonist (−)-propranolol were steeper with Hill number near to one. Gpp[NH]p severely reduced [ 3H]hydroxybenzyl-isoproterenol binding, an effect which apparently resulted from the reduction of the number of both the high and low affinity sites. In membranes which had been previously exposed to (−)-isoproterenol, then number of [ 3H]hydroxybenzylisoproterenol binding sites was reduced by 50%, an effect which apparently resulted from the loss of part of both the high and low affinity state binding sites. Finally, the ability of (−)-isoproterenol to stimulate adenylate cyclase correlate closely with the ability of (−)-isoproterenol to displace [ 3H]hydroxybenzylisoproterenol binding. Comparison of these findings with the binding characteristics of the β-antagonist [ 3H]dihydroalprenolol to rat adipocyte membranes, led to conclude that [ 3H]hydroxybenzylisoproterenol can be successfully used to label the β-adrenergic receptors of rat fat cells and suggests that it might be a better ligand than [ 3H]dihydroalprenolol in these cells.

A search in rat brain cortex synaptic vesicles for endogenous ligands for kainic acid receptors

Conditions were found for stabilizing rat brain cortex kainic acid (KA) receptors. Such receptors had the same Hill number (about 0.6) for KA and for glutamate. The receptors were then used as detectors for endogenous ligands present in brain cortex synaptic vesicle (SV) soluble extracts. When these SV extracts were fractionated by gel filtration on Sephadex G-10, by thin-layer chromatography, or by high voltage electrophoresis, a single endogenous component, that in all cases comigrated with glutamic acid, was found.

Ｐｓｅｕｄｏｃｈｏｌｉｎｅｓｔｅｒａｓｅ変異のＨｉｌｌ ｎｕｍｂｅｒによる解析

The physico-chemical and kinetical properties of abnormal pseudocholinesterase (p-ChE) in two families (J. T. and K. K.) were studied and discussed.The p-ChE levels of the patient J. T. and K. K. were 10% and 3% of the lower limit of normal ranges, respectively. The p-ChE of patient J. T. was classified as homozygote for atypical gene by the inhibition studies. However, the p-ChE of K. K. showed much larger Km-values for several substrates than normal controls and the Hill's number of the patient's p-ChE was 1.10, though the values less than 1.0 were usually obtained from normal control. Therefore, the p-ChE of K. K. have not the allosteric property with negative cooperativity, as was the nature of normal one. Further studies by isoelectric focusing and slab PAG electrophoresis revealed the traces of the activity of p-ChE in the patient J. T., but any activity band was not detected in the patient K. K. These results indicated that the patient J. T. was homozygote of atypical gene and that the patient K. K. was homozygote for silent gene. It was suggested that the analysis of Hill's number, additional to the conventional inhibition studies, might be useful for the detection of the silent gene of p-ChE in details.

Functional properties of lyophilized hemoglobin in the presence of amino acids after 13 months of conservation

Four lyophilisates of hemoglobin, each protected by an amino acid salt, were conserved for 13 months. The determinations carried out (oxyhemoglobin, methemoglobin, p50, Hill's number, and visible spectrum) demonstrated that the hemoglobin had retained its functional properties.

Action of detergents and pre- and postsynaptic localization of 3H-naloxone binding in synaptosomal membranes. A structural approach.

3H-naloxone specific binding was carried out on synaptosomal membranes isolated from basal ganglia of the cat brain. A high- and a low-affinity site with Kd1 = 3.7 nM and Kd2 = 35 nM having B max 1 = 79 pmole/g protein and B max 2 = 224 pmole/g protein were found. The Hill number for the high- and low-affinity sites were, respectively, 1.01 and 0.86. Digitonin and Triton X-100 had an inhibitory effect on the binding at concentrations between 10(-5) and 10(-1)% (w/v). Deoxycholate and Nonidet P-40 also inhibited the binding of 3H-naloxone, but at 10(-4)% produced a 50% enhancement. After the binding to membranes, the 3H-naloxone receptor complex is stable to the action of Triton X-100 and dissociates slowly. In membranes bound with 10 nM 3H-naloxone and then submitted to 0.1-0.2% Triton X-100, in which only the presynaptic membrane disintegrates, the specific radioactivity is decreased. With a more drastic treatment that disintegrates the postsynaptic membrane, the 3H-naloxone binding to synaptosomal membranes is almost completely abolished. These results suggest that opiate receptors may be localized both pre- and postsynaptically in central synapses.

Different degrees of cooperativity of the Ca2+-induced changes in fluorescence intensity of solubilized sarcoplasmic reticulum ATPase.

The tryptophan fluorescence emission of sarcoplasmic reticulum Ca2+-ATPase was studied both in purified ATPase vesicles and in ATPase solubilized with the nonionic detergent dodecyloctaethyleneglycolmonoether (C12E8). Fluorescence intensity changes in purified ATPase were titrated as a function of free Ca2+ in the medium. It exhibited a cooperative pattern, with a Hill number of 2.21 +/- 0.02 and K0.5 = 0.51 microM Ca2+. Upon solubilization of the ATPase, the cooperative pattern of fluorescence change was lost; the Hill number was 0.96 and K0.5 = 1.4 microM Ca2+. When solubilization was carried out in the presence of 0.5 or 1.0 mM CaCl2, followed by the titrations of fluorescence change in the micromolar Ca2+ range, the cooperative pattern was preserved under the same concentrations of C12E8 which would otherwise promote the loss in cooperativity. For the ATPase solubilized in millimolar Ca2+, the Hill number was 1.98 with a K0.5 = 1.5 microM Ca2+. The maximal amount of Ca2+ bound to the high affinity sites corresponded to approximately 1 mol of calcium/mol of polypeptide chains, both in purified ATPase vesicles and in the soluble ATPase. A model is suggested, which involves a minimum of 4 interacting Ca2+ sites (tetramers). Cooperativity is accounted for in the model by the predominance in the absence of Ca2+ of low affinity state (E') of the Ca2+ site (K'D = 5.7 x 10(-4) M), which would be congruent to 90 times more concentrated than (E), the high affinity state (KD = 1.9 x 10(-7) M). Simulations derived from this model fit the experimental data.

Benzodiazepine receptors in rat brain: action of Triton X-100 and localization in relation to the synaptic region.

Membranes were isolated from the cerebral cortices of rat brain and submitted to binding with 3H-flunitrazepam (3H-FNZP). Specific binding gave a hyperbolic saturation curve reaching a maximum between 10 - 12 nM suggesting a single population of sites. By Scatchard analysis a KD = 4 nM and a Bmax = 407 fmol/mg protein was obtained; the Hill number was 0.97. The effect of Triton X-100 was analyzed between concentrations of 6.4 x 10(-8) and 5 x 10(-1)%. With 6.4 x 10(-6) and 6.4 x 10(-3)% there is an activation of the binding without loss of protein. This activation is not reversed by washing. At 6.4 x 10(-2)% or higher concentrations there is an inhibition that is partially reversed and also solubilization of some receptors. The activation with low detergent is due to an increase in Bmax without change in KD. With 2 x 10(-1)% and 5 x 10(-1)% Triton X-100 there is considerable decrease in Bmax and in th latter case, an increase in affinity, as well. The results obtained through the action of Triton X-100 suggest that a proportion of benzodiazepine receptors are localized presynaptically. There findings are discussed in relation to previous studies from this laboratory on the localization of other central receptors with reference to the synaptic region.