Half-maximal Saturation Research Articles

Lipoprotein metabolism by rat hepatomas. Studies on the etiology of defective dietary feedback inhibition of cholesterol synthesis.

In contrast to normal liver, it is known that in vivo hepatomas fail to decrease their rate of cholesterol biosynthesis in response to increased dietary cholesterol. From a consideration of the available data it has been hypothesized that the defect might lie in the delivery of cholesterol to the hepatoma cell. To study this further, lipoprotein interactions with rat hepatoma cells in tissue culture (HTC 7288C) and with the same cell line in vivo were investigated. HTC cells grown in a medium containing 10% calf serum exhibited saturable, specific, calcium-dependent binding of rat 125I-chylomicron remnants at 4 degrees C with half maximal saturation at 4.8 micrograms protein/ml and maximum binding of 96 ng protein/10(6) cells. At 4 degrees C, HTC cells also bound human 125I-low density lipoprotein (LDL) specifically, but bound it with a much lower affinity. These cells also exhibited specific binding for rat LDL and rat hypercholesterolemic very low density lipoprotein (VLDL). All these lipoproteins were degraded by HTC cells. Thus, it was concluded that hepatoma cells possess lipoprotein receptors that recognize and process LDL, VLDL, and chylomicron remnants. Overnight incubation of HTC cells in lipid-depleted medium containing 0.5 microM compactin increased binding of rat chylomicron remnants and of hypercholesterolemic VLDL approximately 1.7-fold without a significant change in binding affinity. LDL binding also increased, by approximately 3.5-fold. These changes were also observed when binding and internalization were measured at 37 degrees C. After HTC cells were incubated in lipid-depleted medium, the rate at which [14C]acetate was incorporated into [14C]cholesterol increased 2.5-fold. Inclusion of rat chylomicron remnants at 5-10 micrograms protein/ml prevented this increase in acetate incorporation or, if added after culture in lipid-depleted medium, reduced the increased levels back to control values. However, the rate of acetate incorporation into cholesterol by cells grown in complete medium was not decreased to levels below base line by rat chylomicron remnants. Inclusion of human LDL only partially prevented the rise or only partially reduced the increased levels back to control and did not reduce control levels below base line. Hypercholesterolemic VLDL, which contain more cholesterol per particle than chylomicron remnants, did reduce [14C]acetate incorporation to below control levels. Therefore, the intracellular mechanism for down regulation of cholesterol synthesis by lipoproteins is intact in these cells. Based on these results we hypothesized that a relative lack of lipoprotein receptors expressed by hepatomas in vivo in comparison with those expressed by normal liver would explain the apparent absence of feedback inhibition of cholesterol synthesis. Consistent with this hypothesis, the binding of chylomicron remnants to liver cell membranes was 3-5 times greater than to membranes from tumors grown in vivo subcutaneously or intramuscularly. Membranes from tumor cells grown in vitro bound remnants least well. It is proposed that the relative lack of receptors places the hepatoma at a disadvantage in competing with the liver for lipoproteins of dietary origin and may account for the lack of feedback regulation of cholesterol synthesis in hepatomas.

Photoaffinity labeling of human chorionic gonadotropin-binding sites in rat ovarian plasma membranes.

A photoaffinity derivative of human chorionic gonadotropin (hCG-N-hydroxysuccinimidyl-4-azidobenzoate (hCG-HSAB) ) was used to identify components of the lutropin (LH)/hCG receptor in plasma membranes prepared from pregnant mare serum-pretreated rat ovaries. In intact plasma membranes, the Kd for hCG (1 X 10(-10) M) was the same with both 125I-hCG and 125I-hCG-HSAB, and the binding capacity for the photoaffinity label (1 pmol/mg of membrane protein) was the same as for native hCG. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions of 125I-hCG-HSAB cross-linked to plasma membranes revealed newly formed complexes of Mr = 106,000, 85,000, and 80,000 (representing membrane protein components of Mr = 86,000, 65,000, and 60,000, respectively). However, only the Mr = 106,000 complex displayed an affinity for hCG (Kd of 1 X 10(-10) M) consistent with the physiological LH/hCG receptor. The other binding sites were nonsaturable. Half-maximal saturation by 125I-hCG-HSAB of the Mr = 106,000 complex (1-2 X 10(-9) M) was similar to the Kd of the specific binding of 125I-hCG-HSAB to intact plasma membranes (8 X 10(-10) M). The present data suggest that among the different components of the LH/hCG receptor, only one component (of Mr = 86,000) possesses the affinity and binding capacity characteristics of the LH/hCG receptor as determined using radiolabeled, underivatized hCG.

Characterization of the interchain and intrachain interactions between the binding sites of the free regulatory moiety of protein kinase I.

The interaction between the four binding sites (two A sites and two B sites) of the regulatory subunit dimer of protein kinase I (RI2) was studied. The rate of association of c[3H]AMP to site B was slower when site A had already been occupied. Occupation of site A also retarded the rate of dissociation of c[3H]AMP from site B. This site A-B interaction was intrachain since it was observed also for a monomeric fragment of RI2. Thus, each monomer of RI2 must have one A site and one B site. Quantitative analysis of the rate constants for cAMP binding to variously liganded RI2 suggested little or no thermodynamic coupling between site A and B. This conclusion was supported by equilibrium binding data. Occupation of one A site retarded the dissociation of c[3H]AMP from the A site of the other subunit (interchain interaction). The rate kinetic constants as well as equilibrium binding data indicated a positively cooperative site A-A interaction. The interaction between cAMP and either site was enthalphy-driven (25 degrees C), the process being accompanied by a loss of entropy. The thermodynamic parameters did not support the occurrence of an abrupt conformational change at a certain level of ligandation of RI2. Half-maximal saturation of either site occurred at 1-2 nM cAMP (37 degrees C, pH 7.0, 0.15 M KCl). The concentration of RI2 did not detectably influence any binding parameters. Aging of RI2 produced a form with minimally, if at all, altered Mr, but which showed a more rapid release of c[3H]AMP bound to site B.

Characterization of forskolin binding sites in rat brain membranes using [14,15-3H]14,15-dihydroforskolin as a ligand.

[14,15-3H]14,15-Dihydroforskolin [( 3H]DHF) has been used as a radioactive ligand to identify forskolin binding sites in rat brain membranes. The binding was saturable and reversible. The binding sites showed positive cooperative properties as evident from an upward convex Scatchard plot and a Hill coefficient of 1.6. The equilibrium dissociation constants (KD) were in the range between 10 microM and 10 nM as estimated from the limiting slopes of the curved Scatchard plot. Half-maximal saturation of the binding sites was observed at a ligand concentration of 225 nM. The binding kinetics were very rapid: Binding equilibrium was reached in less than 2 min and a large excess of cold forskolin displaced 80% of the radioligand within 2 min. The dissociation reaction was not first order, characterized by a decreasing dissociation rate constant. Bound [3H]DHF could be displaced with forskolin (IC50 0.3 microM), 14,15-dihydroforskolin (IC50 0.8 microM) and 7-desacetylforskolin (IC50 3 microM). However, nucleotides (ATP, GTP) and other receptor ligands (adenosine, isoproterenol) had no effect on the binding. Although the density of the forskolin binding sites (3.2 pmole/mg protein) is similar to those of other adenylate cyclase linked receptors, discrepancies between the KD and the ED50 obtained in adenylate cyclase studies and the finding that activation of the enzyme by forskolin is negative cooperative makes it difficult to clearly relate the binding sites to adenylate cyclase.

Voltage-dependent calcium block of normal and tetramethrin-modified single sodium channels

The mechanisms by which external Ca ions block sodium channels were studied by a gigaohm seal patch clamp method using membranes excised from N1E-115 neuroblastoma cells. Tetramethrin was used to prolong the open time of single channels so that the current-voltage relationship could be readily determined over a wide range of membrane potentials. Comparable experiments were performed in the absence of tetramethrin. Increasing external Ca ions from 0.18 to 9.0 mM reduced the single channel conductance without causing flickering. From the dose-response relation the dissociation constant for Ca block at 0 mV was estimated to be 32.4 +/- 1.05 mM. The block was intensified by hyperpolarization. The voltage dependence indicates that Ca ions bind to sodium channels at a site located 37 +/- 2% of the electrical distance from the outside. The current increased with increasing external Na concentrations but showed a saturation; the concentration for half-maximal saturation was estimated to be 185 mM at -50 mV and 204 mM at 0 mV. A model consisting of a one-ion pore with four barriers and three wells can account for the observations that deviate from the independence principle, namely, the saturation of current, block by Ca ions, and rectification in current-voltage relationship. The results suggest that the Ca-induced decrease of the macroscopic sodium current results from a reduced single sodium channel conductance.

The vanadate complex of the calcium-transport ATPase of the sarcoplasmic reticulum, its formation and dissociation.

Vanadate binding to different sarcoplasmic reticulum membrane preparations was determined by measuring bound vanadate colorimetrically and by phosphorylating the vanadate-free enzyme fraction with [gamma-32P] ATP. Colorimetry allowed the study of the dependence of equilibrium vanadate binding on ionized magnesium and the displacing effect of ionized calcium at vanadate concentrations greater than 0.1 mM only. At saturating magnesium concentration the enzyme binds 6-8 nmol vanadate/mg protein and half-maximum saturation is reached at 40 microM. Vanadate is displaced from the enzyme when its high-affinity calcium-binding sites are saturated and conversely calcium is solely displaced from its high-affinity binding sites by vanadate. The phosphorylation procedure allowed the measurement of equilibrium binding as well as the kinetics of vanadate binding and release at vanadate concentrations below 0.1 mM. Half-times of 30s and 3s were observed for vanadate release induced by 0.1 mM and 1 mM calcium respectively. Millimolar concentrations of ATP are required for vanadate displacement. Under equilibrium conditions the enzyme displays an affinity for vanadate of 1.6 X 10(6) M-1. The dependence on the concentration of vanadate of the rate of vanadate binding yielded an affinity of only 1 X 10(4) M-1. Closed vesicles bind vanadate much more slowly than calcium-permeable preparations. The initial rate of calcium-induced vanadate dissociation is accelerated considerably when the vesicles are made calcium permeable. The rate of vanadate dissociation from calcium-permeable vesicles reaches half-maximum values at 1-2 mM calcium indicating that the internal low-affinity calcium-binding sites must first be occupied in order to release bound vanadate. The results suggest that vanadate binding leads to a transition of the external high to internal low-affinity calcium-binding sites.

Increase of serotonin receptors in rat uterus induced by estradiol.

[3H]Spiroperidol was used to label uterine membrane-binding sites that have the characteristics expected of serotonergic receptors. The characteristics of specific [3H]spiroperidol binding to the uterine membrane of 17 beta-estradiol-3-benzoate-treated ovariectomized rats were studied. The specific [3H]spiroperidol binding was rapid and reversible, and the half-maximal saturation, taken as the apparent dissociation constant (KD) for [3H]spiroperidol, was 5.16 +/- 0.24 (n = 12) nM [3H]spiroperidol. Scatchard plots of saturation curves of the specific [3H]spiroperidol binding were convex and the Hill coefficient was 2.06 +/- 0.11 (n = 12). Cinanserin, mianserin, metergoline (which are serotonergic antagonists), and serotonin (5-HT) inhibited the [3H]spiroperidol binding with apparent Ki values of 21.2, 14.1, 14.1, and 176.5 microM, respectively. Concentrations of 1 mM sulpiride (a dopaminergic antagonist) and dopamine reduced [3H]spiroperidol binding only 26 and 23%, respectively. 1 mM GTP reduced the potency of 5-HT (10(-6) - 10(-3) M) to displace bound [3H]spiroperidol. The uterine membranes were treated with various enzymes and protein-modifying reagents, and binding studies on the treated uterine membranes showed that protein(s), phospholipids, and N-acetyl-neuraminic acid in uterine membranes were important as specific binding sites of [3H]spiroperidol. Measurement of the specific binding of [3H]spiroperidol to uterine membranes from untreated and estradiol-treated ovariectomized rats showed that estradiol significantly increased the number of specific binding sites of [3H]spiroperidol, but did not change the apparent affinity of specific [3H]spiroperidol binding. Estradiol also did not change the dissociation constant or the number of binding sites for [3H]3-quinuclidinyl benzilate, which binds to muscarinic acetylcholine receptors. These findings suggest that [3H]spiroperidol mainly binds to 5-HT receptors in the uterine membrane of estradiol-treated ovariectomized rats. The finding that administration of estradiol significantly increased the number of [3H]spiroperidol-binding sites is consistent with the specific increase in the contractile response to 5-HT observed in isolated uterus from ovariectomized rats treated with estradiol.

Age-dependence of polyadenylate stimulation of nuclear-envelope nucleoside triphosphatase

Nuclear envelopes of mammalian cells contain a nucleoside triphosphatase which is probably involved in mRNA transport through the nuclear membrane. The activity of the enzyme, studied in RNA-depleted nuclear ghosts, can be stimulated by poly(A) or by poly(A) (+)mRNA. Using nuclear ghost preparations from mature (8–10 months' old) and old (40–42 months' old) Wistar rats, it was shown that in “old” preparations the basal activity of the enzyme is significantly reduced (by 15%). In addition, the enzyme from old animals responds only very little to poly(A) or poly(A) (+)mRNA, compared to preparations from mature animals. Using a concentration of 6.8 × 10 11 poly(A) (+)mRNA molecules per μg of enzyme preparation, the nucleoside triphosphatase from mature animals is stimulated by 77% and the enzyme from old animals by only 26%. Binding studies of poly(A) to pore laminae revealed that the number of binding sites in unphosphorylated preparations from old animals is significantly reduced (by 24%) compared to “mature” preparations. As a consequence of in vitro phosphorylation, no difference is observable in the number of binding sites between the two age groups. The values for half-maximal saturation binding constants for poly(A) are identical in unphosphorylated and phosphorylated pore-laminae preparations, irrespective of the age group studied. The results presented indicate that in old animals the pathway from the phosphorylated to the dephosphorylated nuclear-envelope protein which is controlled by poly(A) is impaired in the proposed cycle for mRNA efflux from nuclei.

Growth, structure and function of baboon aortic smooth muscle cells in culture

Procedures for the successful culture of aortic medial smooth muscle cells (SMC) from the baboon ( Papio cynocephalus) have been described. Growth rates, plating efficiency, and doubling times were determined. SMC showed increasing growth with increasing concentrations of heat-inactivated homologous serum. Growth on glass was accelerated relative to plastic surfaces, a difference reflected in the more numerous and extensive attachment sites on glass and more extensive cellular spreading. Attachment sites on both glass and plastic were associated with short parallel arrays of thin filaments (50 Å). The fine structure of SMC grown on plastic or glass was examined at intervals from 7 to 74 days in culture. Cells exhibited many of the features traditionally associated with cultured SMC, including basement membrane material and extracellular matrix, predominantly thin cytoplasmic filaments (40–80 Å) with dense bodies, coated pits and vesicles, caveolae and pinocytotic vesicles, and both cell-cell and internalized gap junctions. Changes with increasing time in culture included an increased extracellular matrix, more numerous lipid inclusions, residual bodies, myelin forms, multivesicular bodies, and crystalline lysosomal inclusions. Membranous multivesicular blebs and extruded autophagosomes were also seen with increasing frequency. An increasing culture age was also associated with changes in organelles, including less prominent Golgi profiles, more free ribosomes, dilatation of the cisternae of the RER, and less prominent shorter profiles of mitochondria with increasing electron density. Cultured SMC exhibited both a continuous and a homogeneous ruthenium red-staining glycocalyx around their perimeter, and the ability to respond to the polycationic ligand, CF, with a redistribution of surface-associated anionic binding sites. Additionally, they have been shown to possess high-affinity LDL receptors ( K d = 1 × 10 −7 M) with binding showing half-maximal saturation at 20 μg/ml.

Cholestatic action of somatostatin in the rat: Effect on the different fractions of bile secretion

Somatostatin was administered intravenously to male Wistar rats, recovered for 3 h from an anesthesia during which the common bile duct and jugular vein were cannulated. Different bile acid-secretory rates were obtained by infusion of saline, or of Na+-taurocholate (150 nmol/min/100 g body wt), or by 8-h bile depletion. At the dose of 2 μg/h/100 g body wt, Somatostatin causes a prompt decrease of bile flow (about 30%) and of bile acid secretion (32%-47%). The bile acid-independent fraction of canalicular bile is more decreased than the one associated with bile acid secretion. The changes are dose dependent and show a saturation pattern, with halfmaximal saturation already at 2.2 ng/min/100 g body wt. Despite this cholestasis, endogenous bilirubin secretion remained unchanged, pointing to different secretory mechanisms for bilirubin and bile acids. In the isolated and perfused liver, somatostatin displays an anticholeretic effect, proportional to the amount of Na+-taurocholate present in the system. Hepatic blood flow and O2 consumption remained constant during perfusion, and were not affected by Somatostatin. The hepatic transport of bile acid, and the water and electrolyte secretion are directly affected by Somatostatin, and the experimentally-induced cholestasis seems a new and suitable model for studying mechanisms of bile secretion.

Characterization of beta-adrenergic receptors by (3H) isoproterenol in adipocytes of humans and rats.

The specific binding of (3H) isoproterenol to isolated fat cells of human and rat was characterized. Binding of (3H) isoproterenol to isolated fat cells of rat was saturable with 420 pmol of (3H) isoproterenol bound/100 mg of lipid. Half-maximal saturation occurred at 5 microM providing an estimate of the equilibrium dissociation constant, KD, for the interaction of (3H) isoproterenol with its binding sites. Kinetic analysis of (3H) isoproterenol binding provided a value of 2.01 x 10(4) min-1. M-1 for the forward bimolecular rate constant, k1. Dissociation of (3H) isoproterenol was a first order reaction with a rate constant, k2, of 0.62 x 10(-1) min-1. The ratio k2/k1 = 3.07 microM provides an independent measurement of the KD for the interaction of (3H) isoproterenol with its binding sites which is in agreement with the values obtained by steady state analysis (3 to 5 microM). The apparent equilibrium dissociation constant, KD, for the interaction of (3H) isoproterenol with its receptor in human fat cells obtained by steady state analysis was 1 to 0.9 microM. Scatchard- and Hill-analysis suggest the possibility of different negatively cooperative interactions among the binding sites in human and rat. beta-Adrenergic agonists competed for the binding sites. The order of potency was isoproterenol greater than epinephrine greater than norepinephrine. Compounds such as DOPA, dopamine and (m-Hydroxyphenyl)2-methyl-aminoethanol which are structurally related to catecholamines had little or no affinity for (3H) isoproterenol binding sites.

Calmodulin confers calcium sensitivity on ciliary dynein ATPase.

Extraction of demembranated cilia of Tetrahymena by Tris-EDTA (denoted by the suffix E) yields 14S-E and 30S-E dyneins with ATPase activities that are slightly increased by Ca++. This effect is moderately potentiated when bovine brain calmodulin is added to the assay mixture. Extraction with 0.5 M KCl (denoted by the suffix K) yeilds a 14S-K dynein with a low basal ATPase activity in the presence of Ca++. Subsequent addition of calmodulin causes marked activation (up to 10-fold) of ATPase activity. Although 14S-K and 14S-E dyneins have Ca++-dependent ATPase activities that differ markedly in the degree of activation, the concentration of calmodulin required for half-maximal saturation is similar for both, approximately 0.1 microM. Both 30S-K and 30S-E dyneins, however, require approximately 0.7 microM bovine brain calmodulin to reach half-maximal activation of their Ca++-dependent ATPase activities. Tetrahymena calmodulin is as effective as bovine brain calmodulin in activating 30S dynein , but may be slightly less effective than the brain calmodulin in activating 14S dynein. Rabbit skeletal muscle troponin C also activates the Ca++-dependent ATPase activity of 30S dynein and, to a lesser extent, that of 14S dynein, but in both cases is less effective than calmodulin. The interaction of calmodulin with dynein that results in ATPase activation is largely complete in less than 1 min, and is prevented by the presence of low concentrations of ATP. Adenylyl imidodiphosphate can partially prevent activation of dynein ATPase by calmodulin plus Ca++, but at much higher concentrations than required for prevention by ATP. beta, gamma-methyl-adenosine triphosphate appears not to prevent this activation. The presence of Ca++-dependent calmodulin-binding sites on 14S and 30S dyneins was demonstrated by the Ca++-dependent retention of the dyneins on a calmodulin-Sepharose-4B column. Gel electrophoresis of 14S dynein that had been purified by the affinity-chromatography procedure showed that presence of two major and one minor high molecular weight components. Similar analysis of 30S dynein purified by this procedure also revealed on major and one minor high molecular weight components that were different from the major components of 14S dynein. Ca++-dependent binding sites for calmodulin were shown to be present on axonemes that had been extracted twice with Tris-EDTA or with 0.5 M KCl by the use of 35S-labeled Tetrahymena calmodulin. It is concluded that the 14S and 30S dyneins of Tetrahymena contain Ca++-dependent binding sites for calmodulin and the calmodulin mediates the Ca++-regulation of the dynein ATPases of Tetrahymena cilia.

A micromethod for rapid estimation of trans-N-glycosidase with modified cellulose nitrate membranes

A sensitive and rapid assay of the enzyme nicotinate ribonucleotide:benzimidazole phosphoribosyltransferase (EC 2.4.2.21) was developed. The method combines the use of labeled benzimidazole or its derivatives as substrates with a new kind of chromatography based on the use of modified cellulose nitrate membrane strips. Benzimidazole and some of its derivatives are strongly adsorbed to the membrane in contrast to the corresponding N-α-glycosidic-bound nucleotides produced from them enzymatically. Picomolar quantities of the labeled product and the unconverted substrate may be estimated in less than 10 min. The adsorption of benzimidazole on membrane strips is not affected by up to 1 m concentrations of KCl, K 2HPO 4 and (NH 4) 2SO 4 as well as by the pH range of 6.5 to 9.5. The rate constant of benzimidazole desorption from the cellulose nitrate strips in water is 0.14 min −1. The adsorptive capacity for the modified membranes of 0.45-μm pore size from Millipore is 7.8 μmol/cm 2 at saturating concentrations of benzimidazole (>50 m m). The association constant at half maximal saturation, K 0.5, is 1.4 × 10 2 m −1. The enzyme from Propionibacterium shermanii utilizes as substrates benzimidazole, 5,6-dimethylbenzimidazole, 5,6-dichlorobenzimidazole, and 5(6)-nitrobenzimidazole. The latter is converted at a 2.6 times higher rate than benzimidazole and its product shows higher resistance to dephosphorylation by endogenous phosphatases. The nucleotides obtained enzymatically from the respective benzimidazoles were purified by paper chromatography and dephosphorylated enzymatically to the corresponding nucleosides. The R f values of the benzimidazole bases, the respective nucleosides, and nucleotides are given.

Calmodulin-sensitive ATP-dependent Ca2+ transport across adipocyte plasma membranes.

An ATP-dependent transport system which is active at concentrations of free Ca2+ in the submicromolar range has been identified in adipocyte plasma membranes. The system appears to represent the functional component of the high affinity insulin-sensitive calcium-stimulated, magnesium-dependent adenosine triphosphatase preveiously described in the same preparation (Pershadsingh, H. A., and McDonald, J. M. (1979) Nature 281, 495-497). This ATP-dependent Ca2+ transport pump was stimulated approximately 3-fold by the Ca2+-dependent regulatory protein, calmodulin. This effect was confined to the plasma membrane since a similar effect was undetectable in the fraction enriched in endoplasmic reticulum. Calmodulin stimulation was dose-dependent but saturable with half-maximal activation occurring at 0.72 microgram/ml (43 nM). Calmodulin appeared to stimulate the system primarily by decreasing the apparent half-maximal saturation constant for free Ca2+ from 0.20 +/- 0.04 microM to 0.07 +/- 0.01 microM (n = 3). The Hill coefficient increased from 1.6 +/- 0.2 to 3.2 +/- 0.6 (n = 3), thus showing an increased positive cooperativity which allows the pump to be activated by an exceedingly narrow Ca2+ threshold in the presence of calmodulin. The calmodulin stimulation of the plasma membrane Ca2+ extrusion pump in adipocytes, working in opposition to metabolic signals which increase cytoplasmic Ca2+, could constitute a self-regulating negative feedback device for maintaining a low steady state level of intracellular Ca2+. This feedback system may be of critical importance in regulation of cellular metabolism by insulin.

Interaction of pyrethroids with the Na + channel in mammalian neuronal cells in culture

The interaction of a series of pyrethroids with the Na + channel of mouse neuroblastoma cells has been followed using both an electrophysiological and a 22Na + influx approach. By themselves, pyrethroids do not stimulate 22Na + entry through the Na + channel (or the stimulation they give is too small to be analyzed). However, they stimulate 22Na + entry when used in conjuction with other toxins specific for the gating system of the channel. These include batrachotoxin, veratridine, dihydrograyanotoxin II or polypeptide toxins like sea anemone and scorpion toxins. This stimulatory effect is fully inhibited by tetrodotoxin with a dissociation constant of 1.6 nM for the tetrodotoxin-receptor complex. Half-maximum saturation of the pyrethroid receptor on the Na + channel is observed in the micromolar range for the most active pyrethroids, Decis and RU 15525. The synergism observed between the effect of pyrethroids on 22Na + influx on the one hand, and the effects of sea anemone toxin II, Androctonus scorpion toxin II, batrachotoxin, veratridine and dihydrograyanotoxin II on the other, indicates that the binding component for pyrethroids on the Na + channel is distinct from the other toxin receptors. It is also distinct from the tetrodotoxin receptor. Some of the pyrethroids used in this study bind to the Na + channel but are unable to stimulate 22Na + entry. These inactive compounds behave as antagonists of the active pyrethroids. An electrophysiological approach has shown that pyrethroids by themselves are active on the Na + channel of mammalian neurones, and essentially confirm the conclusions made from 22Na + flux measurements. Pyrethroids are also active on C9 cells in which Na + channels are ‘silent’, that is, not activatable by electrical stimulation. Pyrethroids chemically activate the silent Na + channel in a manner similar to that with veratridine, batrachotoxin, or polypeptide toxins, which are known to slow down the inactivation process of a functional Na + channel.

Protein binding to two-dimensional hydrophobic binding-site lattices: adsorption hysteresis on immobilized butyl-residues

Long-lived metastable states involving multiple binding sites of a protein ligand with immobilized alkyl residues on a solid phase can be observed at high ionic strength between butyl agaroses (5.21 μ mol/ml packed gel) and phosphorylase b by perturbations enforcing either the on-reaction (adsorption) or the off-reaction (desorption). These apparent equilibrium states are suggested because the adsorption isotherms of phosphorylase b on butyl agaroses are not retraced by the desorption isotherms. In this first example of macromolecular adsorption hysteresis on immobilized alkyl residues, it can be shown that the irreversible entropy ( Δ i S) produced in an adsorption-desorption cycle lies between ∼6 (5 μ mol/ml packed gel) and ∼40 (21 μ mol/ml packed gel) J mol 1 K −1. For the latter gel the apparent standard entropy of adsorption ( Δ a S i 0′) is 160 J mol −1 K −1. The metastable state observed during adsorption is probably due to an energy barrier which must be overcome for the nucleation of protein binding on the matrix. Other metastable states may possibly be encountered during desorption when the adsorbed enzyme resists the breakage of hydrophobic interactions. In the transition from the adsorption branch to the desorption branch of the hysteresis loop, the apparent affinity of the enzyme-matrix interaction is enhanced. For the desorption branch, the apparent association constant of half-maximal saturation corresponds to K d,0.5′ = 4.2 × 10 9 ] m −1 as compared to the respective constant of adsorption K a, 0.5′ = 1.6 × 10 5 m −1 (gel: 21μ mol/ml packed gel). Since the area of the hysteresis loops (see also Δ i S) depends strongly on the density of butyl residues on the gel, it is concluded that the number of alkyl residues interacting with the protein molecule is crucial for the metastable states and hysteresis. It is unlikely that hysteresis is due to the pore structure of the agarose or to nearest neighbour interactions of ligand molecules. Since thermodynamic irreversibility and hysteresis may be encountered when macromolecules, such as proteins, are adsorbed to cell membranes or cell organelles: an analysis and understanding of these phenomena should be of general biological significance.

Agonist regulation of the human platelet prostaglandin D 2 receptor

The effect of exposing platelets to prostaglandin D 2 (PGD 2) on hormone binding was studied. Incubation of platelets with PGD 2 for 2 hr resulted in a decrease in [ 3H]PGD 2 binding that was dose dependent. Inhibition of binding was 14% after incubation with 10 −8M PGD 2, 19% after incubation with 10 −7M PGD 2, and 40% after exposure to 10 −6M PGD 2. This decreased binding (desensitization) was specific for [ 3H]PGD 2 as binding to platelets by [ 3H]PGE 1 and the α-adrenergic antagonist [ 3H] dihydroergocryptine (DHEC) was comparable to control platelets. Saturation of [ 3]PGD 2 binding to desensitization platelets was at 27 fmole ligand/10 8 platelets compared to 43 fmoles/10 8 platelets in control platelets. Half-maximal saturation occured at 20 nM PGD 2 both for desensitized and control platelets, suggesting that decreased binding sites rather than altered affinity between ligand and receptor accounted for these results. These platelets had a partial increase in [ 3H]PGD 2 binding a few hours after plasma was washed free of PGD 2 with complete resensitization after 24 hr. Since prostaglandins such as PGI 2, PGD 2, and PGE 1 are potent inhibitors of platelet aggregation, decreased binding of platelets to these hormones after prostaglandin exposure may provide a mechanism for altered responsiveness of platelets to aggregating stimuli.

The β-Adrenergic Receptor of Newborn Mouse Skin

Binding of the potent beta-adrenergic antagonist [125I]iodohydroxybenzylpindolol ([125I]IHYP) to particulate preparations from newborn mouse skin was characterized. A number of criteria were used to establish that binding occurred to specific, high affinity beta-adrenergic receptors in the skin preparations. Thus specific binding (that displaced by 10 micrometer concentrations of the beta-adrenergic antagonist (-)propranolol) reached equilibrium in 15--20 min, was saturable (ligand concentration for half-maximal saturation, 0.25 nM) and freely reversible. Stereoselectivity of binding was demonstrated by the observation that displacement of [125I]IHYP by (-)propranolol occurred at concentrations at least 100 times lower than with (+)isoproterenol. Displacement was also observed with the beta-adrenergic agonists (-)isoproterenol, (-)epinephrine and (-)norepinephrine, but not with the alpha-adrenergic antagonist phentolamine.

Insulin receptor: Interaction with nonreceptor glycoprotein from liver cell membranes

In crude receptor preparations (either particulate or soluble) of rat liver membranes, the insulin receptor exhibits complicated binding kinetics (two binding plateaus, half-saturated at approximately 60 pM and 700 pM insulin) and an apparent chromatographic heterogeneity, suggested by the presence of two detectable, soluble insulin-binding components with apparent Stokes radii of 72 A and 38 A. In contrast, the insulin receptor isolated by affinity chromatography exhibits a simple binding isotherm (half-maximal saturation of binding at 700 pM insulin) without evidence for negative cooperativity and behaves as a single component (apparent Stokes radius of 38 A) upon chromatography on Sepharose 6B. The apparent discrepancies between the properties of the unpurified insulin receptor and the affinity-purified receptor can be attributed to the presence in crude preparations of a nonreceptor constituent(s) having properties consistent with those of a membrane glycoprotein. A glycoprotein fraction from such crude soluble membrane preparations, freed from insulin receptor and subsequently partially purified using concanavalin-A-agarose, when combined with affinity-purified insulin receptor, causes both a reappearance of the complicated binding kinetics and an increase in the receptor's apparent Stokes radius from 38 A to 72 A. Similar results are observed for a glycoprotein fraction obtained from rat adipocyte membranes but are not observed for an identical fraction isolated from human erythrocyte membranes. We conclude that the insulin receptor in rat liver membranes can interact with another nonreceptor membrane glycoprotein that may represent either a nonrecognition moiety of the receptor oligomer or an effector molecule to the biological action of insulin.

Absorption of Colostral Immunoglobulins by the Neonatal Calf

SUMMARY The time course of the appearance of immunoglobulin classes G, M and A were measured in the plasma of suckled newborn calves. Under natural conditions considerable amounts of immunoglobulin had appeared by 3 h after suckling. At least 5 h were required for half maximal saturation levels to be achieved. There were differences in the rate of appearance of the individual immunoglobulin classes, with IgG appearing before both IgM and IgA. The implication of these findings is discussed in relation to protection of the calf against infective agents.