Studies in vitro on the uptake and degradation of sodium hyaluronate in rat liver endothelial cells.

Rat liver endothelial cells in primary cultures at 7 degrees C bind radioactively labelled sodium hyaluronate (HA; Mr 400 000) specifically and with high affinity (Kd = 6 X 10(-11) M). Maximal binding capacity is approx. 10(4) molecules per cell. Inhibition experiments with unlabelled HA and oligosaccharides from HA indicate that each molecule is bound by several receptors acting co-operatively and that the single receptor recognizes a tetra- or hexa-saccharide sequence of the polysaccharide. At 37 degrees C the liver endothelial cells endocytose the HA. The process combines the features of a receptor-mediated and a fluid-phase endocytosis. The rate of internalization does not show any saturation with increasing HA concentration, but is approximately proportional to the polysaccharide concentration at and above the physiological concentration. At 50 micrograms of free HA/l each liver endothelial cell accumulates 0.1 fg of the polysaccharide/min. Fluorescent HA accumulates in perinuclear granules, presumably lysosomes. Degradation products from HA appear in the medium about 30 min after addition of the polysaccharide to the cultures. The radioactivity from HA containing N-[3H]acetyl groups or 14C in the sugar rings is recovered mainly as [3H]acetate and [14C]acetate respectively. Estimations of the capacity of liver endothelial cells to internalize and degrade HA in vitro indicate that these cells may be primarily responsible for the clearance of HA from human blood in vivo.     

Concentration and relative molecular mass of hyaluronate in lymph and blood.

Human lymph was collected from patients with leaking lymph vessels after thoracic surgery. Ovine lymph was obtained from the mesenteric, lumbar, popliteal and prescapular lymph ducts by cannulation. The concentration of hyaluronate varied considerably (between 0.2 and 50 mg/l) and the highest concentrations were found in mesenteric lymph. The Mr of the polysaccharide showed a great polydispersity and variation between individuals and in different regions of the lymphatic system. High-Mr hyaluronate (greater than 10(6) was present in lymph both from man and sheep. Hyaluronate was also isolated by affinity chromatography in 70-80% yield from human serum and plasma obtained from healthy individuals and patients with rheumatoid arthritis and primary biliary cirrhosis. The weight (Mw)- and number (Mn)-average relative molecular masses were roughly the same in the three groups [(1.4-2.7) X 10(5) and (2.1-5.7) X 10(4) respectively]. The low Mr of hyaluronate in blood compared with that in lymph is explained by a preferential uptake of the large molecules by the liver endothelial cells.     

The expressed human hepatic receptor for low-density lipoproteins differs from the fibroblast low-density lipoprotein receptor

The role of the cellular receptor for the low-density lipoproteins (LDL) in cholesterol transport was initially defined through the study of nonhepatic cells in vitro. Since the liver is central in plasma lipoprotein metabolism, an investigation of hepatic lipoprotein receptors is important for understanding normal lipoprotein transport. Utilizing human hepatic and fibroblast membranes, the characteristics of receptors for LDL from hepatic and nonhepatic tissues were directly compared. Human hepatic membranes reversibly bound LDL within 5 min. Although both fibroblast and hepatic membranes saturably bound LDL at 37 degrees C, the fibroblast LDL receptor affinity (Kd = 2.5 X 10(-8) M) and number (5.5 X 10(12) sites/mg membrane protein) were greater than the hepatic receptor affinity (Kd = 10.8 X 10(-8) M) and number (0.5 X 10(12) sites/mg membrane protein). In contrast to the fibroblast LDL receptor which was unable to bind LDL in the presence of EDTA, the hepatic LDL receptor binding of LDL was only partially blocked by EDTA. The binding of LDL to its hepatic receptor is highly temperature-dependent, and studies utilizing both radiolabeled LDL and colloidal gold-labeled LDL indicate that little, if any, binding of LDL hepatic membranes occur at 0-4 degrees C. The hepatic membrane receptor(s) (Mr approximately equal to 270 000 and 330 000) differ from that of the fibroblast LDL receptor (Mr approximately equal to 130 000) and these proteins are present in hepatic membranes from a patient lacking the fibroblast LDL receptor. These data indicate that an expressed hepatic LDL receptor has unique properties different from those of the fibroblast LDL receptor and that the expressed protein(s) is genetically distinct from the fibroblast receptor.     

Affinity and distribution of surface and intracellular hyaluronic acid receptors in isolated rat liver endothelial cells

125I-Hyaluronic acid (HA) uniquely modified only at the reducing end (Raja, R.H., LeBoeuf, R. D., Stone, G.W., and Weigel, P.H. (1984) Anal. Biochem. 139, 168-177) binds specifically to rat liver endothelial cells in suspension or in culture. About 67-85% of the HA binding sites in isolated cells in suspension and 50% in cultured cells were intracellular, since they were exposed after permeabilizing cells with digitonin. Specific 125I-HA binding at 4 degrees C varied from 60 to 80% for intact cells and from 70 to 90% for permeabilized cells. Freshly isolated permeabilized cells bound about 500,000 HA molecules/cell at saturation. Within 5 h of culture, however, total HA binding decreased to 250,000 molecules/cells and then remained constant for at least 36 h. Surface HA receptor activity was essentially the same on cultured cells or cells in suspension (approximately 10(5)/cell). Cultured cells had 1.8 x 10(5) fewer intracellular receptors/cell. The affinities of surface and intracellular receptors of cells in culture and in suspension were essentially the same. The average Kd, determined by equilibrium binding studies, was 5.8 +/- 2.8 x 10(-8) M (n = 12). Dissociation of bound 125I-HA from permeable cultured cells was rapid (t1/2 = 30.9 min;kappa off = 3.7 x 10(-4) s-1). A variety of carbohydrates had essentially identical effects on 125I-HA binding to surface or total cellular receptors in cells in culture or in suspension. Chondroitin sulfate and heparin competed almost as effectively as unlabeled HA for 125I-HA binding at 4 degrees C. Other saccharides including polygalacturonic acid, dextran, glucuronic acid, and N-acetylglucosamine competed poorly or not at all. We conclude that (i) the 125I-HA binding sites within liver endothelial cells are HA receptors, identical in affinity and specificity to those on the cell surface; (ii) the distribution of cellular HA receptors is similar to other receptor systems with about 50-80% being intracellular; (iii) the liver endothelial cell HA receptor recognizes several glycosaminoglycans; and (iv) the liver endothelial receptor is different in function and characteristics than the fibroblast HA receptor.     

Kinetic aspects of hemoglobin.haptoglobin-receptor interaction in rat liver plasma membranes, isolated liver cells, and liver cells in primary culture

125I-Hemoglobin.haptoglobin injected intravenously into rats was incorporated into liver parenchymal cells as evidenced by a cell separation technique. A mixture of freshly isolated liver parenchymal and nonparenchymal cells failed to internalize and degrade the 125I-hemoglobin.haptoglobin added, although it retained the ability to bind the molecule. The liver parenchymal cells in primary culture also lacked the ability to degrade 125I-hemoglobin.haptoglobin, although they bound the molecule more extensively as compared with the freshly isolated liver cells. It was confirmed that the 125I-hemoglobin.haptoglobin which was bound to the freshly isolated liver parenchymal cells localized on the outer surface of liver plasma membranes. Scatchard plots revealed the existence of two binding sites for 125I-hemoglobin-haptoglobin on the isolated liver plasma membrane: an apparent high affinity binding site (Kd = 1.3 X 10(-7) M) and an apparent low affinity binding site (Kd = 4.0 X 10(-6) M) at 37 degrees C. In contrast, freshly isolated liver parenchymal cells had only an apparent low affinity binding site (Kd = 1.4 X 10(-6) M) at 37 degrees C. Impairment of the apparent high affinity binding site during the isolation procedure with collagenase seemed to be related to loss of the ability to internalize and degrade the 125I-hemoglobin.haptoglobin molecules into the freshly isolated liver parenchymal cells or liver parenchymal cells in primary culture.     

Comparative kinetics of phosphomannosyl receptor-mediated pinocytosis of fibroblast secretion acid hydrolases and glycopeptides prepared from them

In a previous report we demonstrated that phosphorylated oligosaccharides isolated from acid hydrolases were subject to pinocytosis by phosphomannosyl receptors present on the cell surface of human fibroblasts [9]. However, limiting quantities of oligosaccharides precluded detailed comparison of the kinetics of pinocytosis of these phosphorylated oligosaccharides to those of the acid hydrolases from which they were derived. In this report we present studies comparing the kinetics of pinocytosis of acid hydrolases from NH4Cl-induced fibroblast secretions with those of concanavalin A-binding glycopeptides prepared from them by pronase digestion. The uptake of both secretion acid hydrolases and 125I-labeled glycopeptides was linear for at least 3 hr, saturable, inhibited competitively by mannose 6-phosphate, and destroyed by prior treatment of the ligand with alkaline phosphatase. The inhibition constants of excess unlabeled glycopeptide for the uptake of 125I-labeled glycopeptides (Ki of 1.5 X 10(-6) M) and for the uptake of secretion acid hydrolases (Ki of 2.2 X 10(-6) M) were remarkably similar. Furthermore, the Ki for mannose 6-phosphate inhibition of pinocytosis of glycopeptide uptake (3 X 10(-5) M) compares closely to that previously determined for the pinocytosis of intact "high-uptake" acid hydrolases (3-6 X 10(-5) M). "High-uptake" fractions of both ligands were prepared and quantified by affinity chromatography on immobilized phosphomannosyl receptors purified from bovine liver. Only 10% of the concanavalin A-binding glycopeptides bound to the immobilized phosphomannosyl receptors, while 80% of the acid hydrolases from which they were prepared bound and were eluted with 10 mM mannose 6-phosphate. However, the fraction of each type of ligand that binds to the immobilized phosphomannosyl receptors accounts for all the uptake activity of that ligand.     

Characterization of the asialoglycoprotein receptor in a continuous hepatoma line

The asialoglycoprotein receptor has been identified on a continuous human hepatoma cell line, HepG2. This receptor requires Ca2+ for ligand binding and is specific for asialoglycoprotein. There are approximately 150,000 ligand molecules bound/cell at 4 degrees C. These receptors represent a homogeneous population of high affinity binding sites with Kd = 7 X 10(-9) M. From the rate of 125I-ASOR binding at 4 degrees C, kon was 0.95 X 10(6) M-1 min-1. Uptake of 125I-ASOR at 37 degrees C was approximately 0.02 pmol/min/10(6) cells.     

Aortic endothelial cells synthesize a large chondroitin sulphate proteoglycan capable of binding to hyaluronate.

Confluent cultures of mouse aortic endothelial (END-D) were incubated with either [35S]methionine or 35SO4 2-, and the radiolabelled proteoglycans in media and cell layers were analysed for their hyaluronate-binding activity. The proteoglycan subfraction which bound to hyaluronate accounted for about 18% (media) and 10% (cell layers) of the total 35S radioactivity of each proteoglycan fraction. The bound proteoglycan molecules could be dissociated from the aggregates either by digestion with hyaluronate lyase or by treatment with hyaluronate decasaccharides. Digestion of [methionine-35S]proteoglycans with chondroitinase and/or heparitinase, followed by SDS/polyacrylamide-gel electrophoresis, indicated that the medium and cell layer contain at least three chondroitin sulphate proteoglycans, one dermatan sulphate proteoglycan, and two heparan sulphate proteoglycans which differ from one another in the size of core molecules. Among these, only the hydrodynamically large chondroitin sulphate species with an Mr 550,000 core molecule was shown to bind to hyaluronate. A very similar chondroitin sulphate proteoglycan capable of binding to hyaluronate was also found in cultures of calf pulmonary arterial endothelial cells (A.T.C.C. CCL 209). These observations, together with the known effects of hyaluronate on various cellular activities, suggest the existence of possible specialized functions of this proteoglycan subspecies in cellular processes characteristic of vascular development and diseases.     

A hepatic reticuloendothelial cell receptor specific for SO4-4GalNAc beta 1,4GlcNAc beta 1,2Man alpha that mediates rapid clearance of lutropin.

We have identified a receptor in hepatic endothelial and Kupffer cells that binds oligosaccharides terminating with the sequence SO4-4GalNAc beta 1,4GlcNAc beta 1,2-Man alpha (S4GGnM). This receptor can account for the rapid removal of the glycoprotein hormone lutropin, which bears unique Asn-linked oligosaccharides terminating in S4GGnM, from the circulation. Hepatic endothelial cells express 579,000 S4GGnM receptors at their surface and bind lutropin with an apparent Kd of 1.63 x 10(-7) M. Bound ligand is rapidly internalized. Binding does not require divalent cations, is reversed by incubation at pH 5.0 or below, and is inhibited by fucoidin but not by hyaluronate, heparin, chondroitin sulfate, or dextran sulfate. We propose that the S4GGnM-specific receptor represents a major mechanism for clearance of certain sulfated glycoproteins from the blood, including members of the glycoprotein hormone family.     

Ligand interactions of the cation-dependent mannose 6-phosphate receptor. Comparison with the cation-independent mannose 6-phosphate receptor

The interactions of the bovine cation-dependent mannose 6-phosphate receptor with monovalent and divalent ligands have been studied by equilibrium dialysis. This receptor appears to be a homodimer or a tetramer. Each mole of receptor monomer bound 1.2 mol of the monovalent ligands, mannose 6-phosphate and pentamannose phosphate with Kd values of 8 X 10(-6) M and 6 X 10(-6) M, respectively and 0.5 mol of the divalent ligand, a high mannose oligosaccharide with two phosphomonoesters, with a Kd of 2 X 10(-7) M. When Mn2+ was replaced by EDTA in the dialysis buffer, the Kd for pentamannose phosphate was 2.5 X 10(-5) M. By measuring the affinity of the cation-dependent and cation-independent mannose 6-phosphate receptors for a variety of mannose 6-phosphate analogs, we conclude that the 6-phosphate and the 2-hydroxyl of mannose 6-phosphate each contribute approximately 4-5 kcal/mol of Gibb's free energy to the binding reaction. Neither receptor appears to interact substantially with the anomeric oxygen of mannose 6-phosphate. The receptors differ in that the cation-dependent receptor displays no detectable affinity for N-acetylglucosamine 1'-(alpha-D-methylmannopyranose 6-monophosphate) whereas this ligand binds to the cation-independent receptor with a poor, but readily measurable Kd of about 0.1 mM. The spacing of the mannose 6-phosphate-binding sites relative to each other may also differ for the two receptors.     

Initial steps in Haemophilus influenzae transformation. Donor DNA binding in the com10 mutant

The com10 mutant of Haemophilus influenzae binds donor DNA reversibly, but is deficient in uptake. The DNA binding has all the characteristics of interaction with a protein receptor; it is saturable, reversible, and specific. However, binding specificity is 6-fold weaker in com10 than is uptake specificity in wild-type. The binding of small (120 base pairs) and large (14,400 base pairs) DNA molecules were compared. For small molecules, binding data fitted a straight line by Scatchard analysis (Bmax = 4.8 DNA molecules/cell, Kd = 0.5 X 10(-9) M). In contrast, for large DNA molecules, the Scatchard plot was not linear. A high affinity binding (Kd = 0.4 X 10(-12) M) and a lower affinity binding (Kd = 1.2 X 10(-11) M) were found with a total number of 3 molecules bound per cell. In wild-type cells, 3.2 large molecules were taken up per cell, whereas up to 40 small 120-base pair DNA fragments were taken up per cell. Uptake of small DNA molecules followed a Michaelis-Menten function with a Km of 0.5 X 10(-9) M and a maximal initial velocity of 1.5 molecules/cell/min at room temperature. For large DNA molecules, maximal initial velocity was approximately 2 molecules/cell/min at room temperature. The analysis of the binding and uptake data suggest to us that a receptor or a receptor complex is responsible for the uptake of either a single large DNA molecule or, successively, a number of small DNA molecules.     

Human fibrinogen specifically binds hyaluronic acid

Fibrin and hyaluronic acid (HA) are macromolecules whose concentrations are elevated at the same time in the extracellular space of damaged tissues. We have investigated whether HA can bind to fibrinogen using solid phase and soluble assays. Purified human fibrinogen specifically bound to HA-Sepharose to a greater extent (greater than 5-fold) than did alpha 1-acid glycoprotein, DNaseI, ovalbumin, haptoglobin, or lysozyme. Fibrinogen did not bind to ethanolamine-Sepharose, a control chromatographic support. Treatment of HA-Sepharose containing bound 125I-fibrinogen with ovine testicular hyaluronidase released 44% of the 125I radioactivity, indicating that fibrinogen was specifically bound to HA. Moreover, 125I-fibrinogen bound to HA-Sepharose could be displaced by free HA but not by either of the monosaccharide components of this polymer, glucuronic acid, or N-acetylglucosamine. Chondroitin sulfate and polygalacturonic acid competed only weakly for bound 125I-fibrinogen. Bound 125I-fibrinogen was also not released by high concentrations of NaCl (up to 4 M), indicating that the interaction is not simply ionic. The apparent affinity of fibrinogen for HA covaried with the molecular weight of the HA. Small HA oligosaccharides (Mr = 3900) were only 50% as effective as larger HA (Mr = 8 X 10(5)) in eluting bound 125I-fibrinogen from HA-Sepharose. The optimal oligosaccharide size for displacement of bound 125I-fibrinogen was greater than or equal to 200 monosaccharides. Additionally, the amount of 125I-fibrinogen bound to HA-Sepharose was directly related to the size of the HA-amine linked to the affinity support. The affinity constant for fibrinogen binding to 125I-HA (approximately 150 monosaccharides) is estimated to be at least 2 X 10(7) M-1. These results demonstrate for the first time a specific, reversible binding between HA and fibrinogen.     

A cytoplasmic oestrogen-binding component in chicken liver.

A macromolecular component in the liver cytosol from laying hens as well as roosters, protein in nature and sedimenting at 4S, was shown to bind oestradiol. The dissociation constant (Kd) of the complex is approximately 5 X 10(-6)M. No binding component with a higher affinity for oestradiol was detectable in the cytosol. The binding is specific for the tissue and hormone, with the exception that progesterone also shows some affinity for this 4S component. The number of binding sites is about 330 pmol/mg cytosol protein. This number is not altered significantly after treatment of a rooster with oestrogen (24 h) or with cycloheximide (3 h). The cytoplasmic complex (oestradiol-4S-component) does not enhance the binding of oestradiol to the chromatin from rooster liver. The nuclear complex (oestradiol bound to the soluble nuclear receptor seems to be more effective in doing so.     

Galanin receptor in the rat pancreatic beta cell line Rin m 5F. Molecular characterization by chemical cross-linking

125I-Galanin was cross-linked to receptor in Rin m 5F cell membranes using the bifunctional reagent disuccinimidyl tartarate. Regardless of the presence of reducing agents, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cross-linked galanin-receptor complexes revealed the presence of a radioactive band at Mr 57,000. Excess unlabeled galanin completely inhibited the labeling of the band while other regulatory peptides had no effect. Labeling of the Mr 57,000 complex was abolished by galanin concentration from 10(-9) to 10(-6) M (IC50 = 5 X 10(-9) M). Initial incubation with 125I-galanin in the presence of increasing concentrations of guanyl-5'-yl imidodiphosphate (GMP-P(NH)P) (10(-7) to 10(-4) M) also inhibited the labeling of the Mr 57,000 complex. Moreover, pretreatment of membranes with pertussis toxin before formation of the covalent galanin-receptor complex, dramatically reduced the labeling of the Mr 57,000 species. Covalent Mr 57,000 galanin-receptor complexes solubilized by Triton X-100 bound specifically to wheat germ agglutinin-concanavalin A-, and soybean-coupled Sepharose, supporting the glycoproteic nature of the galanin receptor. Assuming one molecule of 125I-galanin (Mr 3,000) was bound per molecule of protein, these results suggest that the pancreatic galanin receptor is a glycoprotein with a Mr of 54,000 bearing the recognition site for the ligand and which is coupled with a pertussis toxin-sensitive G protein in the plasma membrane.     

Endothelial binding of transferrin in fractionated liver cell suspensions

Several studies using crude liver cell suspensions incubated with labeled transferrin have led to a conclusion that hepatocytes have transferrin receptors. When a visual probe, which permits evaluation of transferrin binding to individual cells, was used, the binding was unexpectedly found to be limited to endothelial cells in liver cell suspensions. Neither hepatocytes nor Kupffer cells contained transferrin receptors. In the present study, we fractionated liver cell suspensions using metrizamide gradients and centrifugal elutriation to obtain hepatocytes, Kupffer cell and endothelial cell fractions of high purity. Incubation of these fractions with 125I- or 59Fe-labeled transferrin led to exclusive binding to endothelial cells but not hepatocytes nor Kupffer cells. Kinetic analysis demonstrated Kd of 1.9 X 10(-7) M, Bmax of 3.1 pmol/10(6) cells per min, corresponding to 2.1 X 10(5) molecules/cell per min. At 4 degrees C, the binding reached a steady-state plateau within 5 min. Comparison of our data with those of previous investigators demonstrates a consistency if we consider that crude liver cell suspensions are contaminated with 2-3% endothelial cells. Thus, the previously reported findings may be entirely due to the contamination of crude liver cell suspensions with a small number of endothelial cells.     

Characterization and regulation of alpha-interferon receptor expression in interferon-sensitive and -resistant human lymphoblastoid cells

Interferon-sensitive (IFN-S) and IFN-resistant (IFN-R) Daudi lymphoblastoid cells were studied for IFN-alpha receptor expression and regulation by steady state and kinetic procedures, utilizing a homogeneous 125I-IFN-alpha 2 probe. Heterogeneity in the binding of this probe to IFN-S cells was determined to result from negatively cooperative interactions between an initially homogeneous class of IFN receptor. No such heterogeneity was noted in the IFN-R cells, indicating an apparent difference in the interaction of IFN-alpha 2 with these cells. The apparent dissociation constants (Kd) for IFN-S cell receptors were calculated to be 1 X 10(-10) M and 1 X 10(-8) M, for the high and low affinity sites, respectively. The Kd for sites on the IFN-R cells was estimated to be 4 X 10(-9) M. IFN-R and IFN-S cells expressed 2.4 X 10(4) and 3.5 X 10(4) binding sites per cell, respectively, representing an increase of at least 6-fold over previous reports of IFN-S Daudi IFN receptor density. Both IFN-S and IFN-R cells were capable of down-regulating expression of the IFN-alpha receptor in response to low concentrations of IFN-alpha 2. Furthermore, both cell lines were shown to be capable of internalizing specifically bound 125I-IFN-alpha 2 to an equivalent degree. Accordingly, we propose that the relative insensitivity of the Daudi IFN-R phenotype involves the loss of a high affinity interaction between cellular receptors and IFN-alpha 2, in addition to the reduced level of expressed low affinity binding sites.     

Biosynthesis of proteoglycans and their assembly into aggregates in cultures of chondrocytes from the Swarm rat chondrosarcoma.

Cultured chondrocytes from the Swarm rat chondrosarcoma incorporate [35S]sulfate into proteoglycans typical of hyaline cartilage. The movement of newly synthesized proteoglycans from inside the cells into the extracellular matrix and, finally, into the culture medium was examined by measuring the distribution of 35S-labeled proteoglycans in the medium, a 4 M guanidine HCl extract of the cell layer, and in the remaining residue for a number of chase times following a 5-min pulse with [35S]sulfate. When hyaluronate oligosaccharides containing greater than or equal to 10 monosaccharides were included in the chase media, a proportion of newly synthesized proteoglycans were displaced from the matrix (4 M extract) into the culture medium. This displacement was greatest when oligomers were in the chase media between 10 and 20 min after the pulse, approximately the time when the molecules are being secreted from the cells. The proportion of link-stabilized aggregate in the medium was examined by Sepharose 2B chromatography after adding an excess of unlabeled monomer which displaces labeled monomer from complexes with hyaluronate which are not link-stabilized. The proportion of link-stabilized aggregate increased from 12% to about 70% between 12 and 120 min of chase. The presence of 40 micron hyaluronate oligosaccharides of 16 monosaccharides in the chase media retarded but did not prevent aggregate formation. Oligomers of about 50 monosaccharides, which are large enough to bind both a monomer proteoglycan and a link protein, almost completely prevented the formation of the large link-stabilized aggregates. The results suggest: (a) newly synthesized proteoglycans are not bound into link-stabilized aggregates at the time of secretion; (b) hyaluronic acid oligomers which are long enough to interact only with the hyaluronic acid-binding site of proteoglycans will retard but not prevent link-stabilized aggregation; and (c) hyaluronic acid oligomers long enough to accommodate additionally a link protein form a link-stabilized ternary complex and prevent aggregation with larger hyaluronic acid molecules.     

Molecular mechanisms of olfactory reception. VI. Kinetic characteristics of camphor interaction with binding sites of rat olfactory epithelium

Camphor binding to a possible receptor of rat olfactory epithelium has been studied within the ligand concentration range 10(-11)-10(-6) M. At these concentrations camphor is bound by a set of receptors. They are distinguished by both the affinity to the ligand (K1 = 5 X 10(-10) M, K2 = 3.5 X 10(-8) M, K3 approximately equal to 10(-6) M) and their amount in the epithelium. The differences in the affinities are due to different values of the association rate constant of camphor (k1), which varies from 10(6) M-1 X s-1 for the receptors with high affinity up to 2 X 10(2) M-1 X s-1 for those with low affinity. These data are discussed in terms of equilibrium and kinetic models of the receptor-stimulus interaction.     

Increase of (Ca2++Mg2+)-ATPase activity of renal basolateral membrane by parathyroid hormone via cyclic AMP-dependent membrane phosphorylation

Studies were made on the mechanism of the effect of parathyroid hormone (PTH) on the activity of (Ca2++Mg2+)-ATPase, a membrane bound Ca2+-extrusion pump enzyme from the basolateral membranes (BLM) of canine kidney (Km for free Ca2+ = 1.3 X 10(-7) M, Vmax = 200 nmol Pi/mg/min). At 1 X 10(-7) M free Ca2+, both PTH (10(-7)-10(-6) M) and cAMP (10(-6)-10(-4) M) stimulated (Ca2++Mg2+)-ATPase activity dose-dependent and their stimulatory effects were inhibited completely by 5 microM H-8, an inhibitor of cAMP-dependent protein kinase. PTH (10(-7) M) also caused 40% increase in 32P incorporation into the BLM and 5 microM H-8 inhibited this increase too. PTH (10(-7) M) was found to stimulate phosphorylation of a protein of Mr 9000 by cAMP dependent protein kinase and 5 microM H-8 was found to block this stimulation also. From these results, it is proposed that PTH stimulates (Ca2++Mg2+)-ATPase activity by enhancing its affinity for free Ca2+ via cAMP-dependent phosphorylation of a BLM protein of Mr 9000.     

Ca2+-activated ATPase in microsomes from human liver

Human liver microsomal fractions exhibit ATP-supported Ca2+ uptake which is half-maximal at 7 X 10(-7) M free Ca2+ in the presence of oxalate. Ca2+ uptake is coupled to a Ca2+-stimulated ATPase activity, which is half-maximal at 4 X 10(-7) M free Ca2+. Catalysis involves formation of an Mr = 116,000 phosphoprotein with stability characteristics of an acylphosphate compound suggested to represent a phosphoryl protein intermediate of the Ca2+-ATPase. Phosphorylation is half-maximal at about 10(-6) M free Ca2+. The Mr = 116,000 protein is highly susceptible to proteolysis with trypsin. The phosphorylated active site was localized in an Mr = 58,000 primary tryptic fragment and in an Mr = 34,000 subfragment. Analyses on the mechanism of the Ca2+-ATPase suggest the following reaction sequence: formation of an ADP-reactive phosphoenzyme (Mr = 116,000) with bound Ca2+, which can transphosphorylate its Pi to ADP, giving rise to synthesis of ATP; reversible transformation of the ADP-reactive phosphoenzyme into an isomer without bound Ca2+, which cannot further react with ADP; hydrolytical cleavage, probably catalyzed by Mg2+, of the ADP-unreactive phosphoenzyme with liberation of Pi. Comparison with the Ca2+-transport ATPase in sarcoplasmic reticulum of skeletal muscle led us to suggest that the Mr = 116,000 Ca2+-ATPase belongs to the class of E1P . E2P-ATPases and might be operative as a Ca2+-transport ATPase at the level of the endoplasmic reticulum in human liver.