Solubilization and partial purification of the high-affinity gonadoliberin receptor from the bovine pituitary gland

The high-affinity gonadoliberin (GnRH) receptor contained in a membrane preparation from frozen bovine anterior pituitary glands has been solubilized in Triton X-100 and the binding properties of the solubilized product have been examined. The radioreceptor-binding assay, using the GnRH agonist [D-Ser(t-Bu)⁶] des-Gly¹⁰GnRH N-ethylamide (GnRH-A) as radioligand, demonstrated that the kinetics of association and dissociation, the binding constants, as well as the specificity of receptor were not altered in the solubilized receptor preparations. Affinity chromatography on a concanavalin A-Sepharose column, with elution of adsorbed material using a solution of α-methyl-d-mannoside, allowed a 33-fold purification of the receptor. The K_a of the receptor thus purified was of the same order as that of the starting material, although slightly higher values were found. Only about one-half of the total receptor activity applied to the column was retained in spite of several recyclings. The other half was found in the nonadsorbed fraction. It is postulated that the detergent-solubilized fraction contains two forms of the GnRH receptor. The nonadsorbed fraction probably contains a partially or totally deglycosylated form. It is possible that the detergent-solubilization process somewhat alters the physicochemical properties of a part of the GnRH receptor molecules. Electrophoretic analysis of the purified receptor preparations, with a subsequent GnRH-binding assay, suggests that the apparent molecular mass of the high-affinity GnRH receptor, or of its monomeric form, is approximately 60,000 Da.     

Purification of guinea pig liver transglutaminase using a phenylalanine-sepharose 4B affinity column

Guinea pig liver transglutaminase has been purified utilizing an improved protocol. The new steps include QAE-Sephadex ion-exchange, hydroxylapatite adsorption, and affinity chromatography using a phenylalanine-Sepharose column. The overall yield for the enzyme was 31%. This new scheme should enable more laboratories to take advantage of the protein crosslinking and labeling properties of transglutaminase.     

Isolation and biochemical characterization of nuclei from immature and mature guinea pig granulocytes

Intact nuclei were isolated in high yield from enriched fractions of immature and mature guinea pig granulocytic leukocytes. These nuclei were used to determine whether any changes in synthesis and content of nuclear proteins accompany the striking increase in chromatin condensation and the nuclear lobation which occur during granulocyte maturation. The results indicate that the synthesis of nuclear proteins and the nuclear RNA content decrease markedly during granulocyte maturation. The incorporation of l-[U-¹⁴C]leucine into the acid-soluble histone-rich fraction of chromatin from immature cells is about 25 times that of mature cells, and the incorporation into the acid-insoluble, nonhistone proteins of chromatin from immature cells is about 6 times that of mature cells. It appears that there is very little quantitative change with respect to the protein components of nuclei from immature and mature granulocytic leukocytes. No significant differences in the amounts of histone, nonhistone protein, or phosphoprotein between nuclei of immature and mature granulocytes could be detected. No major differences in gel electrophoretic patterns of histones or nonhistone proteins could be detected. The fact that the amount of the chromatin proteins remains relatively constant during cell maturation in spite of the pronounced decrease in the rate of synthesis suggests that the rate of turnover of these proteins decreases significantly as the maturation of granulocytic leukocytes proceeds.     

Induction of acetylcholine receptor synthesis and aggregation: partial purification of low-molecular-weight activity

We have studied the effect of saline and acid extracts of chick brain on the total number of acetylcholine (ACh) receptors and the number of receptor clusters in cultured chick muscle cells. Myotubes in 7-day cultures responded more rapidly to brain extract than did myotubes in 4-day cultures, so the older cells were used in subsequent bioassays. A large percentage of the receptor inducing activity was soluble in 2% trifluoroacetic acid (TFA), and this material appeared by Sephadex G-25 chromatography to be about 1000 daltons in size. Activity was retained on octadecasilyl silica and was further purified by reverse-phase high-pressure liquid chromatography using a TFA-acetonitrile gradient system. Material that eluted between 35 and 40% acetonitrile, termed C4018, was 500- to 1000-fold more potent than saline extract. The receptor accumulation induced by C4018 was associated with an increased rate of receptor incorporation, presumably receptor synthesis, rather than to a decrease in receptor degradation. An increase in incorporation was detected as early as 3 hr after C4018 was added to 7-day cultures and the effect was maximal after 10 hr. C4018 also promoted the aggregation of receptors that were already incorporated in the surface membrane at the time to addition. It is not yet known if aggregation of "old" receptors and increased receptor synthesis are related or if the two phenomena are mediated by the same molecule.     

Effects of several calcium antagonists and dipyridamole in the isolated perfused guinea pig heart

Three calcium (Ca) antagonists and dipyridamole were examined in the isolated perfused guinea pig heart at perfusate Ca concentrations of 1.25 and 3.75 mM. The Ca antagonists: FR 7534, nifedipine and D600 produced similar dose-dependent decreases in left ventricular dp/dt and myocardial oxygen consumption (MV?O₂) at both Ca concentrations. However, dose response curves were shifted significantly to the right by increased perfusate Ca requiring six to ten times more Ca antagonist to produce equivalent effects. Dipyridamole produced only slight negative inotropic effects which appeared to be less dependent on external Ca concentration. All four agents significantly increased coronary blood flow at 1.25 mM Ca but not at 3.75 mM Ca. The Ca antagonists decreased heart rate at 3.75 mM Ca whereas dipyridamole had strong negative chronotropic effects at both perfusate Ca concentrations. These experiments provide evidence that FR 7534 acts as a Ca antagonist. In addition, Ca antagonists of different structure had similar effects on the isolated heart distinct from those of dipyridamole.     

Water-soluble acetylcholine receptor from Torpedo californica. Solubilization,purification and characterization

The nicotinic acetylcholine receptor from electrogenic tissue of Torpedo californica was solubilized by tryptic digestion of membrane fragments obtained from autolysed tissue, without use of detergent. The water-soluble acetylcholine receptor was purified by affinity chromatography on a cobra-toxin-Sepharose resin. The purified receptor bound 4000–6000 pmol per mg protein of α-[¹²⁵]bungarotoxin, and toxin-binding was specifically inhibited by cholinergic ligands. Gel filtration revealed a single molecular species of Stokes radius $\text{125 ± 10}$ Å and on sucrose gradient centrifugation one major peak was observed of 20–22 S. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and β-mercaptoethanol revealed two major polypeptides of mol. wt. 30 000 and 48 000.     

Solubilization and characterization of vitamin K epoxide reductase from normal and warfarin-resistant rat liver microsomes

Two procedures have been developed for the solubilization of vitamin K epoxide reductase from rat liver microsomal membranes using the detergent Deriphat 160 at pH 10.8. The methods are applicable to both normal and Warfarin-resistant-strain rat liver microsomes and yield material suitable for further purification. The preparations retain dithiothreitol-dependent vitamin K quinone reductase activity as well as vitamin K epoxide reductase and are free of vitamin K-dependent carboxylase and epoxidase activities. Optimal epoxide reductase activity is obtained at 0.1 M KCl and pH 9 in the presence of sodium cholate. Artifactual formation of vitamin K metabolites was eliminated through the use of mercuric chloride to remove excess dithiothreitol prior to extraction and metabolite assay. Using the solubilized enzyme, valid initial velocities were measured, and reproducible kinetic data was obtained. The substrate initial velocity patterns were determined and are consistent with a ping-pong kinetic mechanism. The kinetic parameters obtained are a function of the cholate concentration, but do not vary drastically from those obtained using intact microsomal membranes. At 0.8% cholate, the enzymes solubilized from normal Warfarin-sensitive- and Warfarin-resistant-strain rat livers exhibit respective values of Vmax = 3 and 0.75 nmol/min/g liver; Km for vitamin K epoxide = 9 and 4 microM; and Km for dithiothreitol of 0.6 and 0.16 mM.     

Solubilization of ligand-stabilized vasopressin receptors from plasma membranes of bovine kidney and rat liver

The solubilization of vasopressin receptors from plasma membranes of bovine kidney and rat liver by different detergents was investigated. A prerequisite for the extraction of vasopressin receptors retaining binding affinity for their ligand was the stabilization of the receptors by the prior formation of the membrane-bound hormone-receptor complexes. The vasopressin-receptor complexes from both kidney and liver membranes were solubilized in a high yield with dodecyl-beta-D-maltoside and 3-laurylamido-N,N'-dimethylpropylaminoxide. Several other nonionic detergents including octyl-beta-D-glucopyranoside effectively extracted the hepatic vasopressin receptor. For the hormone-receptor complex solubilized from bovine kidney with dodecyl-beta-D-maltoside, a Stokes' radius of 5.8 nm was determined.     

3,4,5,3',4'-Pentachlorobiphenyl as a useful inducer for purification of rat liver microsomal cytochrome P448.

An easy purification of rat liver microsomal cytochrome P448 was performed by using 3,4,5,3′,4′-pentachlorobiphenyl as an inducer. The cytochrome P448, a high spin form, was purified to 18.1 nmoles/mg protein with a good yield by ω-aminooctyl Sepharose 4B column chromatography followed by a hydroxyapatite column chromatography. This hemoprotein cross-reacted with antibody to cytochrome P448 from β-naphthoflavone-treated rats, but not with antibody to cytochrome P450 from phenobarbital-treated rats at all. The results of amino acid analyses suggested that this cytochrome P448 is similar to cytochrome P448 of 3-methylcholanthrene-treated rats.     

Specific binding and pharmacological interactions of apamin,the neurotoxin from bee venom,with guinea pig colon

This paper describes the interaction of apamin, the bee venom neurotoxin, with its receptor in the guinea pig colon. The pharmacological activity of the toxin was assayed by measuring its contracting effect on guinea pig colon preparations that had been previously relaxed by neurotensin. The IC₅₀ value of apamin in this $\text{in vitro}$ bioassay is 7 nM. These pharmacological data are compared to the binding properties of apamin to smooth muscle membranes prepared from guinea pig colon. The highly radiolabeled monoiododerivative of apamin binds to its colon receptor with a dissociation constant $\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}^1\text{= 36}\text{pM}$. The maximal binding capacity of colonic membranes is 30^dfmol/mg of protein. The dissociation constant of the unmodified toxin is 23 pM. The difference between the toxin concentrations that produce half-maximal effects in the binding and pharmacological studies arises from the different experimental conditions used for the two assays.     

Purification and characterization of the individual glutathione S-transferases from sheep liver

The glutathione S-transferases (EC 2.5.1.18) have been purified to electrophoretic homogeneity from 105,000g supernatant of sheep liver homogenate by employing a combination of gel filtration on Sephadex G-150 and affinity chromatography on S-hexylglutathione-linked Sepharose-6B columns. Approximately 70% of the original glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide could be recovered by this purification method. Of particular importance in developing this procedure was the fact that the enzyme preparation obtained after affinity column chromatography represented all the isozymes of sheep liver glutathione S-transferases. Further purification by CM-cellulose and DEAE-cellulose column chromatography resolved the glutathione S-transferases into seven distinct cationic isozymes designated C-1, C-2, C-3, C-4, C-5, C-6, and C-7 and five overlapping anionic transferases designated A-1, A-2, A-3, A-4, and A-5, respectively, in the order of their elution from the ion-exchange columns. The sodium dodecyl sulfate SDS-gel electrophoretic data on subunit composition revealed that cationic enzymes are composed of two subunits with an identical Mr of 24,000 whereas a predominant subunit with Mr of 26,000 was observed in all anionic isozyme peaks except A-1. Cationic isozymes accounted for approximately 98% of the total peroxidase activity associated with the glutathione S-transferase whereas only A-1 of the anionic isozymes displayed some peroxidase activity. Isozyme C-4 was found to be the most abundant glutathione S-transferase in the sheep liver. Characterization of the individual transferases by their specificity toward a number of selected substrates, subunit composition, and isoelectric points showed some similarities to those patterns for human liver glutathione S-transferases.     

Structural glycoprotein from the media of pig aorta. Aggregation of the S-carboxamidomethyl subunits.

Media of pig aorta was extracted with 1 M NaCl and 2 M MgCl2 to remove most of the soluble collagen, proteoglycans and glycoproteins. The glycoproteins remaining in the residue were extracted with 6 M urea-0.1 M mercaptoethanol. The urea soluble proteins were precipitated by dialysis, redissolved in 4 M guanidine-0.05 M DTT and were S-carboxamidomethylated (CM-guanidine extract). This extract was further fractionated by a variety of methods in order to separate a glycoprotein from collagen and proteoglycans. Caesium chloride density-gradient ultracentrifugation of the CM-guanidine extract separated a minor proteoglycan peak from a major glycoprotein fraction still containing some hydroxyproline. This major glycoprotein fraction was excluded as a single peak from Sephadex G 100 and G 200 in 4 M guanidinium chloride or in 6 M urea-0.2 per cent SDS. Sodium dodecylsulphate gel electrophoresis separated this high molecular weight Sephadex fraction into a major low molecular weight (approximately 35000 daltons) component and a minor high molecular weight component. This glycoprotein fraction could also be separated from a collagenous fraction and from proteoglycans by ion exchange chromatography on DEAE cellulose or by gelfiltration on Sepharose 4 B in 6 M urea-0.02 M EDTA-0.2 per cent SDS at pH 7.0. The isolated glycoprotein fraction is rich in dicarboxylic amino acids, contains galactose, mannose, (glucose), N-acetylglucosamine and sialic acid. The S-carboxamidomethyl glycoprotein preparation interacts with acid soluble calf skin collagen on isoelectric focusing in sucrose gradient in urea. This interaction is in favour of the biological role claimed for structural glycoproteins during fibrogenesis and differentiation.     

Special features of Pi transport in pig heart and rat liver mitochondria as revealed by 6,6'-dithiodinicotinic acid (CPDS).

CPDS (6,6'-dithiodinicotinic acid), a non permeant thiol agent which affects several mitochondrial functions in a way different to that of mersalyl [18-19] revealed striking differences between the phosphate translocating systems of pig heart and rat liver mitochondria. Pi entry was measured either by swelling in 0.12 M ammonium phosphate or by rapid centrifugation in 32Pi medium. Pi efflux was measured after preloading of mitochondria with 32Pi, by exchange against Pi or malate; the "ATP-FCCP" system has been tested previously [19]. In pig heart mitochondria, Pi entry seems to proceed exclusively via the Pi/OH- carrier; CPDS completely inhibits this transport and the energy-linked functions. In contrast n-butyl-malonate does not affect the Pi-entry and the energy-linked functions. The Pi efflux is not affected either by CPDS or mersalyl, which do not produce a swelling in the "ATP-uncoupler system". In rat liver mitochondria, CPDS inhibits only the Pi/OH- carrier; both CPDS and n-butylmalonate are necessary to inhibit completely Pi entry. CPDS as well as mersalyl provokes a swelling in the presence of the "APT-uncoupler system". The results suggest two distinct functions of phosphate transport in both types of mitochondria.     

Solubilization,purification, and properties of rabbit brain hexokinase

More than 90% of the total hexokinase activity in rabbit brain was found to be associated with the mitochondrial fraction. The participate enzyme was solubilized in a relatively specific way by glucose 6-phosphate and Triton X-100 and purified to apparent homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and affinity chromatography. The solubilized hexokinase activity has been purified 700-fold in 48% yield with a specific activity of 165 units/mg of protein. The molecular weight was found to be approximately 100,000 both for the native and the denatured enzyme. The isoelectric point, pI, was 6.3 pH units by isoelectric focusing and the enzyme was found to be able to phosphorylate several hexoses with different affinities. Mg · ATP, among the nucleotide substrates, was the most effective as a phosphate donor. The present results indicate considerable similarity between this enzyme and the other mammalian type I hexokinases.     

Isoenzymes of an acyltransferase from rabbit mammary gland: Solubilization of the micelle-specific species with Triton X-100

The micelle-specific palmityl-CoA: monopalmityl-sn-glycerol 3-phosphate palmityltransferase isoenzyme from lactating rabbit mammary gland microsomes is selectively solubilized in Triton X-100 but not Tween 80. Both detergents inactivate the monomer-specific isoenzyme. The possibility that, $\text{in}$$\text{vivo}$, physiological surfactants regulate the relative activities of these two isoenzymes is discussed.     

Isolation and characterization of an anionic glutathione S-transferase from rat liver cytosol

An anionic glutathione S-transferase representing approximately 20% of the total glutathione S-transferase protein and 10% of the total transferase activity toward 1-chloro 2,4-dinitrobenzene has been purified to homogeneity from the 105,000 x g supernatant of rat liver homogenate. The SDS gel electrophoretic data on subunit composition revealed that the anionic isozyme is composed of two subunits with an identical Mr of 26,000. The Km values for 1-chloro 2,4-dinitrobenzene and reduced glutathione were determined to be 0.94 mM and 0.23 mM respectively. A significant amount of glutathione peroxidase activity toward cumene hydroperoxide is associated with the new isozyme.