Characterization of a lipid-associated gonadotropin receptor from the luteinized rat ovary

Gonadotropin receptors which bind luteinizing hormone (lutropin) and human chorionic gonadotropin (hCG) in the ovaries of immature female rats showed a 30-fold increase after treatment of animals with pregnant mare serum gonadotropin (PMSG) and hCG. This marked induction of lutrophin/hCG receptors in the rat ovary was not accompanied by a change in binding affinity for labeled hCG. Such luteinized ovaries have been found consistently to contain a small proportion of soluble receptor sites, which comprised about 5% of the total receptor population. The soluble receptor sites were present in the floating lipid fraction of the 360 000 × g supernatant of homogenate prepared from luteinized ovaries, and could not be detected in similar fractions prepared from interstitial cells or homogenates of the normal rat testis.The physico-chemical properties of the spontaneously soluble ovarian receptors were similar to those derived for detergent-solubilized receptors prepared by extraction of particulate ovarian binding fractions with Triton X-100. The affinity constant to the soluble ovarian receptor sites for [¹²⁵I]hCG was 0.70 · 10¹⁰ M^?1, and that of the receptors solubilized by Triton X-100 was 0.72 · 10¹⁰ M^?1. The sedimentation pattern of the soluble receptors during sucrose density gradient centrifugation showed extensive aggregation into rapidly sedimenting forms. However, centrifugation of the cytosol receptor in the presence of Triton X-100 gave a single 6.5 S component, corresponding to the solubilized receptors previously characterized in detergent extracts of the rat ovary and testis.The pesence of a spontaneously soluble lutropin/hCG receptor in ovarian cytosol fractions suggests that rapid synthesis and assembly of receptors in ovaries of PMSG-hCG-treated rats is accompanied by increased production of cytoplasmic receptor precursors; alternatively, this receptor population may represent a fraction that has been internalized or processed as during receptor turnover in the cell membrane.     

Reconstitution of Solubilized Serotonin-S2 Receptors into Phospholipid Vesicles

Abstract

Serotonin-S₂ receptors from rat frontal cortex were solubilized using CHAPS/sodium chloride. Reconstitution of the solubilized receptors was achieved by dilution of the soluble preparation, followed by centrifugation to remove the detergent. The receptors were truly reconstituted as judged by sedimentation, increased thermostability and electron microscopy. The reconstituted preparation showed high-affinity binding of [³H]7-aminoketanserin. The binding characteristics resembled those obtained for membrane-bound receptors.     

Solubilization and Characterization of High Affinity Follicle-Stimulating Hormone Receptors from Porcine Granulosa Cells

Abstract

Follicle-stimulating hormone (FSH)-receptors were solubilized from immature porcine ovarian granulosa cells with retention of high affinity ¹²⁵I-porcine FSH-binding activity. The optimal concentration of Triton X-100 for solubilization was 0.5% (w/v), and the optimal cellular protein concentration 25 mg/ml. Glycerol (30%) increased recovery of solubilized receptor. ¹²⁵I-pFSH-binding affinity ranged from 4 × 10¹⁰ M^?1 to 8 × 10¹⁰ M^?1 inn either the absence or presence of glycerol. ¹²⁵I-pFSH-binding capacity was 5 fmol/mg protein in the absence of glycerol and 58 fmol/mg protein in the presence of glycerol as determined by equilibrium saturation binding analysis. By gel permeation chromatography, the apparent size of the ¹²⁵I-pFSH-receptor complex was 462 kDa in the absence of glycerol and 762 kDa in the presence of glycerol. Ligand blotting of solubilized receptor yielded a single species with an apparent molecular weight of 200 kDa under nonreducing conditions and a single species with an apparent molecular weight of 60 kDa under reducing conditions. These studies indicated that high affinity FSH-binding activity can be solubilized from membranes of immature porcine granulosa.     

Solubilization of High-Affinity, Guanine Nucjeotide-Sensitive μ-Opioid Receptors from 7315c Cell Membranes

Abstract: High-affinity μ-opioid receptors have been solubilized from 7315c cell membranes. Occupancy of the membrane-associated receptors with morphine before their solubilization in the detergent 3-[(3-cholamidopropyl) dimethyl]-1-propane sulfonate was critical for stabilization of the receptor. The solubilized opioid receptor bound [³H]-etorphine with high affinity (K_D= 0.304 ± 0.06 nM; B_max= 154 ± 33 fmol/mg of protein). Of the membrane-associated [³H]etorphine binding sites, 40 ± 5% were recovered in the solubilized fraction. Both μ-selective and non-selective enkephalins competed with [³H]etorphine for the solubilized binding sites; in contrast, 5- and K-opioid enkephalins failed to compete with [³H]etorphine for the solubilized binding sites at concentrations of <1 μM.The μ-selective ligand [³H][D-Ala²,A/-Me-Phe⁴,Gly⁵-ol]enkephalin also bound with high affinity (K_D= 0.79 rM; B_max= 108±17 fmol/mg of protein) to the solubilized material. Of the membrane-associated [³H][D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin binding sites, 43 ± 3% were recovered in the solubilized material. Guanosine 5′-O-(3-thiotriphosphate), GTP, and guanosine 5′-O-(2-thiodiphosphate), but not adenylylimidodiphosphate, diminished [³H][D-Ala²,N-Me-Phe⁴,Gly⁵-ol]enkephalin binding in a concentration-dependent manner. Finally, μ-opioid receptors from rat brain membranes were also solubilized in a high-affinity, guanine nucleotide-sensitive state if membrane-associated receptors were occupied with morphine before and during their solubilization with the detergent 3-[(3-cholamidopropyl) dimethyl]-1-propane sulfonate.     

TYROSINE HYDROXYLASE IN BOVINE CAUDATE NUCLEUS

Approximately 80 per cent of tyrosine hydroxylase activity in bovine caudate nucleus was particle-bound. The rest of the activity was found in the soluble fraction. The enzyme activity in crude tissue preparations was inhibited, probably by the presence of endogenous inhibitors. Dilution of crude tissue preparations such as the crude mitochondrial fraction caused an increase in the specific activity. The particle-bound enzyme was solubilized by incubation with trypsin. The presence of deoxycholate increased the degree of solubilization. The activity of the solubilized enzyme from the washed particles was also inhibited, but the subsequent purification by ammonium sulphate could eliminate the inhibition. The solubilized enzyme was partially purified by ammonium sulphate fractionation and Sephadex G-150 chromatography. A tetrahydropteridine and ferrous ion were required as cofactors for the partially purified enzyme. Among various divalent cations, only ferrous ion could activate the partially purified enzyme. The enzyme was inhibited by L-α-methyl-p-tyrosine and catecholamines such as dopamine. The optimum pH was found between 5.5 and 6.0. K_m values toward tyrosine, 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine and Fe²⁺, were approximately 5 × 10^?5 M, 1 × 10^?4 M and 4 × 10^?4 M, respectively.     

Water Soluble Receptors for Human Growth Hormone from Rabbit Liver

Abstract

Soluble receptors that bind human growth hormone have been prepared by incubation of liver membranes from pregnant female rabbits in 1 mM Tris buffer (pH 7.5 or 9.0) at 4°C. Up to 29% of the growth hormone binding sites could be solubilized within 48 hours. The kinetics of binding of human growth hormone to the soluble receptor, the hormonal specificity and the binding parameters calculated by Scatchard analysis (K_a 2.2 × 10⁹ M^-1, capacity 409 fmole/mg) were essentially unchanged compared with those for the parent membrane-associated (particulate) receptor. Gel filtration on Ultrogel AcA22 indicated that the major binding peak eluted at a molecular weight of 300,000 daltons. Specificity studies showed that the soluble binding sites had a moderately high affinity for ovine prolactin (K_a ～1 × 10⁸ M^-1), but negligible affinity for insulin. Although aqueous extraction gives a lower yield of binding sites for human growth hormone than detergent extraction, it nevertheless avoids some of the problems associated with use of detergents and should facilitate the subsequent purification of the receptor in a relatively unaltered state. It may also have applicability for solubilization of other hormone receptor systems.     

Solubilization of muscarinic acetylcholine receptor by zwitterionic detergent from rat brain cortex

The muscarinic acetylcholine receptor was solubilized from rat brain cortex by zwitterionic detergent 3-[(3-chloramidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). About 15% of the binding activity was solubilized and 40% of the activity was destroyed by the detergent. Binding of the muscarinic antagonist [³H]-N-methyl-4-piperidyl benzilate (4NMPB) was saturable. Scatchard analysis revealed a single population of binding sites with K_D value of 0.7 nM and a B_max value of 340 fmoles/mg protein. The homogenate and the CHAPS treated pellet and soluble receptors showed similar affinity for the agonists oxotremorine and carbamylcholine and for the antagonists QNB and atropine. The dissociation of 4NMPB from the soluble receptors appears slightly slower than from the membrane bound receptors.     

Solubilized active pituitary and ovarian gonadotropin-releasing hormone receptors retain binding properties for adenosine 3′, 5′-cyclic monophosphate derivatives

The non-denaturing zwitterionic detergent, (3 (3-cholamidopropyl)-dimethyl-ammonio)-1-propane sulfonate (CHAPS), has been used to solubilize membrane gonadotropin-releasing hormone (GnRH) receptors from rat ovaries. The solubilized receptors retain a high affinity (K_a = 1.85 ± 0.3 nM^?1), comparable to the affinity measured in membrane particles (K_a = 3.25 ± 0.7 nM^?1), and a preserved specificity for several analogs and fragments of GnRH. At millimolar concentrations, cyclic AMP derivatives inhibit [125_I] - GnRH analog binding to both membrane particles and soluble receptors from pituitary and ovary. These results support the hypothesis that cyclic AMP may play the role of an extracellular messenger by interacting with the GnRH receptor itself.     

Solubilization of pituitary receptors for thyrotropin-releasing hormone

Receptors for thyrotropin-releasing hormone were solubilized by Triton X-100. Membrane fractions from GH₃ pituitary tumor cells were incubated with thyrotropin-releasing hormone in order to saturate specific receptor sites before the addition of detergent. The amount of protein-bound hormone solubilized by Triton X-100 was proportional to the fractional saturation of specific membrane receptors. Increasing detergent: protein ratios from 0.5 to 20 led to a progressive loss of hormone · receptor complex from membrane fractions with a concomitant increase in soluble protein-bound hormone. The soluble hormone · receptor complex was not retained by 0.22 μm filters and remained soluble after ultracentrifugation. Following incubation with high (2.5–10%) concentration of Triton X-100 and other non-ionic detergents, or following repeated detergent extraction, at least 18% of specifically bound thyrotropin-releasing hormone remained associated with particulate material. Unlike the hormone receptor complex, the free hormone receptor was inactivated by Triton X-100. A 50% loss of binding activity was obtained with 0.01% Triton X-100, corresponding to a detergent: protein ratio of 0.033.The hormone · receptor complex was included in Sepharose 6B and exhibited an apparent Stokes radius of 46 Å in buffers containing Triton X-100. The complex aggregated in detergent-free buffers. Soluble hormone receptors were separated from excess detergent and thyrotropin-releasing hormone by chromatography on DEAE-cellulose. Thyrotropin-releasing hormone dissociated from soluble receptors with a half-time of 120 min at 0°c, while the membrane hormone · receptor complex was stable for up to 5 h at 0°C.     

MONOAMINE OXIDASE (EC 1.4.3.4): ISOLATION AND CHARACTERIZATION OF MULTIPLE FORMS OF THE BRAIN ENZYME

Monoamine oxidase (MAO) in crude mitochondrial preparations from rat brain was solubilized, and different MAO-active fractions were separated by agarose columns and by Sephadex electrophoresis. Any combination of these techniques yielded at least three fractions possessing MAO activity as measured by assays using radioactive serotonin and benzylamine as substrates. The molecular weight of one of the MAO forms was found to be approximately 400,000 daltons while another was at least 1.5 × 10⁶ daltons. The crude mitochondria1 MAO was inhibited by [¹⁴C]-labelled pargyline and then solubilized and the radioactivity of the soluble and particulate MAO was compared to the enzyme activity found in the soluble and particulate fractions. Our studies suggest that appreciable MAO activity is lost upon solubilization and that the conformation of MAO may be altered.     

Beta-Adrenergic receptors: solubilization of (-)(3H)alprenolol binding sites from frog erythrocyte membranes.

The β-adrenergic receptors ((?)[³H]alprenolol binding sites) present in a purified preparation of frog erythrocyte membranes have been solubilized with digitonin and assayed by equilibrium dialysis with (?)[³H]alprenolol. At a concentration of 0.5–1% the detergent solubilizes about 80% of the receptor binding activity. The soluble receptor sites are not sedimented at centrifugal forces up to 105,000 xg for two hours, pass freely through Millipore filters of 0.22 μ pore size and fractionate on Sepharose 6B gel with an apparent molecular weight of 130–150,000 in the presence of digitonin. The soluble receptor sites retain all of the binding characteristics of the membrane-bound receptors. β-adrenergic agonists and antagonists compete with (?)[³H]alprenolol for occupancy of the soluble sites with affinities which are directly related to their β-adrenergic potency on membrane-bound adenylate cyclase.     

Detergent solubilization of mammalian cardiac and hepatic beta-adrenergic receptors.

The solubilization of canine cardiac and hepatic β-adrenergic receptors was characterized with 19 different ionic and nonionic surfactants and surfactant combinations. The effects of alterations in the hydrophobic and hydrophilic moieties of the nonpolar detergents were examined in relation to their efficacy in solubilizing these receptor molecules. Within this group of surfactants the more effective agents contained an average of 9–10 polyoxyethylene units per molecule. The best degree of β-receptor solubilization for both heart and liver was obtained with 1% Brij 96 or a combination of 1% digitonin with 0.25% Triton X-100. Hepatic but not cardiac β-receptors were solubilized by polyoxyethylene ether W-1 or by Triton X-100. Solubilization time courses indicated that the maximum degree of receptor solubilization occurred within 5 min at 0–4 °C. Solubilization temperatures were varied from 0 to 37 °C. Temperatures up to 30 °C increased numbers of cardiac receptors solubilized by 30% over those obtained at 0 °C. The same temperature changes had no significant effects on liver β-receptor solubilization. Increasing the solubilization temperature to 37 °C decreased the detectable number of β-receptors by 91 (liver) and 72% (heart). β-Receptors solubilized in the absence of receptor ligand were extremely labile with a half-life on the order of 90 min at 4 °C for both heart and liver. Occupation of the receptors by [¹²⁵I]-iodohydroxybenzylpindolol prior to solubilization conferred a certain degree of stability on the receptors.     

Opiate Receptors from Different Tissue Sources: Solubilization and Characterization

Abstract: Membrane-bound opiate receptors from neuroblastoma-glioma hybrid cells and from different parts of the rat brain (whole brain minus cerebellum, cortex, thalamus-hypothalamus and cerebellum) were labeled with the methionine-enkephalin analogue, D-[³H]Ala²-Met-enkephalinamide, and solubilized with the nonionic detergent Brij 36T. The protease inhibitors bacitracin, phenylmethylsulfonyl fluoride, Trasylol, and leupeptin were included in the solubilization buffer to minimize proteolysis. Two simple techniques, ammonium sulfate precipitation and activated charcoal absorbence, were adapted to separate the free and the macromolecule-bound ligands. The solubilized receptor-[³H]enkephalin complexes were partially purified by consecutive passages through Sephadex G-75 and Sepharose 6B columns. Of the three peaks of radioactivity that were observed in the effluent of the Sepharose column, two contained proteins, and one of them, with a Stokes radius of 59 Å, seemed to contain the specific opiate receptor, as evidenced by additional experiments. This peak was further purified on thiol-Sepharose or diethylaminoethanol-Sephadex columns that were eluted with a gradient of 0–50 mM dithiothreitol or with 1.0 M KCI, respectively. The receptor-[³H]enkephalin complex from neuroblastoma-glioma cells (apparent δ-type receptors) binds less to the thiol-Sepharose beads than receptor-(³H]enkephalin prepared from the hypothalamus-thalamus, which is rich in μ receptors. The [³H]enkephalin receptor complexes of the various sources also differed in their stability. The dissociation of the ligand from the neuroblastoma-glioma receptor was monophasic, with a half- life of 250 min, whereas that of two brain regions was biphasic, with half-lives of 195–330 min and 10,000 min. The methods described may be of use for further purification of soluble opiate receptors, either active or cross-linked to the ligand.     

Solubilization of High-Affinity,Guanine Nucleotide-Sensitive μ-Opioid Receptors from Rat Brain Membranes

Abstract: High-affinity μ-opioid receptors have been solubilized from rat brain membranes. In most experiments, rats were treated for 14 days with naltrexone to increase the density of opioid receptors in brain membranes. Occupancy of the membrane-associated receptors with morphine during solubilization in the detergent 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate appeared to stabilize the μ-opioid receptor. After removal of free morphine by Sephadex G50 chromatography and adjustment of the 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate concentration to 3 mM, the solubilized opioid receptor bound [³H][d -Ala²,N-Me-Phe⁴,Gly-ol⁵]-enkephalin ([³H]DAMGO), a μ-selective opioid agonist, with high affinity (K_D = 1.90 ± 0.93 nM; B_max = 629 ± 162 fmol/mg of protein). Of the membrane-associated [³H]-DAMGO binding sites, 29 ± 7% were recovered in the solubilized fraction. Specific [³H]DAMGO binding was completely abolished in the presence of 10 µM guanosine 5′-O-(3-thiotriphosphate). The solubilized receptor also bound [³H]diprenorphine, a nonselective opioid antagonist, with high affinity (K_D = 1.4 ± 0.39 nM, B_max = 920 ± 154 fmol/mg of protein). Guanosine 5′-O-(3-thiotriphosphate) did not diminish [³H]diprenorphine binding. DAMGO at concentrations between 1 nM and 1 µM competed with [³H]diprenorphine for the solubilized binding sites; in contrast, [d -Pen²,d -Pen⁵]-enkephalin, a δ-selective opioid agonist, and U50488H, a κ-selective opioid agonist, failed to compete with [³H]diprenorphine for the solubilized binding sites at concentrations of <1 µM. In the absence of guanine nucleotides, the DAMGO displacement curve for [³H]diprenorphine binding sites better fit a two-site than a one-site model with K_Dhigh = 2.17 ± 1.5 nM, B_max = 648 ± 110 fmol/mg of protein and K_Dlow = 468 ± 63 nM, B_max = 253 ± 84 fmol/mg of protein. In the presence of 10 µM guanosine 5′-O-(3-thiotriphosphate), the DAMGO displacement curve better fit a one- than a two-site model with K_D = 815 ± 33 nM, B_max = 965 ± 124 fmol/mg of protein.     

Ultraviolet radiation,thiol reagents,and solubilization enhance AMPA receptor binding affinity via a common mechanism

The binding properties of membrane-bound or solubilized AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid)-type glutamate receptors from rat brain were tested following exposure to ultraviolet (UV)_radiation or incubation with the thiol reagent p-chloromercuriphenyl-sulfonic acid (PCMBS). Brief exposure to UV radiation (254 nm) increased [³H]AMPA binding to brain membranes, while binding to soluble fractions decreased. The increase in brain membrane binding was caused by an apparent interconversion of low-affinity [³H]AMPA binding sites into a higher-affinity state. Incubation with PCMBS caused a significant increase in [³H]AMPA binding to brain membranes but had no significant effect on [³HAMPA binding to solubilized receptors. There was an interaction between the PCMBS and UV effects in the brain membranes such that prior exposure to one of the treatments reduced the relative magnitude of the other's effects. The present results suggest that ultraviolet radiation, PCMBS and solubilization all increase AMPA receptor binding affinity via a common mechanism.     

Nerve growth factor receptors on PC12 cells: Evidence for two receptor classes with differing cytoskeletal association

PC12, an NGF responsive cell line, exhibits two classes of NGF receptors which we designate “Fast” and “Slow.” Fast receptors, accounting for 75% of specific NGF binding, are distinguished by their rapid rates for association and dissociation of ¹²⁵I-NGF. At 37°C, binding of ¹²⁵I-NGF to Fast receptors is 5-fold more rapid than to Slow receptors and dissociation of ¹²⁵I-NGF from Fast receptors is 40-fold more rapid than from Slow receptors. No evidence was obtained for a ligand-induced conversion of receptors from Fast to Slow characteristics. Scatchard analysis of binding experiments indicates that PC12 cells possess 60,000 specific receptors for NGF of which 15,000 are of the Slow class. Despite having very different kinetic constants, Slow and Fast receptors have similar equilibrium binding constants (about 2 × 10^?10 M) due to cancelling effects of differing association and dissociation rates. Brief digestion of PC12 cells with trypsin before addition of NGF inactivates essentially all Fast receptors without significantly affecting Slow receptors. Therefore Fast and Slow classes of receptors must exist prior to addition of NGF, and the observed receptor heterogeneity is not due to ligand-induced changes. ¹²⁵I-NGF bound to Slow receptors is preferentially associated with preparations of Triton X-100 insoluble cytoskeletons, while ¹²⁵I-NGF bound to Fast receptors is solubilized by this procedure. Cytoskeletally associated NGF is almost exclusively associated with the extranuclear cytoskeletal matrix rather than with the nucleus itself. Preparation of nuclei by various methods suggests that the presence of contaminating cytoskeletal elements should be considered in evaluating the existence of translocation and binding of NGF to the nucleus. Inhibition of endocytotic internalization of NGF either by lowering of temperature to O°C or by preincubation of cells with sodium azide in medium lacking glucose does not reduce the slowly released component of bound NGF, nor alter its cytoskeletal association. The possible functional roles of Slow and cytoskeletal receptors are discussed.     

Solubilization of functional and stable follitropin receptors from light membranes of bovine calf testis

Rapid destabilization of FSH receptor after solubilization by detergents is a serious problem complicating its purification and further study. We have developed a procedure for the solubilization of stable and functional FSH receptors with Triton X-100. The new protocol selectively utilizes pure lighter membranes isolated from bovine calf testes by preparative sucrose density gradient centrifugation as the source of receptor. The conditions of detergent solubilization were optimized to reduce the required ratio of Triton X-100 to membrane protein to a minimum. In addition, during detergent extraction the membranes were treated with petroleum ether to remove interfering neutral lipids, thus facilitating solubilization of FSH receptors by the detergent. FSH receptors so obtained appeared to be soluble by criteria such as failure to sediment at 145,000 X g after 90 min, passage through 0.22-micron Millipore filters, and retardation upon chromatography on Sepharose 6B column. Approximately 86% of receptors originally present in the light membranes were recovered after solubilization, with a 24-fold increase in specific activity. The detergent-soluble fraction has several interesting properties not previously reported. It contains only high affinity receptors for FSH (Ka = 1.02 X 10(10) M-1), which are stable in the absence of glycerol for 4 days at 1 degree C or 6 months at -80 degrees C. Luteinizing hormone and human chorionic gonadotropin receptor activity usually associated with detergent-solubilized extracts of testes is low due to incomplete solubility of these receptors under the conditions utilized for solubilization of FSH receptors. Of particular interest is the ability of the receptor in the detergent extract to respond to added FSH with stimulation of adenylate cyclase activity. Adenylate cyclase activity also responds to F- stimulation and the detergent extract retains full guanosine 5'-imidotriphosphate-binding activity. This suggests that under the extraction conditions employed, a high proportion of soluble receptors are associated with related components of the adenylate cyclase system. Our data are consistent with the notion that the solubilized hormone-binding sites represent the physiologically relevant and functional receptors originally present in the light membrane fraction of calf testis. The availability of this detergent-soluble, stable and functional receptor fraction in larger amounts (2.2 g of protein from each batch of 11.5 kg bovine calf testes) than heretofore possible should facilitate further studies on FSH receptor purification and its mechanism of action.     

Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase

The Rhodospirillum rubrum pyridine dinucleotide transhydrogenase system is comprised of a membrane-bound component and an easily dissociable soluble factor. Active transhydrogenase complex was solubilized by extraction of chromatophores with lysolecithin. The membrane component was also extracted from membranes depleted of soluble factor. The solubilized membrane component reconstituted transhydrogenase activity upon addition of soluble factor. Various other ionic and non-ionic detergents, including Triton X-100, Lubrol WX, deoxycholate, and digitonin, were ineffectual for solubilization and/or inhibited the enzyme at higher concentrations. The solubilized membrane component was significantly less thermal stable than the membrane-bound component. None of the pyridine dinucleotide substrate affected the thermostability of the solubilized membrane-bound component, whereas NADP⁺ and NADPH afforded protection to membrane-bound component. NADPH stimulated trypsin inactivation of membrane-bound component to a greater extent than NADP⁺, but inactivation of solubilized membrane component was stimulated to the same extent by both pyridine dinucleotides. The solubilized membrane component appears to have a slightly higher affinity for soluble factor than does the membrane-bound component.Abbreviations AcPyAD⁺ oxidized 3-acetylpyridine adenine dinucleotide - BChl bacteriochlorophyll - C_T-particles chromatophores depleted of soluble transhydrogenase factor and devoid of transhydrogenase activity This work was supported by Grant GM 22070 from the National Institutes of Health, United States Public Health Service. Paper I of this series is R. R. Fisher et al. (1975)     

Influence of magnesium in the homogenization medium on the state of a divalent cation-stimulated,diethystilbestrol-sensitive adenylnucleotidyl phosphatase activity from pea stem microsomal preparations

The amount of divalent cation-activated, diethylstilbestrol-sensitive adenylnucleotidyl phosphatase activity recovered in the ‘microsomes’ ($\text{13 000–80 000 x g}\text{sediment}$) from pea stem tissue is strongly influenced by the concentration of Mg²⁺ in the homogenization medium. The absence of Mg²⁺ during homogenization results in a marked decrease of the activity found in the microsomal fraction, compensated by its increase in the soluble fraction. Part of the solubilized activity becomes sedimentable at $\text{80 000 × g}$ upon addition of 5–10 mM Mg²⁺ (or Mn²⁺, Ca²⁺, Zn²⁺) to the supernatant. This sediment shows a very high specific activity, and can be re-solubilized by treatment with either EDTA or 0.3 M monovalent salts, or deoxycholate. When the supernatant containing the solubilized activity is incubated together with low-adenylnucleotidyl phosphatase microsomes and with 10 mM MgCl₂ the activity recovered in the sediment is much larger than the sum of the activity of the microsomes plus that of the sediment obtained by incubating the same supernatant with Mg²⁺. Microsomes prepared with Mg²⁺ in the homogenization medium do not show this effect. The supernatant/microsomes saturation curves as well as a change of the temperature coefficient of the activity following combination of the soluble preparation with the microsomal particles suggest an at least partial reconstitution of the original enzyme-membrane structure.     

Tea Leaf Polyphenol Oxidase

The large part of the polyphenol oxidase was solubilized from tea leaf homogenate by addition of Tween-80. After filtration of the solubilized polyphenol oxidase fraction through a Sephadex G-25 column and fractionation of the filtrate with ammonium sulfate, the specific activity of the solubilized enzyme increased about 4 to 5 times as much as that of tea leaf homogenate. Optimum pH of the solubilized enzyme was 5.5, and was almost the same as that of water-insoluble enzyme in the acetone powder. The minimum concentrations required for the maximum activity were about 5×10^?3 m, 4.3×10^?3 m, and 3×10^?3 m for d-catechin, l-epigallocatechin, and l-epigallocatechin-gallate, respectively. d-Catechin showed the highest activity among them. The enzyme activity was inhibited by potassium cyanide and sodium diethyldithiocarbamate.