Complex formation between metarhodopsin II and GTP-binding protein in bovine photoreceptor membranes leads to a shift of the photoproduct equilibrium

Cap binding protein (CBP)-related polypeptides were identified in different cytoplasmic RNP particles of embryonic chick muscles using monoclonal antibody to purified CBP. A single immunoreactive peptide (M_r 78000) was present in preparations of both free mRNP particles and a novel 10 S translation inhibitory RNP particle. In contrast, proteins isolated from these particles showed two new low-M_r immunoreactive peptides (M_r 43000 and M_r 29000). No CBP related protein could be detected in polysomal mRNP, although an immunoreactive M_r 43000 CBP-related protein was present in polysomes. The relevance of the association of different CBP-related polypeptides with cytoplasmic RNP particles and polysomes are discussed.     

Active Ca2+ transport by membrane vesicles from pigeon erythrocytes Stimulation by amino acids,ATP, GTP,P1 and some other cell constituents

Pretreatment of pigeon erythrocyte membrane vesicles with amino acids, ATP, GTP, P_i and some other simple cell constituents (singly and in combination) causes an increase in ATP-dependent Ca²⁺-uptake activity of vesicles upon subsequent incubation with ⁴⁵Ca²⁺ after removal of the above agents from the ‘i’ face. Amino acids augment the stimulation by all stimulatory agents and are required for stimulation by P_i. The effects of amino acids, ATP, GTP and P_i all occur at physiological concentrations. Many if not all of the effects of the mixture of amino acids that occur naturally in the cells can be accounted for by the group transported by the ‘ASC’ transport system of Christensen (Christensen H.N. (1975) Biological Transport, 2nd edn., W.A. Benjamin, Inc., Reading, MA), but not by any single amino acid at its physiological concentration. The effects of ATP and GTP are not mimicked by their non-hydrolysable β, γ-imido analogues nor by the corresponding 3′, 5′-cyclic monophosphates. None of the effects described appears to involve calmodulin. We suggest that amino acid transport plays a role in metabolic regulation through effects on cell [Ca²⁺]. Analogous effects on cell [Ca²⁺] may be involved in the action of the many hormones which augment amino acid accumulation by the ‘A’ amino acid transport system.     

Sulfhydryl group modification of photoreceptor G-protein prevents its light-induced binding to rhodopsin

The effect of sulfhydryl modification on the light-induced interaction between rhodopsin and the peripheral GTP-binding protein of the photoreceptor membrane (G-protein) has been investigated by time-resolved near-infrared light-scattering and polyacrylamide gel electrophoresis. It has been found that the modification of rhodopsin with the alkylating agent N-ethylmaleimide (NEM) does not affect its light-induced interaction with the G-protein. Modification of G-protein with NEM or other sulfhydryl agents prevents any light-induced binding to rhodopsin. Dark-association of G to the membrane as well as the light-induced complex with rhodopsin (once formed) is insensitive to NEM.     

Purification and properties of galactokinase from Tetrahymena thermophila

Galactokinase (EC 2.7.1.6; ATP: d-galactose-1-phosphototransferase) was purified 152-fold with an 11% yield from Tetrahymena thermophila maximally derepressed for enzyme synthesis in late stationary phase. The purification procedure utilized sequential acid precipitation, batch DEAE-Sephacel chromatography, differential ammonium sulfate precipitation and narrow range electrofocusing. The apparent molecular weight of the holoenzyme as determined by gel filtration on Sephadex G-200 is 50 000-55 000. The holoenzyme consists of two subunits of approx. 28 000 daltons each, as determined by SDS-polyacrylamide gel electrophoresis. The native enzyme appears to be a single species with an isoelectric point at pH 5.1 Optimal activity was obtained at pH 7.8 and 41°C, with no added monovalent salt. d-Galactose, 2-deoxygalactose and galactosamine all are suitable carbohydrate substrates for the stereospecific galactokinase; only substitution at the C-2 position of galactose retains enzyme recognition. The enzyme utilizes ATP, 2′-dATP and 3′-dATP as phosphate donors; ADP and adenosine-5′-[γ-thio]triphosphate are inhibitory. The K_m values for galactose and ATP were determined to be 0.60 mM and 0.15 mM, respectively. The enzyme requires a divalent cation for activity, with effectiveness being in the order: Mg²⁺ >Co²⁺ >Mn²⁺ >Fe²⁺. Galactokinases from all eucaryotic sources studied thus far seem to be very similar. Based upon the results reported here, the galactokinases from Tetrahymena and yeast appear to be most similar in their biophysical and biochemical properties.     

The ADP- and Mg2+-reactive calcium complex of the phosphoenzyme in skeletal sarcoplasmic reticulum Ca2+-ATPase

The effects of ATP on Ca²⁺ binding in the absence of added Mg²⁺ to the purified sarcoplasmic reticulum Ca²⁺-ATPase were studied at pH 7.0 and 0°C. ATP increased the number of Ca²⁺-binding sites of the enzyme from 2 to 3 mol per mol of phosphorylatable enzyme. The association constant for the ATP-induced Ca²⁺ binding was 4·10⁵ M^?1, which was not significantly different from that obtained in the absence of ATP. AdoP[CH₂]PP has little effect on the Ca²⁺-binding process. The amount of phosphoenzyme formed was equivalent to the level of ATP-induced Ca²⁺ binding. ADP decreased the level of ATP-induced Ca²⁺ binding and phosphoenzyme by the same amount. These results suggest that ATP-induced Ca²⁺ binding exists in the form of an ADP-reactive phosphoenzyme·Ca complex. In addition, the Ca²⁺ bound to the enzyme in the presence of ATP was released on the addition of 1 mM MgCl₂; after the release of Ca²⁺, the phosphoenzyme decayed. These observations suggest that Mg²⁺, added after the ATP-induced Ca²⁺-binding process, may replace the Ca²⁺ on the phosphoenzyme and initiate phosphoenzyme decomposition.     

Importance of disulfide bonds in receptors for vasoactive intestinal peptide and secretin in rat pancreatic plasma membranes

(1) Vasoactive intestinal peptide (VIP), secretin, and C-terminal octapeptide of cholecystokinin (CCK-8) receptors were identified in rat pancreatic plasma membranes by the ability of these peptides to stimulate adenylate cyclase activity. The membrane preparation procedure was conducted through a series of steps including discontinuous sucrose density gradient fractionation. 5 mM β-mercaptoethanol was added stepwise. Membrane preparations obtained stepwise were preincubated for 10 min at 25°C in the presence of various concentrations of β-mercaptoethanol or dithiothreitol before assaying adenylate cyclase. The use of the reducing agents exerted no effect on p[NH]ppG-, NaF-, and CCK-8- stimulated activities. By contrast, stimulation of adenylate cyclase by low VIP concentrations was specifically altered when β-mercaptoethanol was used during tissue homogeneization at 5°C. (2) In addition, both VIP and secretin responses were highly sensitive towards a preincubation of 10 min at 25°C in the presence of dithiothreitol. (3) These results were likely to reflect alterations at the receptor level. ¹²⁵I-VIP binding was, indeed, reduced after dithiothreitol preincubation, low concentrations of the thiol reagent decreasing the apparent number of high-affinity VIP receptors and higher dithiothreitol concentrations reducing the affinity of VIP receptors.     

Evidence for a defect in the number of β-adrenergic receptors and in the adenylate cyclase responsiveness to guanine nucleotides in fat cells after adrenalectomy

Receptor binding studies (?)-[³H]dihydroalprenolol as the ligand revealed, in adrenalectomized rat fat cells, a 50% decrease in the number of β-adrenergic receptors. er cell with no change in the receptor affinity for this ligand. Adrenalectomy caused no change in the binding affinity for isoproterenol of both high affinity and low affinity populations of the β-adrenergic receptors. Guanine nucleotide sensitivity of the agonist binding to β-receptors was also unaltered by adrenalectomy. Adrenalectomy caused a 30–40% decrease in the maximal response of adenylate cyclase to (?)-isoproterenol only when guanine nucleotides were present in the assay, without altering the (?)-isoproterenol concentration giving half-maximal adenylate cyclase stimulation (K_act values). The maximal response of adenylate cyclase to Gpp(NH)p also was lower in adrenalectomized membranes, indicating a defect at the guanine nucleotide regulatory site. Removal of adenosine by addition of adenosine deaminase failed to reverse the decreased adenylate cyclase response to isoproterenol in adrenalectomized rats. However, in intact fat cells, in which cyclic AMP accumulation in response to isoproterenol was decreased by adrenalectomy, removal of adenosine almost completely corrected this defect. These results indicate that the observed changes in the number of β-adrenergic receptors and in the ability of guanine nucleotides to stimulate adenylate cyclase, though explaining the decreased adenylate cyclase responsiveness to catecholamines, do probably not contribute significantly to the mechanism by which adrenalectomy decreases the lipolytic responsiveness of adipocyte to catecholamines. In addition, this study also suggests that the increased sensitivity to adenosine of lipolysis reported in adipocytes from adrenalectomized rats may result from an action of adenosine at a post-adenylate cyclase step, possibly on the cyclic AMP phosphodiesterase.     

Evidence for two types of β-adrenergic-sensitive adenylate cyclase activities in bovine cerebellum

A latent, as well as an expressed form of adenylate cyclase coupled to β-adrenergic receptors is present in intact crude synaptosomal preparations from bovine cerebellum. The latent adenylate cyclase activity was assayed in Krebs-Ringer buffer by [³H]adenine labeling and was found to be coupled to a β₁-like adrenergic receptor. The externally accessible adenylate cyclase assayed in the same with [³H]ATP was stimulated via β₂-adrenergic receptors.     

Regulation of phospholipid-ATPase complex interaction by the adenine nucleotide carrier

(1) The effect of phospholipids on a preparation containing the ATPase complex and the adenine nucleotide carrier is studied in the presence of ligands known to affect the conformation of these components of the mitochondrial inner membrane. (2) When ATPase activity is abolished by phospholipid depletion, the reactivation induced by phosphatidylcholine is prevented by the simultaneous addition of ATP. ADP partially reproduces the ATP effect. AMP, GTP, UTP and P_i are ineffective. (3) The influence of ATP is associated with reduced phospholipid binding to the membrane fragments and is reversible. The ATP effect on reconstitution is not manifest when phosphatidylcholine is added together with negatively charged phospholipids. (4) Carboxyatractyloside does not modify the phospholipid-ATPase complex interaction but bongkrekic acid is as effective as ATP. In the presence of ADP, the influence of bongkrekic acid is considerably increased. (5) It is concluded that the binding of ATP to the adenine nucleotide carrier enables the complex to select between the charged and uncharged phospholipids. As a result of the carrier conformational change, the ATPase complex is induced to prefer a negatively charged phospholipid environment.     

Phospholipid chain immobilization and steriod rotational immobilization in acetylcholine receptor-rich membranes from torpedo marmorata

1. The ESR spectra of both phosphatidylcholine and phosphatidylethanolamine spin labels reveal an immobilized lipid component (τ_R ? 50 ns), in addition to a fluid component (τ_R ～ 1 ns), in acetylcholine receptorrich membranes prepared from Torpedo marmorata electroplax according to the method of Cohen et al. (Cohen, J.B., Weber, M., Huchet, M. and Changeux, J.P. (1972) FEBS Lett. 26, 43–47). 2. The ESR spectra of the androstanol spin label display a component corresponding to molecules which are immobilized with respect to rotation about the long molecular axis (

), in addition to the fluid lipid bilayer component in which the molecules are rotating rapidly about their long axes (

). This immobilized component is observed throughout the temperature range 2–22°C, at an approximately constant relative intensity of approx. 45% of the total, which is quantitatively the same as previously observed with fatty acid spin labels.     

Effects of forskolin on adenylate cyclase,cyclic AMP,protein kinase and intermediary metabolism of the thyroid gland

Forskolin (40 μM) stimulated adenylate cyclase activities of bovine thyroid plasma membranes without pthe addition of guanine nucleotides. GDP had little effect on the forskolin-stimulated adenylate cyclase activity while Gpp[NH]p (0.1–1.0 μM) decreased it. In the presence of TSH (10 mU/0.11), Gpp[NH]p no longer caused inhibition. Forskolin did not affect phosphodiesterase activities of thyroid homogenates. Forskolin (10 μM) rapidly increased cAMP levels in bovine thyroid slices both in the absence and presence of a phosphodiesterase inhibitor. The effect of TSH (50 mU/ml) on cAMP levels was additive or greater than additive to that of forskolin. An initial 2-h incubation of slices with forskolin did not decrease their subsequent cAMP responses to either forskolin and/or TSH while similar treatment of slices with TSH induced desensitization of the cAMP response to TSH, but not to forskolin. Forskolin (10 μM) as well as TSH (50 mU/ml) activated cAMP-dependent protein kinase of slices in the absence of a phosphodiesterase inhibitor. Although forskolin activated the adenylate cyclase cAMP system, it did not stimulate iodide organification or glucose oxidation, effects which have been attributed to cAMP. In fact, forskolin inhibited these parameters and ³²P incorporation into phospholipids as well as their stimulation by TSH. These results indicate that an increase in cAMP levels and cAMP-dependent protein kinase activity in thyroid slices may not necessarily reproduce the effects of TSH on the thyroid.     

Differing responses of the two forms of photosystem II carbonic anhydrase to chloride, cations, and pH

The effects of Cl⁻, Mn²⁺, Ca²⁺, and pH on extrinsic and intrinsic photosystem II carbonic anhydrase activity were compared. Under the conditions of our in vitro experiments, extrinsic CA activity, located on the OEC33 protein, was optimum at about 30 mM Cl⁻, and strongly inhibited above this concentration. This enzyme is activated by Mn²⁺ and stimulated somewhat by Ca²⁺. The OEC33 showed dehydration activity that is optimum at pH 6 or below. In contrast, intrinsic CA activity found in the PSII complex after removal of extrinsic proteins was stimulated by Cl⁻ up to 0.4 M. Ca²⁺ appears to be the required cofactor, which implies that the location of the intrinsic CA activity is in the immediate vicinity of the CaMn₄ complex. Up to now, intrinsic CA has shown only hydration activity that is nearly pH independent.     

The locations of the three cysteine residues in the primary structure of the intrinsic segments of band 3 protein,and implications concerning the arrangement of band 3 protein in the bilayer

The intrinsic domains of band 3 protein contain three cysteine residues, one in a 17 kDa middle segment and two in a 35 kDa C-terminal segment. The latter are retained in an 8 kDa fragment produced by chymotrypsin treatment of ghosts. Cleavage of cysteine residues by 2-nitro-5-thiocyanobenzoic acid (NTCB) allows localization of this amino acid in the primary structure of the 8, 17, 35 and 52 (17 plus 35) kDa segments of band 3 protein. The mapping of these residues taken with other information concerning accessibility of various sites at the two sides of the membrane leads to the conclusion that band 3 protein crosses the membrane at least five times, or ten times in a dimer structure. The implications of this conclusion in terms of band 3 protein structure and function are briefly discussed.     

Requirements for antiribosomal activity of pokeweed antiviral protein

It has been known for some time that pokeweed antiviral protein acts by enzymatically inhibiting protein synthesis on eucaryotic ribosome systems. The site of this action is known to be the ribosome itself. In this paper we show that the pokeweed antiviral protein reaction against ribosomes is a strong function of salt concentrations, where 160 mM K⁺ and 3 mM Mg²⁺ retards the reaction, while 20 mM K⁺ and 2 mM Mg²⁺ allows maximum reaction rate. It is also shown, however, that an unidentified protein in the postribosomal supernatant solution, together with ATP, allows the ribosome to be attacked even in the presence of high salt. Kinetic analysis of the antiviral protein reaction has been carried out under both sets of conditions, and reveals that the turnover number for the enzyme is about 300–400 mol/mol per min. in each case. The K_m for ribosomes is 1 μM in the presence of low salt and 0.2 μM at higher salt in the presence of postribosomal supernatant factors plus ATP. The antiviral protein reaction is also shown to be pH dependent and is controlled by a residue with pK_a value of approx. 7.0, apparently a histidine. Stoichiometric reaction of the enzyme with iodoacetamide results in a significant loss of antiribosomal activity.     

Manganese proteins isolated from spinach thylakoid membranes and their role in O2 evolution: I. A 56 kilodalton manganese-containing protein,a probable component of the coupling factor enzyme

The binding of endogenous manganese (Mn) to proteins released from spinach grana-thylakoid membranes by 2% cholate detergent or by osmotic shock is investigated. A mixture of 15–20 proteins is released by cholate and has been separated by isoelectric focusing in a sucrose gradient or by chromatofocusing. Mn coelutes with several proteins, but is lost upon dialysis. A dramatic redistribution of this Mn occurs in proteins released by osmotic shock in the presence of hydrophobic and hydrophilic oxidants. Maintaining an oxidizing solution potential during extraction apparently precludes reduction of the higher oxidation states of Mn to the labile Mn(II) state by reducing agents released from the membranes during lysing. This allows proteins to be separated which bind non-labile Mn ions. Under these extraction conditions, a protein is isolated which has an apparent molecular weight (M_r) of 65 000 or 56 000 on SDS-polyacrylamide gel electrophoresis depending on the sample buffer system used. The nondissociated protein occurs as a monomer of 58 kDa (90%) and an apparent dimer of 112 kDa (10%) by gel filtration. This protein binds little Mn if extracted by cholate and separated by isoelectric focusing. However, extraction by osmotic shock in the presence of oxidants and separation by chromatofocusing results in the retention of 1.9 ± 0.3 Mn ions per monomer. This protein is identical to that reported by Spector and Winget (Spector, M., and Winget, G.D. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 957–959). Contrary to their result, this protein does not reconstitute O₂ evolution when added to depleted membranes. Rabbit antibody to this purified protein inhibits O₂ evolution by 20% when incubated with intact grana-thylakoid membranes or 10–20% with partially inverted, French-pressed thylakoids. This inhibition is completely removed by 10^?3 M NH₃Cl as an uncoupler of photophosphorylation. These results support a role in Phosphorylation and a location on the outer surface of the thylakoids. This antibody also selectively binds purified coupling factor, CF₁, the multisubunit phosphorylation enzyme which is located on the outer thylakoid surface and which is known to bind two Mn ions tightly (Hochman, Y. and Carmeli, C. (1981) Biochemistry 20, 6293–6297). Thus the β-subunit of CF₁, which has a molecular weight of 56 kDa, can be identified as the locus of Mn binding in CF₁ and as the Mn protein isolated by Spector and Winget. This protein plays no role on O₂ evolution.     

Cytochromes and prenylquinones in preparations of cytoplasmic and thylakoid membranes from the cyanobacterium (blue-green alga) Anacystis nidulans

The cytochrome and prenylquinone compositions were compared for cytoplasmic membranes and thylakoid membranes from the cyanobacterium (blue-green alga) Anacystis nidulans. Reduced-minus-oxidized difference absorption spectra at ?196°C indicated that the thylakoid membranes contained photosynthetic cytochromes such as cytochrome ?, cytochrome b-559 and cytochrome b₆, while cytochromes c-549 and c-552 were detected spectrophotometrically only after their release by sonic oscillation. The cytoplasmic membrane preparation contained one or two low-potential cytochrome(s) with α-band maxima at 553 and 559 nm at ?196°C, which differed from the cytochromes in the thylakoid membranes. A cytochrome specific to the cytoplasmic membranes was also found by heme-staining after lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Both types of membranes contained the three prenylquinones plastoquinone-9, phylloquinone and 5′-monohydroxyphylloquinone, but in different proportions.     

Genetically acquired diabetes: adipocyte guanine nucleotide regulatory protein expression and adenylate cyclase regulation

Adipocyte membranes from diabetic (db/db) animals showed marked elevations in the levels of α-subunits for G_i-1 which were almost twice those in membranes from their normal, lean littermates. In contrast, no apparent differences were noted for levels of the α-subunits of G_i-2 and G_i-3, and 42 and 45 kDa forms of G_s and for G-protein β-subunits. Adenylate cyclase specific activity was similar in membranes from both normal and diabetic animals under basal conditions and also when stimulated by optimal concentrations of either NaF or forsckolin. In contrast, the ability of isoprenaline, glucagon and secretin to stimulate adenylate cyclase activity was greater in membranes from normal animals compared with membranes from diabetic animals. Receptor-mediated inhibition of adenylate cyclase, as assessed using PGE₁ and nicotinate, was similar using membranes from both sources, but PIA (phenylisopropyladenosine) was a slightly more effective inhibitor in membranes from diabetic animals. A doubling in the expression of G₁-1 thus appears to have little discernible effect upon the inhibitory regulation of adenylate cyclase.     

Determination of G-protein levels,ADP-ribosylation by cholera and pertussis toxins and the regulation of adenylyl cyclase activity in liver plasma membranes from lean and genetically diabetic (db/db)

Liver plasma membranes prepared from genetically diabetic (db/db) mice expressed levels of G_i α-2, G_i α-3 and G-protein β-subunits that were reduced by some 75, 63 and 73% compared with levels seen in membranes from lean animals. In contrast, there were no significant differences in the expression of the 42 and 45 kDa forms of G_s α-subunits. Pertussis toxin-catalysed ADP-ribosylation of membranes from lean animals identified a single 41 kDa band whose labelling was reduced by some 86% in membranes from diabetic animals. Cholera toxin-catalysed ADP-ribosylation identified two forms of G_s α-subunits whose labelling was about 4-fold greater in membranes from diabetic animals compared with those from lean animals. Maximal stimulations of adenylyl cyclase activity by forskolin (100 μM), GTP (100 μM), p[NH]ppG (100 μM), NaF (10 mM) and glucagon (10 μM) were similar in membranes from lean and diabetic animals, whereas stimulation by isoprenaline (100 μM) was lower by about 22%. Lower concentrations (EC₅₀-60 nM) of p[NH]ppG were needed to activate adenylyl cyclase in membranes from diabetic animals compared to those from lean animals (EC₅₀-158 nM). As well as causing activation, p[NH]ppG was capable of eliciting a pertussis toxin-sensitive inhibitory effect upon forskolin-stimulated adenylyl cyclase activity in membranes from both lean and diabetic animals. However, maximal inhibition of adenylyl cyclase activity in membranes from diabetic animals was reduced to around 60% of that found using membranes from lean animals. Pertussis toxin-treatment in vivo enhanced maximal stimulation of adenylyl cyclase by glucagon, isoprenaline and p[NH]ppG through a process suggested to be mediated by the abolition of functional G_i activity. The lower levels of expression of G-protein β-subunits, in membranes from diabetic compared with lean animals, is suggested to perturb the equilibria between holomeric and dissociated G-protein subunits. We suggest that this may explain both the enhanced sensitivity of adenylyl cyclase to stimulation by p[NH]ppG in membranes from diabetic animals and the altered ability of pertussis and cholera toxins to catalyse the ADP-ribosylation of G-proteins in membranes from these two animals.     

Effect of light quality on the composition and function of thylakoid membranes in atriplex triangularis

Light quality was shown to exert well-coordinated regulatory effects on the composition and function of the thylakoid membranes as well as on the photosynthetic rates of intact leaves from Atriplex triangularis grown in continuous blue, white and red lights (50 μE · m^?2 · s^?1). The higher photosynthetic rates in plants grown in blue light, as compared to those in white and red lights, resulted from marked changes in both light-harvesting complexes and electron carriers. The concentrations of electron carriers such as atrazine binding sites, plastoquinone, cytochromes b and f and P-700 on a chlorophyll basis were markedly increased in Atriplex grown in blue light; and the apparent light-harvesting antenna unit sizes of Photosystems I and II were greatly reduced. Consequently, the electron transport capacities of Photosystems I and II were also increased as was the coupling factor CF₁ activity. Atriplex grown in red light had lower photosynthetic rates than those grown in blue or white light by incorporating changes in the composition and function of the thylakoids in a direction opposite to those caused by growth in blue light. When these regulatory effects of light quality were compared with those of light quantity [6,7], it is clear that $\text{Chl}\text{a}\text{Chl}\text{b}$ ratios, electron transport capacities of Photosystems I and II, concentrations of plastoquinone, atrazine binding sites, coupling factor CF₁ activity and the apparent antenna unit size of Photosystem II are more affected by light quantity, whereas light quality has a greater influence on the concentration of P-700, the apparent antenna unit size of Photosystem I and the overall photosynthetic rates of intact leaves.