Characterization of GTP binding and hydrolysis in plasma membranes of zucchini.

We have investigated the possibility that G-protein-like entities may be present in the plasma membrane (PM) of zucchini (Cucurbita pepo L.) hypocotyls by examining a number of criteria common to animal and yeast G-proteins. The GTP binding and hydrolysis characteristics of purified zucchini PM are similar to the characteristics of a number of known G-proteins. Our results demonstrate GTP binding to a single PM site having a Kd value between 16-31 nM. This binding has a high specificity for guanine nucleotides, and is stimulated by Mg2+, detergents, and fluoride or aluminium ions. The GTPase activity (Km = 0.49 micromole) of zucchini PM shows a sensitivity to NaF similar to that seen for other G-proteins. Localization of GTP mu 35S binding to nitrocellulose blots of proteins separated by SDS-PAGE indicates a 30-kDa protein as the predominant GTP-binding species in zucchini PM. Taken together, these data indicate that plant PM contains proteins which are biochemically similar to previously characterized G-proteins.     

Interaction of filamentous actin with isolated liver plasma membranes

Actin-membrane interactions have been studied using purified liver plasma membranes and muscular filamentous actin. Despite the large quantity of endogenous actin present in membranes, exogenous muscular filamentous actin cosediments with membranes after a 30 min centrifugation at 30 000 g. The cosedimentation process is time-dependent and exhibits a complex relationship with actin concentration. The cosedimentation of actin with membranes can be partly explained by gelation as shown by low-shear viscosity and electron microscopy. The characterization of the gelation phenomenon as a function of time, actin and membrane concentrations, ionic strength, temperature and Ca2+ concentration is also presented. Gelation alone cannot however account for the overall cosedimentation data, and a more direct mode of association between actin and the membrane must be envisaged. The analogy that exists between the results obtained with liver plasma membranes and those obtained with other membrane systems suggests that a general mechanism may be involved in the interaction of actin with plasma membranes.     

Adenosine receptor-mediated stimulation of GTP hydrolysis in adipocyte membranes

The adenosine derivative, N⁶-phenylisopropyladenosine (PIA), which inhibits adenylate cyclase in adipocyte membranes by a GTP-dependent and sodium-amplified process, was studied on GTPase activity in hamster adipocyte ghosts. PIA stimulated a high affinity GTPase without apparent lag phase. Both unstimulated and PIA-stimulated GTPases exhibited very similar K_m values of about 0.2 μM GTP. PIA-induced low K_m GTPase stimulation was amplified by sodium ions and was half-maximal and maximal at about 0.02 and 0.1 μM PIA, respectively. Stimulations of the low K_m GTPase by PIA and PGE₁, both inhibiting adipocyte adenylate cyclase, were not additive. Similar to PIA-induced adenylate cyclase inhibition, stimulation of the GTPase by PIA but not by PGE₁ was prevented by the adenosine receptor antagonist, 3-isobutyl-1-methylxanthine. The data suggest that PIA-induced stimulation of a high affinity GTPase is an essential mechanism of adenosine receptor-mediated adipocyte adenylate cyclase inhibition.     

GTP hydrolysis by guinea pig liver transglutaminase

Homogeneous guinea pig liver transglutaminase was purified from a commercially available enzyme preparation by affinity chromatography on GTP-agarose. The purified transglutaminase exhibited a single band of apparent Mr = 80,000 on sodium dodecyl sulfate polyacrylamide gel and Western blotting and had enzyme activity of both transglutaminase and GTPase. The guinea pig liver transglutaminase has an apparent Km value of 4.4 microM for GTPase activity. GTPase activity was inhibited by guanine nucleotides in order GTP-gamma-S greater than GDP, but not by GMP. These results demonstrate that purified guinea pig liver transglutaminase catalyzes GTP hydrolysis.     

Interaction between chromium GTP and tubulin. Stereochemistry of GTP binding, GTP hydrolysis, and microtubule stabilization

Chromium GTP (CrGTP) has been used to probe the stereochemistry of metal-GTP binding to exchangeable site of tubulin and to examine the fate and role of nucleotide-bound metal ion in GTP hydrolysis associated with microtubule assembly. The absolute stereoconfiguration of the two pairs of diastereomers of beta,gamma-bidentate CrGTP has been determined by comparison of their visible circular dichroism spectra with those of the beta,gamma-CrATP isomers whose configurations have been established (Lin, I., and Dunaway-Mariano, D. (1988) J. Am. Chem. Soc. 110, 950-956). Tubulin binds metal-GTP preferentially in the delta pseudoaxial configuration. CrGTP-tubulin shows a high propensity to undergo tubulin-tubulin interactions with associated hydrolysis of CrGTP. Hydrolysis of CrGTP in microtubule assembly develops in two consecutive steps: cleavage of the gamma-phosphate followed by release of Pi and chromium. In contrast to other NTPases (actin, hexokinase) tubulin appears able to catalyze the dissociation of the stable chromium-phosphate bonds, which implies a highly nucleophilic environment of the binding site of the metal-triphosphate moiety of GTP. Microtubules assembled from CrGTP-tubulin are made of 90% GDP subunits, and their stability is linked to a 10% proportion of CrGDP-Pi subunits, scattered along the microtubule, from which Pi does not dissociate. The possibility is evoked that some tubulin variants do not catalyze release of Pi and metal ion efficiently, and their presence could affect microtubule dynamics.     

GTP and cytosol stimulate phosphoinositide hydrolysis in isolated platelet membranes

Hydrolysis of polyphosphoinositides by phospholipase C was examined in isolated membranes prepared from [32P]labelled platelets. In the presence of GTP gamma S, thrombin increased the release of inositol triphosphate and inositol biphosphate approximately 500%. GTP gamma S alone stimulated release 2 fold. Maximal activation of thrombin-induced phosphoinositide hydrolysis was observed at 10 uM GTP. Although addition of calcium had no effect, 2 mM EGTA completely inhibited inositolphosphate release. Addition of high speed supernatant to [32P]labelled membranes stimulated the release of inositolphosphates. This hydrolysis was further enhanced by the addition of GTP. These data demonstrate that the breakdown of polyphosphoinositides in isolated platelet membranes is dependent on GTP and stimulated by platelet cytosol.     

Interaction of [3H]AMP with liver cells and their plasma membranes

Specific binding of [3H]AMP to rat hepatocytes and their plasma membranes was studied. It was shown that the time course of this binding reached a maximum within the first 15 seconds. An equilibrium binding study revealed the presence of a single class of binding sites with Kd of 20 microM both in hepatocytes and in plasma membranes. The [3H]AMP binding sites were inactivated by treatment with trypsin as well as by heating. 5'-Phosphorylated derivatives of adenosine (ATP, ADP) effectively competed with [3H]AMP for the binding sites, while adenosine, beta-glycerophosphate and 3'-AMP were inactive. The binding of [3H]AMP increased by 400% in the presence of concanavalin A, a specific inhibitor of plasma membrane 5'-nucleotidase. It was concluded that the catalytic center of 5'-nucleotidase is a receptor for adenine nucleotides.     

Interaction of smooth muscle plasma membranes with flat lipid membranes

The method of implantation of smooth muscle cells from plasma membranes (PM) of the rabbit intestine into flat lipid membranes (FLM) is described. The method is based on the pretreatment of PM vesicles with asolectin liposomes in the ratio that provides the activation of membrane ATPases. Thus modified FLM possesses channel conductivity.     

Interaction of lipoproteins with isolated ovary plasma membranes

Plasma membranes of ovarian luteal and adrenal cortical cells from "microvillar channels," a unique extracellular compartment formed by the close apposition of flattened microvillar surfaces. Microvillar channels have unusual affinity for cholesterol-rich lipoproteins, and, in vivo, may provide an increased surface area for these particles. In this research, we have isolated a plasma membrane-enriched fraction from rat luteinized ovaries, in which closely apposed membrane (i.e. microvillar channels) comprise about 30% of the preparation. Following in vitro incubations (approximately 1 h) of this plasma membrane fraction with different plasma lipoproteins, the closely apposed plasma membrane surfaces widen and become filled with lipoprotein particles (up to about 30 nm), whereas other membranes of the fraction show little binding. Competition experiments show that rat high density lipoproteins have the highest affinity for binding to the plasma membrane fraction. Radiolabeled plasma lipoprotein and the tissue-specific hormone, human chorionic gonadotropin, showed specific and saturable binding to the plasma membrane fraction, whereas other macromolecules used as controls did not. Radioautographic analyses of 125I-labeled lipoproteins and human chorionic gonadotropin indicate that binding occurs predominantly to the closely apposed plasma membranes (i.e. microvillar channels of the fraction). These studies show that microvillar channels of steroid-secreting cells entrap large numbers of plasma lipoproteins, particularly high density lipoproteins particles, presumably functioning in the delivery of cholesterol to these cells.     

Interaction of rat liver lysosomal membranes with actin

Membranes were prepared from lysosomes purified 80-fold by centrifugation in a discontinuous metrizamide gradient. When salt- washed membranes were combined with rabbit muscle actin, an increase in viscosity could be measured using a falling ball viscometer. The lysosomal membrane-actin interaction was actin- and membrane- concentration dependent and appeared to be optimal under presumed physiological conditions (2 mM MgCl2, 1 mM MgATP, neutral pH, and free calcium concentration less than 10(-8) M). The actin cross-linking activity of the membrane was optimal at pH 6.4. The interaction was maximal between 10(-7) and 10(-9) M free calcium ions and inhibited by approximately 50% at concentrations of calcium greater than 0.5 x 10(- 7) M. The actin-lysosomal membrane interaction was destroyed if the membranes were pretreated with Pronase, or if the membranes were purified in the absence of protease inhibitors. The interaction was not destroyed if membranes were washed with high salt or extracted with KCl and urea. In addition, a sedimentation assay for the actin-lysosomal membrane interaction was also performed to corroborate the viscometry data. The results suggest the existence of an integral lysosomal membrane actin-binding protein.     

Interaction of plasma lipoproteins and apolipoproteins with lipid bilayer membranes

It was shown that the interaction of lipoproteins (LP) with bilayer lipid membranes (BLM) resulted in some changes in the physical-chemical properties of the membranes. Adsorption of very low and low density lipoproteins (VLDL and LDL) at concentrations of 5-8 g protein/ml increased the surface potential difference and decreased transversal elasticity module of the bilayer. LP concentrations higher than the mentioned ones increased BLM conductance and caused instability and disruption of the membranes. The same effects were revealed for high density lipoproteins (HDL) at higher concentrations--15-20 micrograms protein/ml. The effect of apolipoproteins in the interaction of LP with BLM was investigated. It is proposed that apolipoproteins and especially apo B are the main factor which affects the nonreceptor interactions of LP with the membranes.     

Inside-out Z rings--constriction with and without GTP hydrolysis

The bacterial tubulin homologue FtsZ forms a ring-like structure called the Z ring that drives cytokinesis. We showed previously that FtsZ-YFP-mts, which has a short amphipathic helix (mts) on its C terminus that inserts into the membrane, can assemble contractile Z rings in tubular liposomes without any other protein. Here we study mts-FtsZ-YFP, where the membrane tether is switched to the opposite side of the protofilament. This assembled 'inside-out' Z rings that wrapped around the outside surface of tubular liposomes. The inside-out Z rings were highly dynamic, and generated a constriction force that squeezed the tubular liposomes from outside. This is consistent with models where the constriction force is generated by curved protofilaments bending the membrane. We used this system to test how GTP hydrolysis by FtsZ is involved in Z-ring constriction. Without GTP hydrolysis, Z rings could still assemble and generate an initial constriction. However, the constriction quickly stopped, suggesting that Z rings became rigidly stabilized in the absence of GTP hydrolysis. We propose that remodelling of the Z ring, mediated by GTP hydrolysis and exchange of subunits, is necessary for the continuous constriction.     

Guanine nucleotide and pyrophosphate activate exogenous phosphatidylinositol 4,5-bisphosphate hydrolysis in rat liver plasma membranes

5'-guanylylimidodiphosphate (GppNHp) in the presence of deoxycholate, stimulated the phospholipase C-mediated hydrolysis of exogenous [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) to myo-[3H]inositol 1,4,5-trisphosphate in rat liver plasma membranes. Activation was not specific for guanine nucleotides as 5'-adenylylimidodiphosphate, imidodiphosphate and pyrophosphate stimulated the enzyme with similar efficacies and potencies. Enzyme activation by GppNHp was most pronounced when [3H]PIP2 was used as substrate. No added Ca++ was required for [3H]PIP2 breakdown but hydrolysis was inhibited by divalent ion chelators. GppNHp stimulation was apparent in the presence of Ca++ or Mg++ as well as chelator concentrations that partially inhibited the enzyme, indicating that this effect was not attributed to changes in affinity of these divalent cations for the enzyme or substrate. These results suggest that guanine nucleotides can stimulate the hydrolysis of exogenous [3H]PIP2 in rat liver membranes by a non-specific effect probably due to the interaction of the diphosphate moiety with the enzyme or substrate.     

Interaction of mitogenic lectins with lymphocyte plasma membranes]

It is shown that mitogenic stimuli from concanavalin A and phytohemagglutinin are summed up in the time. Under given experimental conditions each of the mitogens used did not induce mitogenesis when the interaction with lymphocytes continued for 14 and/or 20 hours. The results obtained are discussed from the point of view of the cell-to-cell interaction between the lymphocytes. It is assumed that the ability of the stimuli to sum up in the time underlies the nonspecific mechanism of defense from tolerance.     

Change of coupling system of receptor-adenylate cyclase induced by epinephrine and GTP in plasma membranes of rat liver.

1. The binding of [3H]epinephrine to plasma membranes was affected (temporary release of bound epinephrine and characteristic retardation of epinephrine binding) not only by GTP but also by dGTP and guanylylimidodiphosphate, whereas the binding of [3H]dihydroalprenolol was not affected by GTP. GTP affected the binding of [3H]epinephrine in the presence of alpha-antagonists, but not in the presence of beta-antagonists, suggesting that the GTP effects are specific to beta-agonists and beta-receptors. 2. The half-maximal release of bound [3H]epinephrine was found at 8.8 . 10(-6) M GTP in the absence of ATP, whereas it was found at 1.6 . 10(-6) M GTP in the presence of 0.3 mM ATP in coincidence with the half-maximal activation of adenylate cyclase by GTP in the presence of 0.3 mM ATP (as measured at 30 s of incubation). 3. In the presence of 4 . 10(-5) M GTP, adenylate cyclase activity as measured at 30 s of incubation (State I) tended to increase with epinephrine concentration, showing no saturation tendency even at 1 . 10(-4) M epinephrine. The activity of State II, which is established at 4 min of incubation, was much lower than that of State I but was found to reach a plateau as the epinephrine concentration increased, showing half-maximal activation at an epinephrine concentration between 2 . 10(-6) and 2 . 10(-7) M. 4. Apparent kinetic parameters (Km and V) for State I as assayed at 30 s of incubation suggested that GTP alone may increase V slightly, whereas epinephrine plus GTP may increase the V to a further extent and simultaneously decrease the Km. 5. Adenylate cyclase of plasma membranes pretreated with epinephrine plus GTP was stimulated by GTP alone similarly to untreated membranes, but it was no longer responsive to the synergistic activation by epinephrine plus GTP. Accordingly, the binding of [3H]epinephrine to the pretreated plasma membranes was no longer affected by GTP. 6. The results of the present study seem to support the idea that the most active and coherently coupling state (State I) of the beta-receptor-adenylate cyclase system generated in the presence of epinephrine plus GTP is very labile and degenerates before reaching equilibrium. In turn, State II, in which the coherently coupling mechanism is largely impaired, seems to be established in due time. The characteristic biphasic kinetics of [3H]epinephrine binding in the presence of GTP seem to be related to the above change occurring in the beta-receptor-adenylate cyclase system.