Properties of LH-sensitive adenylate cyclase in purified plasma membranes from rat ovary

Mintz, Y.; Amir, Y.; Amsterdam, Avraham; Lindner, H. R.; Salomon, Yoram

doi:10.1016/0303-7207(78)90013-8

Cited by 34 publications

(7 citation statements)

References 35 publications

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“…In these experiments the homogenization was carried out in isotonic phosphate buffer using a Potter homogenizer. However, in subsequent experiment using the conditions described by Mintz et al (1978) for ovarian cells i.e. homogenization in hypotonic bicarbonate buffer and also using a Dounce homogenizer, both the homogenates and plasma membrane fractions retained adenylate cyclase activity.…”

Section: Resultsmentioning

confidence: 97%

“…The source of the Leydig cell tumour and preparation of the cells were described previously (Cooke et al 1979;Dix & Cooke 1981). The purified cells were then re-suspended in bicarbonate buffer (Mintz et al 1978) (1.0 nm NaHC03, 1.0 mM dithiothreitol and 0.2 mM MgCl2, pH 7.0) transferred to a Wheaton-Dounce homogeniser, left to swell for 2.5 min at 4°C and then homogenized with 5 strokes of a tight fitting pestle. The homogenate was immediately brought to iso-osmolarity (0.25 M with respect to sucrose).…”

Section: Preparation Of Plasma Membranesmentioning

confidence: 99%

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Isolation and characterization of plasma membranes containing LH sensitive adenylate cyclase from a Leydig cell tumour

et al. 1982

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An LH sensitive adenylate cyclase from a tumour Leydig cell has been investigated. The plasma membranes, prepared by a 2 phase (dextran-polyethylene glycol) centrifugation method were found to have the following properties: In the presence of LH plus p(NH)ppG (guanosine 5'-beta, gamma-imido triphosphate) or fluoride ions, maximum adenylate cyclase activity was obtained in the plasma membranes with 4 to 6 mM Mg2+ plus 0.33 to 2 mM ATP. LH alone stimulated adenylate cyclase activity 2-fold when compared with basal activity and the time course of cyclic AMP production was linear up to 45 min. With GTP (10(-5)M) and GTP plus LH, adenylate cyclase activity was increased 3 and 6-fold, respectively, for up to 20 min and thereafter declined. In contrast p(NH)ppG (10(-5)M) and p(NH)ppG plus LH increased adenylate cyclase activity 7 and 14-fold which was maintained for at least 45 min. Fluoride ions increased the enzyme activity linearly over 45 min approx 18-fold. When GTP or p(NH)ppG were added alone there was a lag time of activation of approximately 10 min which was abolished by the addition of LH. GTP but not p(NH)ppG at concentrations greater than 10(-4) inhibited basal and LH stimulated adenylate cyclase when compared with 10(-5)M GTP. The tumour Leydig cell adenylate cyclase is thus essentially similar to other hormone sensitive somatic cells. The present study makes it feasible to prepare plasma membranes by a simple method from large quantities of pure Leydig cells.

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Section: Resultsmentioning

confidence: 97%

Section: Preparation Of Plasma Membranesmentioning

confidence: 99%

Isolation and characterization of plasma membranes containing LH sensitive adenylate cyclase from a Leydig cell tumour

et al. 1982

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“…Ovarian plasma membranes (24) and mouse S49 lymphoma plasma membranes (25) were prepared as described. Rod outer segment (ROS) membranes were prepared from frog (Rana ridibunda) eyes (26).…”

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confidence: 99%

Selective labeling of proteins in biological systems by photosensitization of 5-iodonaphthalene-1-azide.

Raviv¹,

Salomon²,

Gitler³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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The apolar azide of 5-iodonaphthalene-1-azide (Ina) partitions into the lipid bilayer of biological membranes. Upon photolysis at 314 nm, it is rapidly converted into the reactive nitrene, which efficiently attaches covalently to lipid-embedded domains of proteins and, to a lesser extent, to membrane phospholipids. Above 370 nm, Ina absorption is negligible and photolysis at these wavelengths does not occur. However, on addition of the photosensitizing molecule 3-aminopyrene, trifluoperazine, or 8-anilinonaphthalene-1-sulfonate, followed by irradiation at 380 nm, efficient conversion of Ina to reactive species was observed, as measured by ['251]Ina-labeling of membrane proteins and inactivation of the hormonal response of adenylate cyclase. Irradiation at 480 nm in the presence of a fluorescein derivative of n-undecylamine also resulted in a pattern of [125] Klip and Gitler (1, 2) introduced the use of highly lipid-soluble azides for the labeling of proteins in membranes. Various reagents of this type have been developed and their properties have been reviewed (3-7). 5-Iodonaphthalene-1-azide (Ina) was used frequently and proved to be efficient in labeling hydrophobic domains of membrane proteins (8-12).Ina is highly hydrophobic (partition coefficient >105) (11) and partitions efficiently into the lipid bilayer of various cellular membranes. Upon photolysis (Xmax = 310 nm, E -21,400M-1 cm-1) with the 314-nm line of a mercury lamp, it is rapidly and efficiently converted into the reactive nitrene, which inserts covalently into intrinsic membrane proteins that contain lipid-embedded domains and, to a lesser extent, into phospholipids. When photoactivated in the plasma membrane, Ina blocks transduction of hormonal stimulation of adenylate cyclase by uncoupling of the hormone receptor from the enzyme (13).It was of interest to determine whether energy-transfer processes could be used to photoactivate a discrete fraction of the azide molecules present in a membrane or cell.Resonance energy transfer could not be used because Ina absorbs light down to 260 nm and any donor molecules chosen for its photoactivation would have to have absorption maxima below this wavelength, this clearly being impractical. Aromatic azides, however, are capable of photosensitized activation by donors that absorb at longer wavelengths than those absorbed by the azides themselves (14, 15). Photosensitization occurs by various mechanisms (16, 17) in a process that requires the approach of the photosensitizer and acceptor pair to within the collisional range in order for intermolecular exchange of excitation energy to take place (18). The nature of the reactive intermediates generated by photosensitization of aryl azides in different solvents is not completely understood. However, they seem to be reactive in hydrogen-abstraction, dimerization, and insertion reactions (19,20). Since many sensitizers are aromatic apolar molecules, it is likely that they will parallel the distribution of Ina into lipophilic regions of the membrane. This sh...

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“…Nitrogen cavitation and Percoli gradient centrifugation provide a number of significant advantages over previously reported techniques for obtaining plasma membranes from granulosa cells (Mintz et al, 1978). Cavitation uniformly disrupts all the cells in the suspension, requires no enzymic treatment, and provides a more homogeneous population of vesicles with fewer contaminants from other regions of the cell.…”

Section: Discussionmentioning

confidence: 99%

Properties of heparin binding to purified plasma membranes from bovine granulosa cells

Winer¹,

Ax²

1989

Reproduction

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Properties of LH-sensitive adenylate cyclase in purified plasma membranes from rat ovary

Cited by 34 publications

References 35 publications

Isolation and characterization of plasma membranes containing LH sensitive adenylate cyclase from a Leydig cell tumour

Isolation and characterization of plasma membranes containing LH sensitive adenylate cyclase from a Leydig cell tumour

Selective labeling of proteins in biological systems by photosensitization of 5-iodonaphthalene-1-azide.

Properties of heparin binding to purified plasma membranes from bovine granulosa cells

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