Differential Degradation of Different Benzodiazepine Binding Proteins by Incubation of Membranes from Cerebellum or Hippocampus with Trypsin

Eichinger, A.; Sieghart, Werner

doi:10.1111/j.1471-4159.1985.tb05496.x

Cited by 21 publications

(23 citation statements)

References 19 publications

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“…A similar trypsin-generated fragment has been reported when photolabeled rat brain homogenates were exposed to proteolytic enzymes (Eichinger and Sieghart, 1985;Klotz et al, 1984). In these studies, trypsin only partially inactivated the benzodiazepine binding sites.…”

Section: Discussionsupporting

confidence: 73%

“…The vesicles, once formed, can be disrupted to varying degrees by different experimental conditions, e.g., warming, vigorous mechanical agitation, sonication, freezing-thawing, and hypo-osmotic treatment (Whittaker, 1969). Thus, the difference in protease sensitivity observed by Eichinger and Sieghart (1985) using rat brain homogenates compared to our results using culture and 20-d-old chick brain homogenates may be due to different experimental conditions during membrane homogenate preparation, resulting in varying amounts of sealed and unsealed vesicles. In the present study, exhaustive trypsinization of intact cells, saponin-treated cells, and homogenates prepared from cell cultures was examined.…”

Section: Discussionmentioning

confidence: 99%

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Transmembrane topology and subcellular distribution of the benzodiazepine receptor

Czajkowski¹,

Farb²

1986

J. Neurosci.

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The distribution and transmembrane topology of benzodiazepine receptors were investigated in situ using intact and saponin-treated neurons of brain cell cultures. Reversible ligand binding and photoaffinity labeling, using 3H-flunitrazepam as a probe, were employed in conjunction with trypsin-induced exhaustive proteolysis and competition binding using a novel benzodiazepine (Ro7-0213) that contains a quaternary ammonium moiety bearing a full positive charge. About 80% of the benzodiazepine receptors, with apparent subunit molecular weights of 48,000 and 51,000, are located in the surface membrane and contain one or more trypsin-sensitive sites exposed at the extracellular surface. The ligand recognition sites of the surface receptors are orientated toward the extracellular space and are sensitive to both competition by Ro7-0213 and trypsin attack. Approximately 20% of the receptors are intracellular or sequestered and are insensitive to both trypsin and competition by Ro7-0213 in intact cells. Strikingly, all of the 3H-flunitrazepam photolabeled sites inaccessible to competition by Ro7-0213 are also resistant to extracellular trypsin, but are sensitive to trypsin and Ro7-0213 in saponin-treated cells. This result provides strong evidence for protected receptors. Surface receptors are differentially sensitive to trypsin-induced inactivation such that 57% (of total) are inactivated, while 24% (of total) are cleaved but, surprisingly, retain the capacity to bind 3H-flunitrazepam. The observation that the ability of the receptor fragment to bind ligand is unaltered demonstrates that the integrity of the benzodiazepine binding site is not contingent upon the presence of about half of the intact polypeptide chain. The receptor fragment, with an apparent molecular weight of 24,000, is produced upon exposure to extracellular trypsin and remains anchored in the plasma membrane. The fragment also contains at least one trypsin-sensitive site that is exposed at the intracellular face of the plasma membrane. The results indicate that this receptor fragment is a transmembrane segment of the GABA/benzodiazepine receptor complex, in which the ligand recognition site faces extracellularly.A question of central importance to understanding the structure and function of the GABA/benzodiazepine receptor complex (GABA/BZD-R) concerns its transmembrane topology and subcellular distribution. It is known that benzodiazepine drugs potentiate GABA responses determined on intact neurons in culture Choi et al., 1977;Macdonald and Barker, 1978) or on isolated patches of adrenal chromaffin cell surface membrane (Bormann and Clapman, 1985) and that

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Section: Discussionsupporting

confidence: 73%

Section: Discussionmentioning

confidence: 99%

Transmembrane topology and subcellular distribution of the benzodiazepine receptor

Czajkowski¹,

Farb²

1986

J. Neurosci.

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“…The significance of the 41-kDa protein band is unclear. Previous degradation studies on the GABA/ BDZ receptor with trypsin (Klotz et al, 1984;Eichinger and Sieghart, 1985) suggest a 39-40-kDa band as the major breakdown product of the receptor. It is, therefore, conceivable that this 41-kDa band seen in our preparation corresponds to such a degraded protein.…”

Section: Discussionmentioning

confidence: 93%

Biochemical Characterization of an Isolated and Functionally Reconstituted γ‐Aminobutyric Acid/Benzodiazepine Receptor

Bristow¹,

Martin²

1990

Journal of Neurochemistry

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We have solubilized, affinity-purified, and functionally reconstituted the gamma-aminobutyric acid/benzodiazepine (GABA/BDZ) receptor from rat brain into natural brain lipid liposomes. The detergent, 3-[(3-cholamidopropyl)-dimethylammonio] 1-propanesulphonate, was employed for the isolation of the receptor in the presence of a whole rat brain lipid extract supplemented with cholesteryl hemisuccinate. The soluble and reconstituted protein showed a homogeneous [3H]flunitrazepam binding population and the allosteric modulation of this binding site by GABA, by the pyrazolopyridine, cartazolate, and by the depressant barbiturate, pentobarbital. The purified GABA/BDZ receptor when incorporated into liposomes has been visualized by electron microscopy and reveals rosette structures, 8-9 nm in diameter, which appear to have a central pore. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the reconstituted GABA/BDZ receptor reveals three major protein bands of 41, 52-56, and 59-62 kDa, the latter two of which appears as doublets. Functional receptor reconstitution is demonstrated by the measurement of GABA-stimulated 36Cl- flux into the purified GABA/BDZ receptor incorporated liposomes and its modulation by the BDZs, barbiturates, and pyrazolopyridines.

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“…Membranes treated with trypsin were still able to reversibly bind 3H-flunitrazepam with an affinity similar to that of membranes not previously treated with trypsin. When these membranes were irradiated with UV-light, the same proteolytic peptides were detected as in membranes first photolabeled and then digested with trypsin (Eichinger and Sieghart, 1985). These results suggest a close association between reversible and irreversible benzodiazepine binding sites and indicate that membrane associated proteins Psi and P55 are differentially protected against degradation by trypsin.…”

Section: Differences In the Molecular Properties Of Benzodiazepine Rementioning

confidence: 70%

“…Recently, additional evidence for the difference in molecular structure of benzodiazepine receptors associated with protein Psi or P55 has been obtained (Eichinger and Sieghart, 1985). On digestion of photolabeled membranes with increasing concentrations of trypsin, up to 40% of 3H-flunitrazepam radioactivity irreversibly bound to protein was removed from the membranes.…”

Section: Differences In the Molecular Properties Of Benzodiazepine Rementioning

confidence: 98%

Benzodiazepine receptors: Multiple receptors or multiple conformations?

Sieghart

1985

J. Neural Transmission

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Several lines of evidence from reversible binding studies seem to indicate there are at least two "central" benzodiazepine receptor subtypes, the BZ1 and BZ2 receptors. Irreversible binding studies, using 3H-flunitrazepam as a photoaffinity label for benzodiazepine receptors, not only are in perfect agreement with the data from reversible binding studies but extend these studies by identifying P51, a protein with apparent molecular weight 51,000, as a protein associated with the BZ1 receptor and by suggesting that the BZ2 receptor might actually consist of several different benzodiazepine receptors associated with different and distinct proteins irreversibly labeled by 3H-flunitrazepam. Other reversible binding studies have accumulated indicating the existence of several different conformations of benzodiazepine receptors. Irreversible binding studies support this conclusion and in addition suggest the existence of four different benzodiazepine binding sites within the GABA-benzodiazepine receptor complex. It is therefore hypothesized that there are several different GABA-benzodiazepine receptor subtypes all of which have four distinct benzodiazepine binding sites which can exist in at least three different but freely interconvertible conformations. This hypothesis can account for all experimental observations obtained so far and might partially explain the distinct clinical effects of structurally similar benzodiazepines.

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Differential Degradation of Different Benzodiazepine Binding Proteins by Incubation of Membranes from Cerebellum or Hippocampus with Trypsin

Cited by 21 publications

References 19 publications

Transmembrane topology and subcellular distribution of the benzodiazepine receptor

Transmembrane topology and subcellular distribution of the benzodiazepine receptor

Biochemical Characterization of an Isolated and Functionally Reconstituted γ‐Aminobutyric Acid/Benzodiazepine Receptor

Benzodiazepine receptors: Multiple receptors or multiple conformations?

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