Ultrastructure and cholinergic binding capacity of junctional complexes isolated from rat brain

Robertis, E. De; Azcurra, Julio M.; Fiszer, Sara

doi:10.1016/0006-8993(67)90218-1

Cited by 107 publications

(14 citation statements)

References 10 publications

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“…The next problem is to isolate the proteins and determine their role in the function and organization of this highly specialized region . This method, which is an outgrowth of earlier efforts (de Robertis et al ., 1967 ;Davis and Bloom, 1970 ;Cotman et al ., 1971), should be an important step toward this end.…”

Section: Discussionmentioning

confidence: 99%

“…The initial breakthrough came from an observation by de Robertis and coworkers (de Robertis et al ., 1967) that Triton X-100 selectively released the synaptic complex from adjoining pre-and postsynaptic membranes . This work was confirmed and extended by Davis and Bloom (1970) and Cotman and coworkers (1971) who showed that synaptic complexes could readily and unambiguously be identified by staining with E-PTA both in synaptic plasma membrane (SPM) fractions and after treatment of SPM fractions with Triton X-100 .…”

Section: Introductionmentioning

confidence: 99%

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Isolation and Structural Studies on Synaptic Complexes From Rat Brain

Cotman

Taylor

1972

The Journal of Cell Biology

266

135

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A fraction enriched in synaptic complexes has been isolated from rat brain . The major structural elements of synaptic complexes after isolation are a sector of pre-and postsynaptic plasma membranes joined together by a synaptic cleft and a postsynaptic density (PSD) located on the inner surface of the postsynaptic membrane . On its outer surface, the postsynaptic membrane has a series of projections which extend about halfway into the cleft and which occur along the entire length of the PSD . Proteolytic enzymes at high concentrations remove the PSD and open the synaptic cleft ; at low concentrations the PSD is selectively destroyed . By contrast, the structural integrity of the PSD is resistant to treatment with NaCl, EGTA, and low concentrations of urea . Pre-and postsynaptic membranes also remain joined by the synaptic cleft after NaCl, EGTA, or mild urea treatment . High concentrations of urea cause the partial dissociation of the PSD . We conclude that polypeptides are probably one of the major components of the PSD and that the structural integrity of the PSD depends on polypeptides because disruption of the covalent or hydrophobic bonding of these polypeptides leads to a progressive loss of PSD structure .

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Isolation and Structural Studies on Synaptic Complexes From Rat Brain

Cotman

Taylor

1972

The Journal of Cell Biology

266

135

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“…(19). Triton X-100 was applied in order to solubilize the labeled components from membrane structures (6,(20)(21)(22). Triton increased the degree of purification of the receptor as substances.…”

Section: Discussionmentioning

confidence: 99%

“…The ratio of Triton to the fraction (idl 100' Triton/mg protein) was 1.1 (6). During addition of Triton, samples were rapidly mixed and the mixtures were centrifuged down at 105,000 x g after incubation at 4C for 30 min.…”

Section: Methodsmentioning

confidence: 99%

Purification of Histamine Receptor

Uchida

Takagi

1973

Jpn.J.Pharmacol

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Abstract-By means of differential centrifugation and density gradient separation, fractionation of histamine receptors of cat small intestinal smooth muscle was under taken. The radioactivity differences were investigated between the muscle labeled with radioactive dibenamine under drug protection and the muscle without the pro tective drug.The fraction was obtained in which specifically labeled histamine receptors were concentrated.The specificity of the fraction was proved by examining the pattern of distribution of radioactivity differences in subcellular fractions of the muscle protected with specific drugs (diphenhydramine, chlorpheniramine and hista mine) or with non-specific drugs (aniline, phenethylamine and benzylamine). The labeled components were of high molecular weight and degradated to smaller molecules by solubilization of the fraction with Triton X-100. A good correlation was found between radioactivity differences and pharmacological observations.

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confidence: 99%

Catecholamine Secretion From the Adrenal Gland Treated With Neuraminidase, Protamine or Alkylating Agent

Iwatsubo

Yamamoto

1972

Jpn.J.Pharmacol

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It has 1-ccn shown that brain gangliosides are concentrated in microsomal fractions and in synaptic membranes (1)(2)(3) and that the increased respiratory responses of cere bral cortex slices to stimulation are inhibited by basic proteins such as histones and protannines and all:ylating agents which form covalent linkages in aqueous solutions with carboxylic and other acidic groupings (4,5). inhibition of the responses can be restored by polyacidic substances such as ga:iglioside and phosphatidylserine (4). On the other hand a possible role of ganglioside in release of acetyl~eholine from synaptic vesicles was also shown (6). It may be of interest to determine the intracellular distribution of gangliosides in the adrenal medulla as well as the possible effect of protamines, neuraminidase and nitro gen mustard of the adrenal medullary secretion.Soon after death bovine adrenal glands obtained from the local slaughterhouse were placed on ice, then prepared for medullary cell-fractionation and perfusion. The medullae were honsoge=?izcd in two volumes of 0.25 M sucrose. Each 0.8 iii] of the homogenate was centrifuged in a 0,?-2.3 iM sucrose gradient in the 5-ml Spinco preparative ultracentri fuge tubes at 125,000 x g for 30 min according to the method of Potter and Axerlod (7).The subceliular fr tctions were dialized for 18 hr in running tap water, dried in vacuum, and suspended in a homogenizer in [00 ml of cl-rloroform-methanol (2:1, v/v). The ganglio side fraction was extracted and estimated as described by Burton (8). Succinic dehydro genase activity was estimated by the method of Slater and Bonner (9). Catecholainines (CA) in the subcellular fractions and the perfusa`e were assayed by the ethylenediamine condensation method (10). Method of perfusion of bovine adrenal glands was similar to that described by Trifaro et al. (11). CA secretion was evoked by switching perfusion fromLocke's solution to Locke's containing 5 x l0 M of acetylcholine (ACh), or from Ca-free Locke's solution to Locke's, using perfusion pump. Flor rate was 15 ml/min. For the determination of the effect of neuraminidase, protamine or nitrogen mustard on the induced CA secretions, the following calculation was used.Percent of initial secretion -st, J sp2 x 100, st, sp2where spy and sty were initial spontaneous release during incubation and the initial stimulated release of CA, respectively, sp, and sty were spontaneous and stimulated release of CA

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Ultrastructure and cholinergic binding capacity of junctional complexes isolated from rat brain

Cited by 107 publications

References 10 publications

Isolation and Structural Studies on Synaptic Complexes From Rat Brain

Isolation and Structural Studies on Synaptic Complexes From Rat Brain

Purification of Histamine Receptor

Catecholamine Secretion From the Adrenal Gland Treated With Neuraminidase, Protamine or Alkylating Agent

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