scholarly journals The incorporation in vitro of sulphate ions into synaptic-membrane glycoproteins

1981 ◽  
Vol 200 (2) ◽  
pp. 373-377 ◽  
Author(s):  
C J B White
Keyword(s):  
Plasma Membranes ◽  
Specific Binding ◽  
Synaptic Membranes ◽  
Synaptic Membrane ◽  
Membrane Glycoproteins ◽  
Synaptic Plasma Membranes ◽  
Molecular Weights ◽  
Carrier Free ◽  
Sheep Brain

Synaptosomes from sheep brain cortex were incubated with carrier-free Na235SO4 and the synaptic plasma membranes were isolated. The membranes were free of contamination from cytosol, mitochondria and microsomal material and accounted for 30% of the radioactivity present in the synaptosomal particulate fraction. Control experiments demonstrated that the radioactivity present in the preparation was not due to non-specific binding of sulphate ions. The synaptic membranes contained at least six 35S-containing protein bands with molecular weights between 160 000 and 16 000. Analysis showed that the radioactivity was located in the carbohydrate moiety of a glycopeptide.

Sodium affinity of brain Na(+)-K(+)-ATPase is dependent on isozyme and environment of the pump

1990 ◽  
Vol 258 (5) ◽  
pp. C803-C811 ◽  
Author(s):  
J. L. Brodsky ◽  
G. Guidotti
Keyword(s):  
Plasma Membranes ◽  
Fibroblast Cell ◽  
Mouse Fibroblast ◽  
Synaptic Plasma Membranes ◽  
Apparent Dissociation Constant ◽  
Low Sodium ◽  
Physiological Relevance ◽  
Mouse Fibroblast Cell Line

The sodium affinities for the two forms of the Na(+)-K(+)-ATPase in brain were characterized. To mimic physiological conditions, synaptosomes, which are pinched off presynaptic nerve termini, were used. Examination of the pump in vitro was performed by preparing synaptic plasma membranes (SPMs). It was first shown that synaptosomes contain the two forms of the Na(+)-K(+)-ATPase, alpha 1 and alpha 2, and that these forms have markedly different affinities for the inhibitory cardiac glycoside ouabain. The apparent dissociation constant (K0.5) of alpha 1 for sodium changed from 12 to 9 mM when going from synaptosomes to membranes. For alpha 2, however, a shift from 36 to 12.5 mM was evident. The conclusion is that in vivo alpha 2 exists as a low sodium affinity species but can be altered to a high-affinity form simply by vesicle disruption. By comparison, the Na(+)-K(+)-ATPase from the mouse fibroblast cell line, 3T3-F442A cells, expressed only the alpha 1-isozyme, as shown by immunoblotting and by measurement of its ouabain and sodium affinities. The physiological relevance of these observations is also presented.

Protein–glycolipid interactions during spermatogenesis. Binding of specific germ cell proteins to sulfatoxygalactosylacylalkylglycerol, the major glycolipid of mammalian male germ cells

1985 ◽  
Vol 63 (10) ◽  
pp. 1077-1085 ◽  
Author(s):  
C. A. Lingwood
Keyword(s):  
Germ Cell ◽  
Germ Cells ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Immune Serum ◽  
Spermatogenic Cells ◽  
Molecular Weights ◽  
Testicular Spermatozoa ◽  
Antibody Staining ◽  
A New Technique

Specific binding of membrane proteins extracted from rat spermatogenic cells to the major glycolipid of the male germ cell has been demonstrated by affinity chromatography. A new method for the production of affinity matrices, using photoactivatable heterobifunctional cross-linking agents, has been used to immobilize sulfatoxygalactosylacylalkylglycerol. Three proteins of apparent molecular weights 68 000, 34 000, and 24 000 from spermatogenic cells have been shown to selectively and reversibly bind to this affinity matrix. Antiserum raised against the major of these species (68 000) was demonstrated to be specific for this protein by immunoblotting. A new technique for the reduction of background nonspecific antibody staining using this method is described. The immune serum has been used to localize the antigen in frozen testicular sections. The protein is present in the plasma membranes of all germ cells, but the expression is elevated for testicular spermatozoa and cells in or near the basal compartment of the seminiferous epithelium. The relevance of these findings to intercellular communication and spermatogenesis is discussed.

Kinetic analysis of rat parotid gland muscarinic receptors in vivo: comparison with brain and heart

1993 ◽  
Vol 264 (3) ◽  
pp. G541-G552
Author(s):  
Y. Hiramatsu ◽  
R. Kawai ◽  
R. C. Reba ◽  
T. R. Simon ◽  
B. J. Baum ◽  
...  
Keyword(s):  
Parotid Gland ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Muscarinic Acetylcholine Receptor ◽  
Binding Potential ◽  
Specific Binding Sites ◽  
Rat Parotid Gland ◽  
Rat Parotid

(RR)- and (SS)-quinuclidinyl iodobenzilate enantiomers [(RR)- and (SS)-IQNB, active and inert, respectively] have been synthesized for quantitative evaluation of muscarinic acetylcholine receptor (mAChR) binding. Pharmacokinetic approaches have not been used previously to assess in vivo IQNB binding in nonexcitable tissues. We have applied this method to examine mAChRs in rat parotid gland in comparison to those in brain and heart. Short-term infusion studies in vivo showed that the "instantaneous" reversible binding of (RR)- and (SS)-IQNB was high in the parotid (greater nonspecific binding potential), intermediate in the heart, and lowest in cortex and cerebellum. Long-term bolus injection experiments showed that the parotid gland mAChRs possessed a binding potential for receptor specific sites (380), which was intermediate between that of parietal cortex (930) and cerebellum (10) and greater than that of heart (165). In vitro binding to plasma membranes was generally consistent with the in vivo findings. In aggregate, these studies show that mAChRs can be evaluated in vivo in a nonexcitable tissue with the use of stereospecific ligands and a pharmacokinetic approach. The data suggest that IQNB, a mAChR antagonist, can identify characteristics of specific binding sites, which may reflect tissue differences.

Na+/Ca2+ Exchange in Plasma Membranes and Microsomal Fraction of Brain Cortex

1985 ◽  
Vol 43 ◽  
pp. 490-491
Author(s):  
O.P. Coutinho ◽  
W.N. Norton ◽  
C.A.M. Carvalho ◽  
A.P. Carvalho
Keyword(s):  
Plasma Membranes ◽  
Brain Cortex ◽  
Microsomal Fraction ◽  
Primary Objective ◽  
Synaptic Plasma Membranes ◽  
Intracellular Ca ◽  
Sheep Brain ◽  
Gradient Separation ◽  
Density Gradient Separation

The Na+/Ca2+ exchange system is one of the mechanisms responsible for regulating intracellular Ca2+ concentrations in neurons. However, the precise mechanism of Na+/Ca2+ exchange among synaptic plasma membranes and microsomes of neurons has yet to be resolved. The primary objective of the present investigation was to determine the specific transport properties with regard to Na+/Ca2+ exchange among neural synaptic plasma membranes and the various fractions of a microsomal density gradient separation.Synaptic plasma membranes were obtained by hypotonic disruption of sheep brain cortex synaptosomes (Fig. 1). Enzymatic characterization of the synaptic plasma membranes revealed high Na+/K+ ATPase activity and low RNA content, which suggest that the material is predominantly of plasma membrane origin. The most common constituents consisted of membrane fragments and resealed vesicles (Fig. 2).

scholarly journals The influence of plasma on basal and ACTH-stimulated in vitro adrenocortical steroidogenesis

1999 ◽  
Vol 162 (1) ◽  
pp. 155-161 ◽  
Author(s):  
PJ Jenkins ◽  
TA Cross ◽  
LA Perry ◽  
SA Medbak ◽  
GM Besser ◽  
...  
Keyword(s):  
Specific Binding ◽  
Steroid Production ◽  
Vycor Glass ◽  
Molecular Weights ◽  
Plasma Factor ◽  
Glass Column ◽  
Unidentified Component ◽  
The Media ◽  
Two Peaks

Early descriptions of in vitro ACTH bioassays all emphasised the need to use extracted plasma samples due to interference by an unidentified component. The aim of these studies was to elucidate the effects of whole plasma on ACTH steroidogenic activity in vitro and to identify the responsible factor. A sensitive in vitro dispersed bovine adrenocortical cell bioassay was established. The addition of 10% ACTH-depleted human pooled plasma to the incubation media resulted in basal steroidogenesis equivalent to that achieved with 10(-9) M ACTH1-24 and potentiated the steroidogenic activity of 10(-9) M ACTH1-24 by 7.8-fold. This potentiation was dependent on the concentration of both ACTH and plasma in the media, but did not result from the mitogenic effect of plasma. A pituitary source was excluded and the potentiating activity was not extractable by Vycor glass. Column chromatography demonstrated two peaks of activity corresponding to molecular weights of 650 and 220x10(3) Da. These peaks did not correspond to the plasma binding of 125I-ACTH which resulted from non-specific binding to albumin. Lipoprotein-deficient serum had no effect on either basal or ACTH-stimulated steroidogenesis, but both were restored by the addition of purified lipoproteins. However, novel findings demonstrated a differential effect of low (LDL) and high (HDL) density lipoproteins on basal and ACTH-stimulated steroid production; thus, LDL exerted a greater effect on the former, whilst HDL potentiated the steroidogenic activity of added ACTH more than LDL. The addition of the lipoproteins to lipoprotein-deficient serum restored its basal and ACTH potentiating effects, the cholesterol concentrations of the chromatographic fractions exactly paralleling their ACTH potentiating effect. These findings suggest that not only are lipoproteins the plasma factor(s) which potentiates ACTH steroidogenic activity in in vitro bioassays, but also that they exert differential effects on basal and ACTH-stimulated steroid production.

Effects of hypophysectomy and GH administration on bovine and human GH binding to rat liver membranes

1985 ◽  
Vol 249 (1) ◽  
pp. E56-E62
Author(s):  
J. L. Messina ◽  
S. Eden ◽  
J. L. Kostyo
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Human Growth ◽  
Male Rats ◽  
Normal Male ◽  
Number Of Binding Sites ◽  
Liver Membranes ◽  
Isolated Liver

Experiments were conducted to investigate the specific binding of highly purified bovine and human growth hormones (bGH and hGH) to purified liver plasma membranes of male rats at various times after hypophysectomy and after the acute intravenous administration of bGH. Liver membranes prepared from hypophysectomized male rats showed a two- to threefold increase in the specific binding of either [125I]iodo-bGH or [125I]iodo-hGH, when compared with membranes prepared from the livers of age-matched normal male rats. The increase in GH binding was apparent within 3 days after hypophysectomy and persisted for a number of weeks after the operation. The increase in GH binding produced by hypophysectomy appeared to be due to an increase in the number of binding sites present on the membranes. The intravenous injection of 200 micrograms of bGH into hypophysectomized male rats 5-60 min before they were killed markedly reduced the ability of liver membranes prepared from these animals to bind [125I]iodo-bGH specifically. This decrease in GH binding seen after the injection of bGH may have been due to the development of a slowly dissociating hormone-binding site complex, which thereby reduced the number of available binding sites. This conclusion is supported by the finding that bGH, which is bound in vitro to isolated liver membranes, dissociates slowly and incompletely in the presence of an excess of unlabeled hormone. Moreover, the degree to which the bound hormone can dissociate appears to depend on the length of time that association is allowed to occur.(ABSTRACT TRUNCATED AT 250 WORDS)

Sensitivity of ATPase-ADPase activities from synaptic plasma membranes of rat forebrain to lipid peroxidation in vitro and the protective effect of vitamin E

1996 ◽  
Vol 21 (3) ◽  
pp. 299-304 ◽  
Author(s):  
Marion Vietta ◽  
Silvana S. Frassetto ◽  
Ana M. O. Battastini ◽  
Adriane Bello-Klein ◽  
Cleci Moreira ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Vitamin E ◽  
Protective Effect ◽  
Plasma Membranes ◽  
Synaptic Plasma Membranes ◽  
Rat Forebrain
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