Plasma membranes from oats prepared by partition in an aqueous polymer two-phase system : on the use of light-induced cytochrome B reduction as a marker for the plasma membrane

Presumptive plasma membrane fractions have been prepared from oat (Avena sativa L. cv. Brighton) roots and shoots, respectively, by partition of microsomal fractions in a dextran-polyethylene glycol two-phase system. The plasma membranes had a high affinity for the polyethylene glycol-rich upper phase, whereas membranes from mitochondria and other organelles partitioned in the dextran-rich lower phase or at the interface. Thus, relatively pure plasma membranes were obtained by only two partition steps, and within 3 hours from homogenization of the material.

The plasma membranes from both organs were enriched in K⁺-stimulated Mg²⁺-dependent ATPase and glucan synthetase II, two tentative markers for the plant plasma membrane. Silicotungstic acid, an indicative stain for the plasma membrane, stained the vesicles recovered from the upper phase, but failed to stain the membranes partitioning in the lower phase or at the interface.

The plasma membranes were also enriched in a light-reducible b-cytochrome. This b-cytochrome can be measured by its light-induced absorbance change and may serve as a marker for the plant plasma membrane.

     

Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins.

A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures.     

Characterization of ferric reducing activity in roots of Fe-deficient Phaseolus vulgaris

Light-induced absorbance changes [LIAC; measured as Δ( A ₄₂₈– A ₄₁₀)] reflecting the reduction of a b -type cytochrome and mediated by an endogenous blue light absorbing receptor have been proposed to be related to blue light physiology of fungi and higher plants. It has also been suggested that the same cytochrome specifically can be reduced by red light in the presence of methylene blue. We have investigated the distribution of LIAC between different membrane fractions from corn ( Zea mays L.) coleoptiles and cauliflower ( Brassica oleracea L.) inflorescences. The membrane fractions were obtained by differential centrifugation followed by partition in an aqueous polymer two-phase system. By this procedure fractions rich in plasma membrane were obtained from both mitochondrial and microsomal fractions obtained by centrifugation. LIAC was by far most enriched in fractions also enriched in plasma membranes (identified by silicotungstic acid staining), but LIAC could be obtained also in other fractions. Our conclusion is that LIAC undoubtedly is caused by a b -cytochrome bound to the plasma membrane, but that LIAC also may be due to other b -cytochromes, one of which is probably located in the endoplasmic reticulum. Thus, the two assay procedures used for LIAC (blue and red light induced) could not disciminate between different b -cytochromes giving rise to LIAC.     

The effect of amantadine on nicotinic acetylcholine receptor (nAchR) in reconstituted membranes

The antiviral drug amantadine is also a potent neuromuscular blocking agent. When the nicotinic receptor from a Torpedinidae species is reconstituted into soybean liposomes, the binding of α-bungarotoxin is not altered although the carbamylcholine induced radioactive cation influx is blocked.By studying cation fluxes in amantadine preincubated membranes previously exposed to different concentrations of carbamylcholine for different periods of time, we have shown that the drug accelerates the conversion of the nicotinic acetylcholine receptor from a state of low affinity to a state of high affinity for carbamyalcholine, a phenomenon correlated with receptor desensitization. The drug did not induce such a shift by itself.The present data and those by Earnest et al. (Biochemistry22, 5523–5535, 1984) show that the nicotinic acetylcholine receptor reconstituted into liposomes is a good model for studying the effects of noncompetitive blockers of nicotinic acetylcholine receptor function.     

Fractionation by velocity sedimentation of Torpedo nicotinic post-synaptic membranes

Velocity sedimentation on sucrose gradients containing Torpedo physiological saline has been utilized to fractionate Torpedo (Torpedo californica and T. nobiliana) post-synaptic membranes isolated initially on the basis of their density by equilibrium centrifugation. Membranes are separated into two populations: (1) those retained within the gradient (referred to as gradient pool); and (2) membranes sedimenting rapidly through the gradient (referred to as f 22, fraction 22 of the gradient). Comparison of their polypeptide compositions by sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicates that the gradient pool consists of highly purified nicotinic post-synaptic membranes containing the peptides of the acetylcholine receptor and a peptide of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 43 000, while f 22 contains the contaminating membranes present in the initial suspension as well as a small fraction of the nicotinic post-synaptic membranes. On the basis of the kinetics of efflux of ²²Na⁺ from the membrane fractions, it is concluded that the gradient pool contains most of the sealed vesicles with functional nicotinic receptors. The internal volume (μl/mg protein) of those membranes exceeds that of f 22 by a factor of 4, and greater than 85% of that internal volume is equilibrated by the nicotinic agonist carbamylcholine, while for f 22 only 40% is equilibrated. Thin-section electron microscopy has been used to estimate the distribution of vesicle sizes. The observed distribution for the gradient pool indicates that these vesicles are a size homogeneous population of diameter 0.3 μm, while f 22 contains a number of smaller and larger vesicles. Torpedo post-synaptic membranes have been treated with alkali to remove the non-receptor peptide of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 43 000. After alkaline extraction, velocity sedimentation permits the isolation of a population of size-homogeneous and well-sealed vesicles containing only the peptides of the nicotinic receptor. It is concluded that upon homogenization, the innervated surface of the Torpedo electroplax tends to form vesicles of uniform size (0.3 μm) which can be readily isolated by velocity sedimentation and that the peptide of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 43 000 is not required for the maintenance of bilayer structure.     

Partition profile of the nicotinic acetylcholine receptor in lipid domains upon reconstitution

The nicotinic acetylcholine receptor (AChR) is in intimate contact with the lipids in its native membrane. Here we analyze the possibility that it is the intrinsic properties of the AChR that determine its partition into a given lipid domain. Torpedo AChR or a synthetic peptide corresponding to the AChR γM4 segment (the one in closer contact with lipids) was reconstituted into “raft”-containing model membranes. The distribution of the AChR was assessed by Triton X-100 extraction in combination with fluorescence studies, and lipid analyses were performed on each sample. The influence of rapsyn, a peripheral protein involved in AChR aggregation, was studied. Raft-like domain aggregation was also studied using membranes containing the ganglioside GM1 followed by GM1 crosslinking. The γM4 peptide displays a marked preference for raft-like domains. In contrast, AChR alone or in the presence of rapsyn or ganglioside aggregation exhibits no such preference for raft-like domains, but it does cause a significant reduction in the total amount of these domains. The results indicate that the distribution of the AChR in lipid domains cannot be due exclusively to the intrinsic physicochemical properties of the protein and that there must be an external signal in native cell membranes that directs the AChR to a specific membrane domain.     

The nicotinic acetylcholine receptor from Discopyge tschudii: purification, characterization and reconstitution into liposomes

The nicotinic acetylcholine receptor from Discopyge tschudii electroplax was purified by affinity chromatography on Affi-Gel 401 using bromoacetylcholine as the ligand. Its specific activity was about 4000 pmol 125I-alpha-bungarotoxin/mg protein. SDS-polyacrylamide gel electrophoresis revealed four bands of apparent molecular weights: 41,200, 49,500, 60,000 and 66,300. The amino acid composition of each individual subunit was determined. Native membranes, rich in nicotinic receptor exhibited carbamylcholine-catalysed cation transport (blocked by curare and desensitized by prior incubation with the cholinergic agonist). The functional activity of the purified material could be reconstituted into soybean lecithin liposomes. Our data show that the Discopyge tschudii nicotinic receptor is similar to that from Torpedo californica.     

Concanavalin A-Sepharose Affinity Chromatography of Axon Plasma Membrane Proteins. Heterogeneity of Cholinergic Binding Sites

Abstract: Lysolecithin-solubilized proteins from axon plasma membranes of lobster walking leg nerve bundles were chromatographed on concanavalin A (Con A)-sepharose. Bound glycoproteins were eluted with α-methyl-D- mannoside. Near quantitative recovery of total protein was observed, 20–30% of the total protein being eluted in the Con A-binding glycoprotein fraction. A 5-fold enrichment of acetylcholinesterase (AChE) activity was achieved, demonstrating the glycoprotein nature of the axonal enzyme. The chromatographed fractions were characterized for binding of [³H]quinuclidinyl benzilate (QNB), [³nicotine (Nic), and [1251]α-bung arotoxin (BgTx) in an attempt to distinguish possible "muscarinic" and "nicotinic" binding sites in axonal membranes. All of the high-affinity "muscarinic" [³H]QNB binding activity appeared in the non-Con A-binding protein fractions, while binding of the two "nicotinic" ligands, [³Nic and ¹²⁵I-BgTx, was found in both the glycoprotein and non-Con A-binding protein fractions. BgTx interaction with the Con A-binding glycoproteins could be blocked with dtubocurarine, but BgTx binding in the non-Con A-binding proteins was not inhibited by curare. The significance of multiple cholinergic binding sites in axonal membranes is discussed. These data suggest a closer similarity between the cholinergic ligand binding proteins of peripheral nerve membrane and ganglia than between the axonal cholinergic binding sites and the ACh receptor of the neuromuscular junction.     

Localization of a prostaglandin F2alpha receptor in bovine Corpus luteum plasma membranes.

The distribution of a prostaglandin F2alpha receptor in various subcellular fractions from bovine corpora lutea obtained by differential and gradient centrifugation paralleled very closely the distribution in these fractions of 5'-nucleotidase, a marker enzyme for plasma membranes. The fractions most enriched in the receptor and 5'-nucleotidase were relatively free of mitochondria and lysosomes but were contaminated to some extent by elements of the endoplasmic reticulum. From these results it can be concluded that the prostaglandin F2alpha receptor is localized on the plasma membranes of the corpus luteum cells. A simple method is described for the purification of plasma membranes from bovine corpora lutea by differential centrifugation.     

STUDIES ON NICOTINIC ACETYLCHOLINE RECEPTORS IN MAMMALIAN BRAIN. CHARACTERIZATION OF A MICROSOMAL SUBFRACTION ENRICHED IN RECEPTOR FUNCTION FOR DIFFERENT NEUROTRANSMITTERS

Abstract— A subfraction, derived from the microsomal fraction of rat cerebral cortex, with a buoyant density of 1.112 g μ ml⁻¹ appears to be enriched in receptor sites for a number of potential neurotrans-mitters. These include the cholinergic (nicotinic and muscarinic) and ß-adrenergic receptors. This microsomal subfraction (P₃B₂) has been isolated on a preparative scale by two sequential isopycnic sedimentations in discontinuous sucrose gradients.
We have studied the morphology, enzymatic markers and protein composition of this fraction and have compared them with the properties of other subcellular fractions from the same source. Synaptic plasma membranes resembled P₃B₂ by exhibiting the same high extent of enrichment in receptors. However, the synaptic membranes appear to contain more mitochondrial and presynaptic (axonal and cell surface) membranes than does P₃B₂, and the postsynaptic membranes in the two fractions appear morphologically distinct since P₃B₂ does not contain the characteristic postsynaptic densities. Thus these membranes may be derived from Gray's type II synapses.     

Lipid-protein interactions at the nicotinic acetylcholine receptor. A functional coupling between nicotinic receptors and phosphatidic acid-containing lipid bilayers.

The structural and functional properties of reconstituted nicotinic acetylcholine receptor membranes composed of phosphatidyl choline either with or without cholesterol and/or phosphatidic acid have been examined to test the hypothesis that receptor conformational equilibria are modulated by the physical properties of the surrounding lipid environment. Spectroscopic and chemical labeling data indicate that the receptor in phosphatidylcholine alone is stabilized in a desensitized-like state, whereas the presence of either cholesterol or phosphatidic acid favors a resting-like conformation. Membranes that effectively stabilize a resting-like state exhibit a relatively large proportion of non-hydrogen-bonded lipid ester carbonyls, suggesting a relatively tight packing of the lipid head groups and thus a well ordered membrane. Functional reconstituted membranes also exhibit gel-to-liquid crystal phase transition temperatures that are higher than those of nonfunctional reconstituted membranes composed of phosphatidylcholine alone. Significantly, incorporation of the receptor into phosphatidic acid-containing membranes leads to a dramatic increase in both the lateral packing densities and the gel-to-liquid crystal phase transition temperatures of the reconstituted lipid bilayers. These results suggest a functional link between the nicotinic acetylcholine receptor and the physical properties of phosphatidic acid-containing membranes that could underlie the mechanism by which this lipid preferentially enhances receptor function.     

Effect of lipid membranes on the apparent pK of the local anesthetic tetracaine spin label and titration studies

Electrometric titrations and spin label data demonstrate changes in the experimentally determined apparent pK of an ionizable drug in the presence of membranes. This effect is attributed to the difference in partition coefficients for the charged and uncharged forms of the drug. Investigation of the binding of a local anesthetic, tetracaine, to egg phosphatidylcholine membranes indicates that the drug apparent pK decreases in the presence of membranes, the decrease being a function of membrane concentration. The agreement between titration and spin label studies is very good and could be simulated by calculating membrane-bound and free populations of charged and uncharged tetracaine from the independently-measured partition coefficients for the two forms.     

Immobilized receptor- and transporter-based liquid chromatographic phases for on-line pharmacological and biochemical studies: a mini-review

This review addresses the synthesis and characterization of two different types of receptor-based liquid chromatographic supports, one based upon a trans-membrane ligand gated ion channel receptor (the nicotinic acetylcholine receptor) and the other a soluble nuclear receptor (the estrogen receptor). In addition, studies with the P-glycoprotein transporter are also reported. The nicotinic receptor was immobilized via hydrophobic insertion into the interstitial spaces of an immobilized artificial membrane (IAM) stationary phase. the estrogen receptor was tethered to a hydrophilic stationary phase and the membranes containing the Pgp transporter were coated on the surface of the IAM stationary phase. The stationary phases were characterized using known ligands and substrates for the respective non-immobilized proteins. The results from zonal and frontal chromatographic experiments demonstrated that the stationary phases could be used to determine binding affinities (expressed as dissociation constants, Kd,'s) and to resolve mixtures of ligands according to their relative affinities. In addition. competitive ligand binding studies on the P-glycoprotein-based stationary phase have established that this phase can be used to identify and characterize competitive displacement and allosteric interactions. These studies demonstrate that immobilized-receptor phases can be used for on-line pharmacological studies and as rapid screens for the isolation and identification of lead drug candidates from complex biological or chemical mixtures.     

Postsynaptic membranes in the electric tissue of Narcine: III. Isolation and characterization

Postsynaptic membranes in homogenates of the electric tissue of Narcine were identified by labelling nicotinic acetylcholine receptors in the membranes with radioactive alpha-bungarotoxin. Various media and centrifugation conditions were examined in an attempt to obtain highly purified postsynaptic membranes. The main criterion for purification was approach towards the specific activity of the pure receptor protein, 9–10 nmol toxin-sites/mg protein. Isolation of tissue microsomes with Tris buffer, EDTA and the protease inhibitor phenylmethylsulfonylfluoride (PMSF), conditions which preserve the receptor molecules optimally, yielded about 50 % of the tissue toxin-sites, 5 % of the protein, 4 % of the ATPase and less than 2 % of the acetylcholinesterase (AChE). Further separation of vesiculated membranes in continuous density gradients of sucrose showed that the major contaminants of postsynaptic membrane vesicles were damaged mitochondria and tubular vesicles of dorsal electroplaque membranes rich in ATPase. Mitochondria were effectively removed from homogenates by ‘differential’ centrifugation, and ATPase-rich vesicles could be largely removed by causing their agglutination with calcium ions, or by controlled proteolysis in the absence of PMSF. Partially purified postsynaptic membranes were obtained having about 7 nmol toxin-sites/mg membrane protein. Further purification appears possible by affinity techniques.     

NUCLEAR MEMBRANES FROM MAMMALIAN LIVER : I. Isolation Procedure and General Characterization

Nuclear membranes were isolated from rat and pig liver by sonication of highly purified nuclear fractions and subsequent removal of adhering nucleoproteins in a high salt medium. The fractions were examined in the electron microscope by both negative staining and thin sectioning techniques and were found to consist of nuclear envelope fragments of widely varying sizes. Nuclear pore complex constituents still could frequently be recognized. The chemical composition of the nuclear membrane fractions was determined and compared with those of microsomal fractions prepared in parallel. For total nuclei as well as for nuclear membranes and microsomes, various enzyme activities were studied. The results indicate that a similarity exists between both fractions of cytomembranes, nuclear envelope, and endoplasmic reticulum, with respect to their RNA:protein ratio and their content of polar and nonpolar lipids. Both membranous fractions had many proteins in common including some membrane-bound enzymes. Activities in Mg-ATPase and the two examined cytochrome reductases were of the same order of magnitude. The content of cytochrome b₅ as well as of P-450 was markedly lower in the nuclear membranes. The nuclear membranes were found to have a higher buoyant density and to be richer in protein. The glucose-6-phosphatase and Na-K-ATPase activities in the nuclear membrane fraction were very low. In the gel electrophoresis, in addition to many common protein bands, some characteristic ones for either microsomal or nuclear membranous material were detected. Significant small amounts of DNA and RNA were found to remain closely associated with the nuclear envelope fragments. Our findings indicate that nuclear and endoplasmic reticulum membranes which are known to be in morphological continuity have, besides a far-reaching similarity, some characteristic differences.     

Plasma membranes from candida tropicalis grown on glucose or hexadecane. I. Isolation,identification and purification

Plasma membranes from Candida tropicalis grown on glucose or hexadecane were isolated using a method based on the difference in surface charge of mitochondria and plasma membranes.After mechanical disruption of the cells, a fraction consisting of mitochondrial and plasma membrane vesicles was obtained by differential centrifugation.Subsequently the mitochondria were separated from the plasma membrane vesicles by aggregation of the mitochondria at a pH corresponding to their isoelectric point. Additional purification of the isolated plasma membrane vesicles was achieved by osmolysis. Surface charge densities of mitochondria and plasma membranes were determined and showed substrate-dependent differences.The isolated plasma membranes were morphologically characterized by electron microscopy and, as a marker enzyme, the activity of Mg²⁺-dependant ATPase was determined.By checking for three mitochondrial marker enzymes the plasma membrane fractions were estimated to be 94% pure with regard to mitochondrial contamination.     

The sucrose carrier of the plant plasmalemma. III. Partial purification and reconstitution of active sucrose transport in liposomes.

The proteins from plasma membranes from sugar beet leaves were solubilized by 1% CHAPS and separated by size exclusion chromatography and by ion-exchange chromatography. The fractions enriched in sucrose transporter were monitored in three ways: differential labeling, ELISA, and reconstitution in proteoliposomes. When the plasma membranes were differentially labeled by N-ethylamaleimide in the presence of sucrose, a major peak of differential labeling was found at 120 kDa upon gel filtration. When this peak was recovered, denaturated by sodium dodecyl sulfate and reinjected on the gel filtration column, it yielded a peak of differential labeling at 42 kDa. When unlabeled membranes were used, the fractions eluted from the column were monitored by ELISA for their ability to recognize a serum directed against a 42 kDa previously identified as a putative sucrose carrier. The results paralleled those obtained by differential labeling, i.e. a major ELISA-reactive peak was found at 120 kDa upon gel filtration, and this peak yielded a peak most reactive at 40 kDa after denaturation. The 120 kDa peak prepared from unlabeled membranes was further separated on a Mono-Q column. The fractions were monitored by ELISA as described above, and reconstituted into proteoliposomes using asolectin. Active transport of sucrose, but not of valine could be observed with the reconstituted 120 kDa fraction. When the eluates from the Mono-Q column were reconstituted, the fractions exhibiting highest transport activity were enriched with a 42 kDa band. The data provide the first report concerning reconstitution of sucrose transport activity and confirm the involvement of a 42 kDa polypeptide in sucrose transport.     

Regulation of the affinity state of the N-formylated peptide receptor of neutrophils: role of guanine nucleotide-binding proteins and the cytoskeleton

Previous studies have indicated that the receptor for N-formylated peptides present on human neutrophils can exist in several ligand- dissociation states at least one of which is sensitive to guanine nucleotides. Human neutrophil membranes rich in cell surface enzyme markers have been isolated from cells pretreated at 37 degrees C with 5 nM fluoresceinated chemotactic peptide (N-formyl-Nle-Leu-Phe-Nle-Tyr- Lys-fluorescein; Fl-peptide) or a buffer control and analyzed for receptor-ligand dissociation states using a previously published fluorescence assay for estimating ligand binding and dissociation rates (Sklar, L. A., et al. 1984. J. Biol. Chem. 259:5661-5669). Fractionation of crude microsomes derived from homogenates of unstimulated cells by ultracentrifugation on linear D2O gradients yielded two plasma membrane-rich fractions termed fast and slow microsomes. Analysis of Fl-peptide dissociation rates from receptor present in fast membrane fractions of unstimulated cells yielded data that could be best fit by assuming that the receptor exists in three distinct ligand-dissociation states. The intermediate ligand- dissociation state (state B) accounted for 47% of the total and was converted to the fastest ligand-dissociation state (state A) by incubation of membranes with GTP or GTP-gamma-S. The remainder of the receptor (17%) present in unstimulated membranes was in a state from which ligand was virtually nondissociable (state C). This form of the receptor was insensitive to GTP-gamma-S. When cells were stimulated with Fl-peptide, most of the receptor present in slow and fast membranes was of the state C type. In contrast to unstimulated cells, slow membranes derived from cells exposed to Fl-peptide contained the majority of the recoverable receptor indicating that receptor was transferred to a physically isolatable membrane domain after ligand binding to the intact cell. The ligand-induced formation of state C in both fast and slow microsome fractions was inhibited by treatment of cells with dihydrocytochalasin B. However, the drug had no effect on translocation of the receptor to slow membranes. Pertussis toxin treatment of intact cells had no effect on ligand-induced formation of state C in either fraction even though other cellular responses were inhibited. Both slow and fast membranes contained a 41-kD G protein as assayed by immunoblot analysis. The data suggest that ligand induces a segregation of receptor-ligand complexes into a membrane domain in which the receptor is functionally uncoupled from the 41-kD neutrophil G protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

COMPOSITION OF CELLULAR MEMBRANES IN THE PANCREAS OF THE GUINEA PIG : IV. Polyacrylamide Gel Electrophoresis and Amino Acid Composition of Membrane Proteins

Two methods of polyacrylamide gel electrophoresis (the acid method of Eytan and Ohad and the Na dodecylsulfate (SDS) disc method of Maizel) have been used for analyzing the proteins of gel fractions isolated from the guinea pig pancreatic exocrine cells and in particular the proteins bound to the membranes involved in the synthesis, intracellular transport, and discharge of secretory enzymes: rough (RM) and smooth microsome (SM) membranes, zymogen granule (ZG) membranes, and plasma membranes (PM). Since in the two systems the electrophoretic mobility of proteins depends on different factors (size, shape, and net charge of molecules in the acid system; size only in the SDS system) a deeper insight into the protein composition of the fractions could be obtained. The gel patterns of RM, SM, and ZG membranes turned out to be accounted for mainly by segregated secretory enzymes (in rough microsomes also by ribosome proteins) and thus were found to share most of the bands. In contrast, with highly purified membrane fractions different patterns were obtained: RM and SM membrane proteins turn out to contain a large number of different proteins with molecular weights varying between ~150,000 and 15,000 daltons. The pattern of ZG membranes was greatly different in the two systems: only two bands were separated by the acid method and as many as 23 by the SDS method. PM gave a rather complex pattern in either system. Both ZG membranes and PM were found to contain a large proportion of low molecular weight proteins. Nothing appears in common between the proteins of SM membranes (primarily of Golgi origin) and those of ZG membranes, while the latter and PM exhibit a certain degree of similarity. By amino acid analysis we found only slight differences: relative to the other fractions: RM membranes were higher in basic amino acids and ZG membranes contained a larger amount of methionine. Taken together with recent data on lipid composition and enzyme activities of the same fractions, these results indicate that the membranes of the pancreatic exocrine cells are chemically and functionally distinct, and hence do not mix randomly with one another during the transport of secretory products.     

Gonadotropin and prostaglandins binding sites in rough endoplasmic reticulum and Golgi fractions of bovine corpora lutea.

Highly purified rough endoplasmic reticulum and three subfractions of golgi were prepared from 105,000g pellet of the homogenate by centrifugation in floatation and sedimentation discontinuous sucrose gradients. Highly purified plasma membranes were also prepared from 9,000g pellet of the same homogenates for assessment under the same experimental conditions. Although 5′-nucleotidase, a marker for plasma membranes, was markedly enriched in plasma membranes, very little or none of this enzyme activity was found in other fractions. Very little or no NADH cytochrome c reductase activity, a marker for rough endoplasmic reticulum, was found in fractions other than rough endoplasmic reticulum. Galactosyl transferase, a marker for golgi, was found and enriched in all the fractions; however, enrichment in golgi fractions was higher than in other fractions. Very little or no lysosomal marker activity, i.e., acid phosphatase, was found in rough endoplasmic reticulum or golgi fractions as compared to lysosomes. These marker enzyme data suggested that rough endoplasmic reticulum and golgi fractions were relatively pure with little or no cross contamination with other organelles. The [¹²⁵I]human choriogonadotropin ([¹²⁵I]hCG), [³H]prostaglandin (PG)E₁, and [³H]PGF_2a specifically bound to rough endoplasmic reticulum and golgi fractions in addition to plasma membranes. The enrichments of binding in the former two fractions, in some cases, were as high as plasma membranes itself. The specific binding of some of the ligands was found to be partially latent in rough endoplasmic reticulum and golgi fractions but not in plasma membranes. Marker enzyme data, ratio between bindings and marker enzyme activities (an index of organelle contamination), and partial latency of binding suggest that rough endoplasmic reticulum and golgi fractions intrinsically contain gonadotropin and PGs binding sites.