Organization of connexons in isolated rat liver gap junctions.

Gap junction plaques from rat liver plasma membranes have been subjected to a range of detergent treatments in order to evaluate systematically the influence of different isolation procedures on their structure. The separation of the connexons was found to vary depending on the conditions used. In the absence of detergent the center-to-center separation of the connexons is, on average, approximately 90 A, and they are arranged on a hexagonal lattice so that the symmetry of the double-layered structure approximates to p6m in projection (or p622 in three-dimensions). Exposure to increasing concentration of detergent reduces the connexon separation to values below 80 A. More severe detergent treatment leads to disintegration of the gap junction plaques. Specimens with center-to-center separations smaller than 86 A show progressively larger deviation from p6m symmetry, seen as apparent rotations of the connexon assemblies within the crystal lattice. This reorganization occurs with both ice-embedded and negatively-stained specimens, using ionic or nonionic detergents, and therefore is probably a packing readjustment caused by depletion of intervening lipid molecules.     

Multichannel recordings from membranes which contain gap junctions.

We have studied multichannel patch-clamp recordings in earthworm axon septal membranes that contain gap junctions. Though all channels have the same conductance and selectivity, the probabilities of the conductance levels in the majority of the recordings could not be fit by assuming independent and identical channels; in these cases, we found that at least two different open probabilities were required to explain the data. The data thus suggest that, within one junctional membrane complex, there exists a heterogenous channel population of similar but not identical channel types. The analysis also revealed cases where cooperativity between individual channels was the only explanation for the amplitude histograms of the observed multichannel activity. The conclusions drawn are based on a theoretical analysis of multichannel current-amplitude histograms. We derive two tests for independent and identical channels. We analyze the effects of mode shifting. These results are based on the ratio of peaks in the histograms; they are independent of the number of channels in the patch and the model of channel gating. In some cases failure to fulfill the criteria of these tests implied an interdependence or cooperativity between channels. Lastly, we have devised statistical tests for stability of the recording in the presence of variance due to finite sample size.     

Restoration of gap junction-like structure after detergent solubilization of the proteins from liver gap junctions

Gap junctions isolated from rat liver were partially solubilized with a mixture of digitonin and octyl glucoside. After supplementation with lecithin and cholesterol, the octyl glucoside was removed from the soluble fraction by dialysis. The membranes of the reconstituted vesicles, observed in freeze-fracture, contained particles ranging from 7 to 12 nm diameter, more or less aggregated depending on the protein-to-lipid ratio. At every protein concentration, the arrangement of particles in contact areas between adjacent membranes closely resembles the organization of intact gap junctions. We conclude that the mixture of digitonin and octyl glucoside is able to solubilize the proteins of the liver gap junctions while preserving their property of restoring a gap junction-like structure.     

A detergent-independent procedure for the isolation of gap junctions from rat liver

In this paper, the isolation of rat liver gap junctions from alkali-extracted rat liver plasma membranes is described. The purification is significantly more rapid than the commonly used detergent-based approaches and is subject to less variability. The gap junctions isolated by this method are comprised of a 27,000-Da polypeptide previously identified as the major gap junction polypeptide. The isolated gap junctions have the characteristic double-membrane organization and subunit structure observed in vivo. The protein yield is from 8 to 10 micrograms/g of liver (wet weight), about a 10-fold increase in recovery over that of earlier isolation procedures. With the availability of increased amounts of material, antibodies were raised to the liver gap junction polypeptide. Immunofluorescence localization of these antibodies on rat liver sections revealed a distribution consistent with that expected from electron microscopic analysis of liver thin sections. Double diffusion of antibody against solubilized gap junctions in detergent-containing gels resulted in the formation of precipitin arcs, suggesting response to multiple determinants. Antibody binding to the 27,000-Da gap junction polypeptide was demonstrated by immunoblot analysis of sodium dodecyl sulfate-polyacrylamide gels containing rat liver plasma membranes and isolated gap junctions. These results confirm the identification of the 27,000-Da polypeptide as the major protein component of gap junctions.     

Fluorescence anisotropy from diphenylhexatriene in rat liver plasma membranes

Rat livers were fractionated to obtain intracellular membrane preparations and a highly purified preparation of bile canaliculi. The fraction containing bile canaliculi was homogenized and subfractionated to give fractions representing fragments of contiguous membrane and of canalicular microvilli. The relative purity and extent of contamination of each preparation was determined. When the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene was incorporated into aliquots of each fraction at the same probe: lipid ratio and the steady-state anisotropy of its fluorescence measured, it was found that the plasma membrane preparations were much more ordered than the intracellular membrane preparations. Of the plasma membrane preparations, that containing the canalicular microvilli was the most ordered, even allowing for any contribution of contaminants. Thus the microvillus membrane of the bile canaliculus appears to be the most ordered domain of the plasma membrane of the hepatocyte. The high order in this domain may be a factor in reducing the susceptibility to bile salt damage during bile secretion, since it is this region which is exposed to high concentrations of bile salts in vivo.     

Characterization of rat liver cell plasma membranes.

A method is described by which bile canalicular membranes (BCM) can be prepared, together with canaliculus-free plasma membrane (PM), both essentially free of contamination. The recovery of both fractions together was estimated to be 46%. The concentrations of total lipid, total phospholipid and cholesterol were substantially greater in the BCM, and polyacrylamide gel electrophoresis revealed differences in protein composition. The differences in lipid and protein composition of these two plasma membrane fractions are presumably related to their very different physiological functions.     

Rectangular arrays of particles on freeze-cleaved plasma membranes are not gap junctions

Freeze-cleave replicas of adult rat diaphragm have revealed the presence of numerous small rectangular arrays of 60 Å particles (respectively pits) on the fracture faces of the sarcolemmas of the myofibers. Since these fibers are separated by thick basal laminae and are not electrically coupled we conclude that the rectangular arrays are not morphological equivalents of gap junctions as suggested by Staehelin [14]. The term “type III gap junctions” for these arrays therefore should be discontinued.     

The fluidity of plasma membranes from ethanol-treated rat liver

Summary Male Wistar rats were maintained for 35–40 days on a liquid diet containing 36% of calories as ethanol. Ethanol was replaced by carbohydrates in the isocaloric diet fed to control animals. The effect of ethanol consumption has been studied on the fluorescence polarization of rat liver plasma membranes and artificial lipid vesicles and on the lipid composition of the membranes. Fluorescence polarization in both membranes and vesicles was determined using DPH and TMA-DPH as fluorescence markers; from these data, the polarization term (r_o/r–1)^–1 and flow activation energy (E) were calculated. The ethanol consumption induces a more fluid environment within the membrane core of liver plasma membranes; the ethanol-fed rat membranes are more resistant to the in vitro effect of ethanol disordering the membrane structure. Vesicles obtained with lipids from either control membranes or ethanol-fed rat membranes were treated with ethanol and the changes in polarization paralleled to those exhibited by the membranes. The absence of phase transitions and of E changes was also shown in temperature-dependence studies. The lower cholesterol content found in ethanol-fed rat plasma membranes might be responsible for observed variations in the microviscosity.Abbreviation OG octyl -D-glucopyranoside     

Gangliosides of plasma membranes from normal rat liver and Morris hepatoma.

Plasma membranes were isolated from normal rat liver and Morris hepatoma 5123tc by discontinuous sucrose gradient centrifugation. There was an average two and one-halffold enrichment of gangliosides in plasma membranes from normal liver and hepatoma as compared with their respective whole cells. The amount of total gangliosides in plasma membranes from hepatoma was eight times greater than that found in normal liver. This increase resulted from a fivefold increase in hematosides, an eightfold increase in monosialogangliosides and a twenty-twofold increase in disialogangliosides. Trisialogangliosides were present in normal liver but not in hepatoma.     

Properties of anion-sensitive ATPase from plasma membranes of rat liver

The effects of bivalent cations, pH, anions, 2,4-dinitrophenol, dicyclohexylcarbodiimide and roseofungine on the anion-sensitive ATPase from rat liver plasma membranes were studied. It was found that ATPase from plasma membranes is similar to the anion-sensitive ATPase of rat liver nuclei and differs in some features from mitochondrial ATPase.     

Purification of NADH-cytochromeb 5 reductase from rat liver plasma membranes

Summary An NADH-cytochromeb ₅ reductase was purified from rat liver plasma membranes. Rat liver plasma membranes were prepared by aqueous two-phase partition. Peripheral proteins were removed by EDTA extraction and integral membrane proteins were solubilized with Triton X-100. The NADH-cytochromeb ₅ reductase was purified by hydroxyapatite, anion exchange, and gel filtration chromatographies. The purified preparation was homogeneous and estimated to have an apparent molecular weight of 32 kDa on SDS-polyacrylamide gel electrophoresis. Two tryptic peptides of the purified enzyme had sequence homologies with rat, human, and bovine NADH-cytochromeb ₅ reductases.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate - BCA bicinchonicic acid - EDTA ethylenediamine tetraacetate acid disodium salt - FeCN ferricyanide - HPLC high-performance liquid chromatography - NADH nicotinamide adenine dinucleotide reduced form - PMSF -phenylmethylsulfonyl fluoride     

Gap junctions in several tissues share antigenic determinants with liver gap junctions.

Using affinity-purified antibodies against mouse liver gap junction protein (26 K), discrete fluorescent spots were seen by indirect immunofluorescence labelling on apposed membranes of contiguous cells in several mouse and rat tissues: pancreas (exocrine part), kidney, small intestine (epithelium and circular smooth muscle), Fallopian tube, endometrium, and myometrium of delivering rats. No reaction was seen on sections of myocardium, ovaries and lens. Specific labelling of gap junction plaques was demonstrated by immunoelectron microscopy on ultrathin frozen sections through liver and the exocrine part of pancreas after treatment with gold protein A. Weak immunoreactivity was found on the endocrine part of the pancreas (i.e., Langerhans islets) after glibenclamide treatment of mice and rats, which causes an increase of insulin secretion and of the size as well as the number of gap junction plaques in cells of Langerhans islets. Furthermore, the affinity purified anti-liver 26 K antibodies were shown by immunoblot to react with proteins of similar mol. wt. in pancreas and kidney membranes. Taken together these results suggest that gap junctions from several, morphogenetically different tissues have specific antigenic sites in common. The different extent of specific immunoreactivity of anti-liver 26 K antibodies with different tissues is likely due to differences in size and number of gap junctions although structural differences cannot be excluded.