Architecture of the outer membrane of Escherichia coli K12. II. Freeze fractur morphology of wild type and mutant strains

Freeze fracturing electron microscopy of Escherichia coli K12 cells showed that the outer fracture face of the outer membrane is densily occupied with particles. On the inner fracture face of the outer membrane, pits are visible, which are probably complementary to the particles at opposite fracture face. This observation suggests that the particles are micelle-like. In some mutants which lack one or more major outer membrane proteins the density of particles is reduced. The loss of protein d appeared to a prerequisite for this phenomenon. However, mutants which lack all glucose and heptose-bound phosphate in their lipopolysaccharide also have a reduction in particle density whereas, the amount of protein d is normal. Moreover, loss of lipopolysaccharide by EDTA treatment also caused a reduction in the density of particles. From these results it is hypothesized that the particles consist of lipopolysaccharide aggregates stabilized by divalent cations and probably complexed with protein and/or phospholipid.     

Transbilayer distribution and movement of lysophosphatidylcholine in liposomal membranes

Single bilayer vesicles were prepared by sonication of 5 mol% 1-palmitoyl lysophosphatidylcholine and 95 mol% egg phosphatidylcholine. Incubation with lysophospholipase results in a fast hydrolysis of 80–90% of lysophosphatidylcholine. The remaining lysophosphatidylcholine is only very slowly hydrolysed. There results are interpreted as lysophosphatidylcholine being asymmetrically distributed over the two halves of the bilayer. The slow phase of lysophosphatidylcholine hydrolysis sets an upper limit to the rate of transbilayer movement of lysophosphatidylcholine. The half time of this process at 37° C is estimated to be about 100 h. Incorporation of cholesterol in the vesicles reduces the distributional asymmetry of lysophosphatidylcholine to the extent of an outside-inside ratio of 60 : 40. [¹⁴C]Lysophosphatidylcholine introduced into the outer monolayer of such vesicles by intervesicular transfer of lysophosphatidylcholine remains virtually completely available for hydrolysis by lysophospholipases, corroborating the interpretation that transbilayer movement of lysophosphatidylcholine in these vesicles is an extremely slow process.In handshaken liposomes consisting of 5 mol% 1-palmitoyl lysophosphatidylcholine and 95 mol% egg phosphatidylcholine 15–20% of lysophosphatidylcholine is readily available for exogenous lysophospholipase. This pool may represent lysophosphatidylcholine in the outer monolayer of the liposomes.     

Photosensitization by hematoporphyrin: ESP evidence for free radical induction in unsaturated fatty acids and for singlet oxygen production.

Hematoporphyrin ability to photoreact by type I and type II mechanisms was investigated in some model systems. At room temperature, visible irradiation of hematoporphyrin-unsaturated fatty acids and hematoporphyrin-cholesterol systems resulted in the Electron Spin Resonance (ESR) spectrum of the hematoporphyrin free radical. Triplet state hematoporphyrin is shown to be involved in the electron transfer from the lipid moiety. Moreover an ESR method to monitor the singlet oxygen production by hematoporphyrin was used. β-carotene effect on both mechanisms (type I and type II) was tested.     

Assay for protein by dye binding

A proposed assay for protein [Bradford, M. M. (1976) Anal. Biochem. 72, 248–254] by binding of Coomassie brilliant blue G-250 has been evaluated. Some proteins give standard curves similar to those reported by Bradford, but others deviate widely; caution is urged in application of the procedure to general assay for protein concentration.     

Acetylene Reduction by Soil Cores of Maize and Sorghum in Brazil

Nitrogenase activity was measured by the C₂H₂ reduction method in large soil cores (29 cm in diameter by 20 cm in depth) of maize (Zea mays) and sorghum (Sorghum vulgare). The activity was compared to that obtained by a method in which the roots were removed from the soil and assayed for nitrogenase activity after an overnight preincubation in 1% O₂. In a total of six experiments and 28 soil cores, the nitrogenase activity of the cores was an average of 14 times less than the activity of roots removed from the same cores and preincubated. Nitrogenase activity in the cores was very low and extrapolated to an average nitrogen fixation rate of 2.8 g of N/hectare per day. It was shown that inadequate gas exchange was not a reason for the lower activity in the soil cores, and the core method gave satisfactory results for nitrogenase activity of soybeans (Glycine max) and Paspalum notatum.     

A comparison of membrane components of normal and transformed BALB/c cells.

Membrane glycolipids, glycoproteins, and surface proteins of normal and transformed BALB/c cell lines have been compared. Several virally and spontaneously transformed cell lines showed differences in membrane components compared to normal A31 cells. These differences consisted of increased amounts of simpler gangliosides, absence of the large external transformation sensitive (LETS) protein, and the appearance of a major new glycoprotein band of about 105 000 molecular weight. In contrast, the spontaneously transformed cell line that caused the fastest growing tumors in vivo and the most rapid animal death (3T12T) did not have these changes. A31 and 3T12T glycolipid profiles appear similar as did glycoproteins and cell surface proteins detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When Pronase-generated glycopeptides were analyzed by Sephadex G-50 chromatography, and enrichment in faster-eluting species was seen in two killing tumor lines (c5T and 3T12T) compared to A31. Regressing tumor lines (MSC, c5) did not show this change. Isolated membrane glycoproteins yield glycopeptides of different sized after Pronase digestion. In addition, several 3T12T glycoproteins yield glycopeptides that are larger than those from the corresponding glycoproteins of A31 cells. It appears that glycopeptide alterations associated with transformation occur in several membrane glycoproteins.     

CMP-N-acetylneuraminic acid hydrolase, an ectoenzyme distributed unevenly over the hepatocyte surface.

The regional localization of CMP-N-acetylneuramic acid hydrolase at the hepatocyte surface was studied by using plasma membranes and hepatocytes isolated from rat liver. 1. By homogenization of the rat liver plasma membrane preparations and subsequent discontinuous sucrose gradient centrifugation, one light and two heavy membrane fractions were obtained. The origin of these three subfractions is discussed based on the specific activities in the three fractions of 5'-nucleotidase, alakaline phosphatase and Mg2+-ATPase and on electron microscopic examination of the fractions. Evidence is given suggesting that the light fraction is derived from the bile canalicular surface of the plasma membrane, and that the heavy fractions are derived predominantly from the sinusoidal and lateral surfaces of the liver cell membrane. CMP-AcNeu hydrolase was present at highest specific activity in one of the heavy subfractions. Therefore it is concluded that CMP-AcNeu hdyrolase is located preferentially in the sinusoidal and/or lateral plasma membrane parts of the liver cell. 2. Experiments with intact and disintegrated hepatocytes isolated from rat liver indicated that CMP-AcNeu hydrolase is located at the surface of the cell membrane, with its functional group directed to the outside.     

Is there a plasma-membrane-located anion-sensitive ATPase? II. Further studies on rabbit kidney.

A study has been made to determine whether renal plasma membranes contain an HCO3 stimulated, ouabain insensitive Mg ATPase. Purified mitochondrial, microsomal and brush border membrane fractions have been isolated from rabbit kidney. The microsomal anion-sensitive ATPase activity appears to be entirely of mitochondrial origin on the basis of the effects of inhibitors of mitochondrial Mg ATPase. The brush border membrane fraction is contaminated with mitochondrial fragments and contains an Mg ATPase activity with low anion-sensitivity. Further purification of this fraction causes parallel decreases in anion-sensitivity of the Mg ATPase activity and in cytochrome c oxidase activity. These results indicate that conclusions previously reached by other investigators for a role of anion-sensitive Mg ATPase in the bicarbonate reabsorption of the proximal tubule may no longer be tenable.     

Histiocytic sarcoma of the scrotum in a rat

A 225-day-old male fourth generation rat from a developing recombinant inbred line (Lewis x Brown Norway) had a bilaterally symmetrical enlargement of the scrotum. Palpation indicated the presence of a firm lobulated mass extending from the tip of the scrotum to the abdominal wall. Bilateral nodular masses totally occupied the scrotal sacs, surrounded the testicles, and extended along the spermatic cords into the abdominal cavity. Tumor nodules also were present in the intestinal mesentery, omentum, mesenteric lymph nodes, pancreas, and lung. Histologically, the neoplasm presented a spectrum of characteristics varying from that of a granuloma with giant cells to a diffuse proliferation of spindle-shaped mononuclear cells.     

Discriminative stimulus,antagonist, and rate-decreasing effects of cyclorphan: Multiple modes of action

The discriminative effects of cyclorphan were studied in pigeons trained to discriminate 0.32 mg/kg ethylketazocine, 1.8 mg/kg cyclazocine, or 32 mg/kg naltrexone from saline. A fourth group of pigeons was administered 100 mg/kg/day morphine and trained to discriminate 0.1 mg/kg naltrexone from saline. Cyclorphan produced dose-related ethylketazocine-appropriate responding that reached a maximum of 83% of the total session responses at 0.3 mg/kg. Higher cyclorphan doses produced less ethylketazocine-appropriate responding. In pigeons trained to discriminate cyclazocine from saline, maximum drug-appropriate responding of greater than 90% occured at 5.6–10.0 mg/kg cyclorphan. In narcotic-naive pigeons trained to discriminate 32 mg/kg naltrexone from saline, cyclorphan produced a maximum of less than 50% drug-appropriate responding. In contrast, in pigeons chronically administered morphine and trained to discriminate 0.1 mg/kg naltrexone from saline, 1.0 mg/kg cyclorphan resulted in 100% drug-appropriate responding. In pigeons responding under a multiple fixed-interval, fixed-ratio schedule of food delivery, cyclorphan produced a complete dose-related reversal of the rate-decreasing effects of 10 mg/kg morphine, the maximally effective antagonist doses being 1.0–3.2 mg/kg. Higher cyclorphan doses (10 mg/kg) resulted in response rate decreases that were not reversed by naloxone (1 mg/kg). Thus, cyclorphan has discriminative effects that are similar to those of both ethylketazocine and, at 20-fold higher doses, cyclazocine. In addition, in morphine-treated pigeons, cyclorphan, across the same range of doses that produce ethylketazocine-appropriate responding, has discriminative effects that are similar to those of naltrexone, an effect that is probably related to the antagonist action of the drug.