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SUMMARY The preparation and characterization of a distribution of gold balls on a thin, flat carbon film is described. The relation of the platinum carbon shadow width distribution means to a gold ball size is reported. Freeze‐etched cytochrome oxidase vesicles and gold ball calibration grids were simultaneously shadowed with platinum/carbon. The shadow width distribution of the cytochrome oxidase located in and spanning the membrane was measured. The membrane fracture face edge and cross‐fractured bilayer membrane edge were also measured. Dimensions of the cytochrome oxidase were found to be 5·8 ± 0·3 nm in diameter parallel to the membrane and 8·2 ± 0·3 nm long across the membrane. The bilayer membrane dimensions were 3·0 ± 0·3 nm for the half bilayer and 5·8 ± 0·3 nm for the cross‐fractured bilayer membrane edge thickness. The length of the cytochrome oxidase was observed to span the bilayer membrane. Previous X‐ray diffraction measurements on similar hydrated liquid crystalline artificial membranes were found to be in good agreement with the freeze‐etched results. Membrane widths from thin‐sectioned cytochrome oxidase vesicles were measured and found to be 5·8–5·9 nm in non‐post‐stained sections. Post‐staining with uranyl acetate and/or lead citrate was shown to increase this average thickness. The technique of freeze‐etching electron microscopy in conjunction with the gold ball shadow width calibration experiment has been shown to provide accurate and precise measurements of membranes and a functional intramembrane protein in a hydrated non‐crystalline sample.

Gs and adenylate cyclase from bovine brain cortices were co-reconstituted into asolectin iiposomes with or without 1000-fold transmembrane Ca 2÷ gradient. Obtained results showed that Gs activities of both binding GTPTS and stimulating adenylate cyclase were the highest in proteoliposomes, with a transmembrane Ca 2+ gradient similar to the physiological situation and the lowest while the transmembrane Ca 2÷ gradient was in the inverse direction. Such a difference could be diminished following the dissipation of the transmembrane Ca 2+ gradient by A23187. Time-resolved fluorescence anisotropy of diphenylhexa-triene (DPH) has been used to compare the physical state of phospholipids among those proteoliposomes. It is suggested that a proper transmembrane Ca 2+ gradient is essential for higher membrane fluidity, which may favor G s function with higher GTP-binding activity and stimulation of adenylate cyclase.

Antibodies to the disulfide knot fragment of bovine fibrinogen have been used to locate the site of this fragment within the intact fibrinogen molecule. The antibodies were isolated from rabbit antifibrinogen antisera by affinity chromatography. Electron micrographs of reaction mixtures of bovine fibrinogen and antibodies against the disulfide knot fragment showed pairs of fibrinogen molecules crosslinked by antibody molecules as well as higher order antibody-fibrinogen complexes. From an electron microscopic investigation of the crosslinked material, we conclude that the disulfide knot lies within the central nodule of the trinodular fibrinogen molecule. Antibodies to fragment H were used in the same manner to locate this fragment within the outer nodules of the human fibrinogen molecule.In connection with our studies of the interaction of thrombin with fibrinogen (1, 2) and of the polymerization of fibrin monomer (3, 4), it is important to locate the sites of the fibrinogen molecule that are involved in various functions such as thrombin-binding, polymerization, fibrin stabilization, and plasmin degradation. From a series of physicochemical (5, 6) and electron microscopic (4, 7-10) observations, the linear trinodular structure of fibrinogen appears established. In addition, electron microscope observations of dimers, trimers, tetramers, etc. of fibrin monomer (4) have verified the conclusions from earlier physicochemical studies (11) that these intermediate polymers arise by a lateral association with partial overlapping, giving two parallel end-to-end chains with staggered junctions. In this paper, we use the technique of immunoelectron microscopy (12-16) to locate two peptide fragments within the trinodular structure of fibrinogen. These two fragments are the "disulfide knot" (N-DSK) (17) and fragment H (18). The former is the disulfide-linked NH2-terminal portions of the Aa, Bf3, and -y chains (Mr -58,000) produced by CNBr cleavage of fibrinogen (17), and fragment H (Mr 20,000) is a plasmin-digestion product of fibrinogen (18). By comparison of the NH2-terminal sequence of fragment H given by Harfenist and Canfield (18) with the amino acid sequence reported by Doolittle et al. (19), it appears that fragment H corresponds to residues 240-424 of the Aa chain. MATERIALS AND METHODSBovine fibrinogen (96% clottable) was prepared from Miles fraction I (lot 90, 75% clottable) or Sigma fraction I (lot 117C-0140, 82% clottable) as described (20) and was dialyzed against 10 mM Na2HPO4/3 mM KH2PO4/120 mM NaCl, pH 7.5 (phosphate-buffered saline), 5 mM H3B04/1.25 mM Na2B4O7.10H20/140 mM NaCl, pH 8.5 (borate/saline buffer), or 50 mM ammonium formate (pH 8.5) for immunization, quantitative precipitation, or electron microscopy, respectively. The reduced and alkylated material exhibited three bands (corresponding to the Aa, BB, and y chains) on NaDodSO4/ 7.5% polyacrylamide gel electrophoresis (21). All other chemicals were of reagent grade or better.Human fibrinogen was purchased from AB Kabi (Stockholm, lot 1981-...

High-level synthesis of the periplasmic protein I8-lactamase in Escherichia coli caused the formation of insoluble protein precipitates called inclusion bodies. (-Lactamase inclusion bodies differed from those reported previously in that they appeared to be localized in the periplasmic space, not in the cytoplasm. The inclusion bodies contained mature I8-lactamase and were solubilized more easily than has been reported for cytoplasmic inclusion bodies. In contrast, overproduction of the periplasmic protein alkaline phosphatase caused the formation of cytoplasmic inclusion bodies containing alkaline phosphatase precursor.