Enzymatic detection of phospholipid biosynthetic enzymes after electroblotting.

The membrane-associated enzymes phosphatidylinositol synthase (CDPdiacylglycerol:myo-inositol 3-phosphatidyltransferase; EC 2.7.8.11) and phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase; EC 2.7.8.8) from Saccharomyces cerevisiae were detected enzymatically after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting. Enzyme activities were measured on nitrocellulose blots by using pure enzyme preparations as well as Triton X-100-solubilized membranes. Phosphatidylinositol synthase activity migrated to Mr 34,000, and phosphatidylserine synthase activity migrated to Mr 23,000.     

Association of an extracellular protein (chondrocalcin) with the calcification of cartilage in endochondral bone formation

We examined bovine fetal epiphyseal and growth plate cartilages by immunofluorescence microscopy and immunoelectron microscopy using monospecific antibodies to a newly discovered cartilage-matrix calcium-binding protein that we now call chondrocalcin. Chondrocalcin was evenly distributed at relatively low concentration in resting fetal epiphyseal cartilage. In growth plate cartilage, it was absent from the extracellular matrix in the zone of proliferating chondrocytes but was present in intracellular vacuoles in proliferating, maturing and upper hypertrophic chondrocytes. The protein then disappeared from the lower hypertrophic chondrocytes and appeared in the adjoining extracellular matrix, where it was selectively concentrated in the longitudinal septa in precisely the same location where amorphous mineral was deposited in large amounts as demonstrated by von Kossa staining and electron microscopy. Mineral then spread out from these "nucleation sites" to occupy much of the surrounding matrix. Matrix vesicles were identified in this calcifying matrix but they bore no observable morphological relationship to these major sites of calcification where chondrocalcin was concentrated. Since chondrocalcin is a calcium-binding protein and has a strong affinity for hydroxyapatite, these observations suggest that chondrocalcin may play a fundamental role in the creation of nucleation sites for the calcification of cartilage matrix in endochondral bone formation.     

Modification of the conductance,selectivity and concentration-dependent saturation of Pseudomonas aeruginosa protein P channels by chemical acetylation

Protein P, an anion-specific channel-forming protein from the outer membrane of Pseudomonas aeruginosa was chemically modified by acetylation and syccinylation of its accessible amino groups. The chemically modified protein retained its ability to form oligomers on sodium dodecyl sulfate polyacrylamide gels, whereas only the acetylated protein formed channels in reconstitution experiments with lipid bilayers. Acetylated protein P demonstrated a substantially reduced mean single channel conductance (25 pS at 1 M KCl) compared to the native protein P channels (250 pS at 1 M KCl) when reconstituted into black lipid bilayer membranes. The homogeneous size distribution of single-channel conductances suggested that all of the protein P molecules had been acetylated. Zero-current potential measurements demonstrated that the acetylated protein P channel was only weakly selective for anions and allowed the permeation of cations, in contrast to the native protein P channels, which were more than 100-fold selective for anions over cations. The dependence of conductance on salt concentration was changed upon acetylation, in that acetylated protein P demonstrated a linear concentration-conductance relationship, whereas native protein P channels became saturated at high salt concentrations. These data strongly suggested that the basis of anion selectivity for native protein P channels is fixed amino groups. In agreement with this, we could demonstrate a 2.5-fold decrease in single-channel conductance between pH 7 and pH 9, between which pH values the ?-amino groups of amino acids would start to become deprotonated. Two alternative schemes for the topography of the protein P channel and localization of the fixed amino groups are presented and discussed.     

An anion-selective channel from the Pseudomonas aeruginosa outer membrane

Protein P from Pseudomonas aeruginosa outer membrane was reconstituted in lipid bilayer membranes from diphytanoylphosphatidylcholine. The reconstitution resulted in the formation of anion-selective channels with a conductance of 160 pS for 0.1 M chloride solution. The channels were at least 100-times more selective for anions than for cations as judged from zero-current membrane potentials. The single-channel conductance was dependent on the size of the different anions and saturated at higher salt concentrations suggesting single ion occupancy of the protein P channel.     

The reaction with oxygen of cytochrome oxidase (cytochrome d) in Escherichia coli K12: optical studies of intermediate species and cytochrome b oxidation at sub-zero temperatures

Optical changes in d- and b-type cytochromes, following initiation of the reaction of cytochrome oxidase d with O2, have been studied in cells and derived membrane particles from oxygen-limited cultures of Escherichia coli K12. At successively higher temperatures between -132 and -88 degrees C, the first scan after photolysis of the Co-liganded, reduced oxidase in the presence of O2 and a slow increase in absorbance at 675 to 680 nm due to an unidentified chromophore. A similar sequence occurs when a single sample is scanned repetitively at -91 degrees C. At higher temperatures, oxidation of at least two spectrally distinct cytochromes b occurs. Selective photolysis of the cytochrome d-CO complex with a He-Ne laser shows that neither of these cytochromes is the CO-binding cytochrome o436. In all oxidation states examined, no absorbance in the 720 to 860 nm region was observed; it is concluded that both cytochromes d and o436 lack redox-active copper that has an environment similar to the copper(s) in mitochondrial cytochrome c oxidase. The amount of cytochrome d650 (but not the amount of reduced cytochrome o436) formed after photolysis is directly proportional to the oxygen concentration in the sample at the time of freeze trapping. The results are discussed in relation to the composition and mechanism of action of cytochrome d.     

Hydration-induced conformational and flexibility changes in lysozyme at low water content

Using laser Raman spectroscopy, we are able to study conformational changes that occur as previously-dried hen egg-white lysozyme is sequentially rehydrated. Parallel n.m.r. exchangeability studies enable us to monitor flexibility changes also during this rehdyration. The results are consistent with a general loosening up of the protein at a water content of ～0.08 g water/g protein, followed by (probably small) local conformational changes. The enzyme regains its activity only after both these processes have gone to completion; thus these solvent-related changes may be necessary before activity can recommence.     

Cytochromes ofAcetobacter pasteurianus NCIB 6428: Reaction with oxygen of cytochromeo in cells,membranes, and nonsedimentable subcellular fractions

Cytochromeo ofAcetobacter pasteurianus NCIB 6428, grown with either vigorous or limiting aeration, was located in both membranes and a fraction nonsedimentable by centrifugation (13.5×10⁶ g min) of a French press extract. In oxygen-limited cells, the reaction with oxygen of the oxidase at low temperatures resembled that inEscherichia coli with respect to the spectrum and formation kinetics of an oxygenated intermediate. Both membranous and nonsedimentable cytochromeso formed this intermediate, but faster and at lower temperatures in the latter. The role of this cytochrome, particularly in the light of other bacterial, soluble, ligand-binding hemoproteins, and the kinetic constraints on its reaction are discussed.     

Formation of the 680 nm-absorbing form of the cytochrome bd oxidase complex of Escherichia coli by reaction of hydrogen peroxide with the ferric form

Reduced minus aerated difference spectra of membranes from Escherichia coli (grown under oxygen-limited conditions) show, in addition to the 650 nm trough attributed to the oxygenated form of cytochrome d, a smaller trough centred at about 680 nm of unknown origin. When the reference spectrum is that of a sample oxidized with ferricyanide and to which hydrogen peroxide was added, the trough proportions changed, the 680 nm species being more dominant. Similarly, when 8.8 mM hydrogen peroxide is added to a persulphate-oxidized sample, a peak at 680 nm is immediately formed. No such compound is observed when peroxide is added to persulphate-oxidized membranes from a cytochrome d-deficient mutant. It is concluded that the 680 nm species represents a peroxy form of haem d, which is stable at room temperature and is probably an intermediate in the reaction mechanism of this oxidase.     

Influence of growth temperature and lipopolysaccharide on hemolytic activity of Serratia marcescens.

Log-phase cells of Serratia marcescens cultured at 30 degrees C were approximately 10-fold more hemolytic than those grown at 37 degrees C. By using a cloned gene fusion of the promoter-proximal part of the hemolysin gene (shlA) to the Escherichia coli alkaline phosphatase gene (phoA), hemolysin gene expression as a function of alkaline phosphatase activity was measured at 30 and 37 degrees C. No difference in alkaline phosphatase activity was observed as a function of growth temperature, although more hemolysin was detectable immunologically in whole-cell extracts of cells grown at 30 degrees C. The influence of temperature was, however, growth phase dependent, because the hemolytic activities of cells cultured to early log phase at 30 and 37 degrees C were comparable. Given the outer membrane location of the hemolysin, lipopolysaccharide (LPS) was examined as a candidate for mediating the temperature effect on hemolytic activity. Silver staining of LPS in polyacrylamide gels revealed a shift towards shorter O-antigen molecules at 37 degrees C relative to 30 degrees C. Moreover, there was less binding of O-antigen-specific bacteriophage to S. marcescens with increasing growth temperature, a finding consistent with temperature-mediated changes in LPS structure. Smooth strains of S. marcescens were 20- to 30-fold more hemolytic than rough derivatives, a result confirming that changes in LPS structure can influence hemolytic activity. The alkaline phosphatase activity of rough strains harboring the shlA-phoA fusion was threefold lower than that of smooth strains harboring the fusion plasmids, a result consistent with a decrease in hemolysin gene expression in rough strains. The absence of a similar effect of temperature on gene expression may be related to less-marked changes in LPS structure as a function of temperature compared with a smooth-to-rough mutational change.