Influence of cell shape and surface charge on attachment of Mycoplasma pneumoniae to glass surfaces.

We had previously separated the ribosome-complexed and -free membrane fractions of Bacillus subtilis by sedimentation in a biphasic sucrose gradient. We now have found that the complexed fraction is contaminated with ribosome-free vesicles and that these can be removed by equilibrium density centrifugation. With this improved preparation, it could be shown that the penicillin-binding proteins are present almost exclusively in the ribosome-free membrane fraction. It thus appears that the fragmentation of the membrane in the lysing protoplast yields separate vesicles for the domains involved in protein translocation and for those involved in the synthesis and reshaping of the peptidoglycan. An enzyme of lipid synthesis (phosphatidylserine synthase) and also H+-ATPase were similarly found to be concentrated, but less exclusively, in the ribosome-free membrane fraction.     

Structural properties of myosin from the particulate fraction of human blood platelets

Fractionation of human blood platelets has revealed that myosin, a contractile and mechanochemical protein, is present in both the soluble and particulate fraction. The aim of this study was to elucidate whether platelets contain more than one myosin isoform, especially in view of the fact that in other cellular systems (cardiac muscle, amoeba) several myosin isoenzymes were found. The particulate fraction was solubilized by Triton X-100, and the myosin was purified by the same procedure used for the cytoplasmic myosin. The final preparation contained, in addition to myosin, a 130-kDa polypeptide, which was observed also in myosin preparations obtained from the soluble fraction. The electrophoretic mobilities of the two myosins were identical under both dissociating and nondissociating conditions. Comparison of the molecular structure of the heavy chain of the two myosins by limited proteolysis with Staphylococcus aureus V8 protease showed that the proteolytic fragments of the two myosins were rather similar, with only minor alterations in the quantitative distribution of the products. Two-dimensional peptide mapping of the iodinated tryptic peptides of the myosin heavy chains indicated that at least one peptide is missing in the map of the particulate myosin, as compared to its soluble counterpart. According to the two-dimensional peptide map, the 130-kDa polypeptide seems to be a proteolytic fragment of the myosin heavy chain and most probably the rod portion of the molecule. The observed minor variations in the structure of myosins isolated from the soluble and the fractions of human platelets may reflect differences in their respective physiological functions.     

Preliminary X-ray diffraction results on co-crystals of wheat germ agglutinin with a sialoglycopeptide from the red cell receptor glycophorin A

Diffraction-quality crystals have been obtained for complexes of each of the major wheat germ agglutinin (WGA) isolectins with the tryptic sialoglycopeptide T-5 from the WGA red cell receptor glycophorin A. This octa-glycopeptide possesses a Thr-linked carbohydrate moiety (GalNAc(NeuNAc)-Gal-NeuNAc) with specificity for the WGA binding site. The crystals belong to the orthorhombic space group P2(1)2(1)2 and have unit cell dimensions: a = 112.2 A, b = 51.0 A, c = 63.5 A (isolectin 1); a = 109.0 A, b = 52.3 A, c = 62.4 A (isolectin 2). There are two monomer complexes in each asymmetric unit.     

Effect of antitranspirant treatment on the water balance of pine seedlings,under different climatic and soil moisture conditions

Summary Antitranspirant treatment with Tag — a polyethylene based emulsion-reduced transpiration and increased height of potted pine seedlings under simulated hot and dry conditions when soil was moist. Under cool, humid conditions reduction of transpiration was very much less and growth was slightly reduced. Application of antitranspirants to pine seedling transplants, under hot dry field conditions, failed to improve, their water balance or to reduce mortality. Physiological tests made during the field trial and experiments with potted seedlings showed that when the soil is dry xeromorphic plants efficiently reduce their water loss and under such conditions anti-transpirants are of no benefit. It is calculated that antitranspirants would only be advantageous under moist soil and high evaporative demand conditions.     

Isolation of membrane glycoproteins by affinity chromatography in the presence of detergents

Wheat germ agglutinin has been used in a one-step preparative method to isolate the major sialoglycoprotein (glycophorin A) from the human erythrocyte membrane. The conditions for isolation and purification of the sialoglycopeptide included low concentration of sodium dodecyl sulfate in the presence of relatively high salt concentration. This medium caused complete solubilization of the membrane but still allowed almost quantitative binding of the sialoglycopeptide to wheat germ agglutinin-Sepharose. The eluted protein from such affinity systems was found to be chemically comparable to glycophorin A, as prepared by other procedures.     

Mechanism of Excretion of a Bacterial Proteinase: Demonstration of Two Proteolytic Enzymes Produced by a Sarcina Strain (Coccus P)

A Sarcina strain (Coccus P) produces two proteolytic enzymes. One is found only extracellularly, is far more prevalent, and is actively excreted during exponential growth. It is the enzyme responsible for the known strong proteolytic activity of the cultures of this strain. A second protease is, however, produced which remains associated with the intact cells but is released by the protoplasts. The two enzymes appear unrelated in their derivation. Calcium ions play an essential role in preventing autodigestion of the excreted enzyme.     

Cholesterol-phosphatidylcholine dispersions as donors of cholesterol to Mycoplasma membranes

Growing cells of sterol-requiring Mycoplasma hominis and sterol non-requiring Acholeplasma laidlawii were used to test the ability of cholesterol-dipalmitoyl phosphatidylcholine dispersions to serve as cholesterol donors to these organisms. Dispersions with high cholesterol to phosphatidylcholine ratios were more effective than dispersions with low cholesterol to phosphatidylcholine ratios in donating cholesterol to the membranes of both mycoplasmas and in promoting growth of the sterol-requiring species. M. hominis took up almost three times as much cholesterol as did A. laidlawii. In addition, significant quantities of the phosphatidylcholine component of the dispersions were found to be associated with M. hominis membranes as against none in the A. laidlawii membrane preparations. In all cases, the percentage of cholesterol taken up by M. hominis from the dispersions exceeded that of phosphatidylcholine by a factor of 3–5. These results were interpreted to suggest that all the cholesterol taken up by A. laidlawii is transferred from the dispersion to the membranes by a process which involves only a transient contact between the organisms and the lipid dispersions, whereas a certain amount of the cholesterol taken up by M. hominis may also be derived from lipid dispersions adhering to or fusing with the cell membranes.     

The steady state activity of succinate dehydrogenase in the presence of opposing effectors

The extent of the deactivation of the mitochondrial succinate dehydrogenase by oxaloacetate is a function of the redox state of the enzyme. Oxidized enzyme is deactivated by much lower concentrations of oxaloacetate than those needed to deactivate reduced enzyme. An accurate method for measuring this relationship is the redox titration of the enzymic activity of succinate dehydrogenase, carried out in the presence of oxaloacetate. For each concentration of oxaloacetate a different redox titration curve was reported with the apparent mid-potential decreasing with increasing oxaloacetate. These results are compatible with a model which proposes that both oxidized and reduced enzymes can form the catalytically non-active complex with oxaloacetate, but that the complex formed the the oxidized enzyme is more stable than that formed by the reduced enzyme. When the oxaloacetate concentration is low, reduction of the enzyme will lower the fraction of the succinate dehydrogenase-oxaloacetate complex, a reaction which we observe as reductive activation of the enzyme. If this experiment is repeated in the presence of high concentration of oxaloacetate, no activation of the enzyme takes place, but the low stability of the reduced enzyme oxaloacetate complex is revealed by the rapid exchange of the enzyme-bound oxaloacetate with the free ligand. The rate of this exchange is extremely slow at high positive potential and becomes faster upon lowering of the poise potential. The reductive activation of the succinate dehydrogenase is regarded as a two step reaction. In the first step the reduced non-active complex releases the oxaloacetate and in the second step the active form of the enzyme is evolved. These two steps can be observed experimentally; Reductive activation at a redox potential higher than the mid-potential of the oxaloacetate-malate couple (minus 166 mV) is characterized by Ea = 18 Kca/mole, the final equilibrium level of activation decreases upon lowering of the temperature. Reduction activation of the enzyme at minus 240 mV is a very rapid reaction which goes to completion at all temperatures tested and has an activation energy of 12.5 Kcal/mole. The mechanism of the reductive activation and its possible role in the regulation of succinate dehydrogenase in the mitochondria is discussed.     

Bacterial lipopolysaccharides and mycoplasmal lipoglycans: A comparison between their abilities to induce macrophage-mediated tumor cell killing and Limulus amebocyte lysate clotting

The ability of various bacterial lipopolysaccharides and mycoplasmal lipopolysaccharides (lipoglycans) to induce macrophage-mediated tumor cell killing and $\text{Limulus}$ amebocyte lysate clotting was determined. Lipoglycans from the mycoplasma $\text{Acholeplasma}\text{axantum}$ or $\text{Acholeplasma}\text{granularum}$ had no activity or 10⁴ to 10⁵ less activity than lipopolysaccharides from $\text{Escherichia}\text{coli}$ 0128:B12, $\text{Escherichia}\text{coli}$ K235, or $\text{Salmonella}\text{minnesota}$ R595 in causing $\text{Limulus}$ lysate clotting and tumor cell killing by peritoneal macrophages from normal or bacillus Calmette-Guérin-infected mice. Previous studies have shown that the lipid A portion of bacterial lipopolysaccharide is responsible for the effects on macrophage-mediated tumor cell killing and $\text{Limulus}$ lysate clotting. The known differences in the lipid structures of bacterial lipopolysaccharides and mycoplasmal lipopolysaccharides (lipoglycans) may account for the noted differences in the biologic potencies observed here.