Interaction between the gene 5 protein, gene 5 protein/single stranded fd DNA complex and gene 8 protein of the filamentous phage fd.

An affinity column consisting of gene 8 protein, the major coat protein of fd phage, bound to Sepharose was prepared. Isolated gene 5 protein/single stranded fd DNA complex was found to bind to this column and was eluted with fd phage single stranded fd DNA. pH changes, and 1 M CaCl2 were not effective in eluting the protein from the affinity column. Gene 5 protein/single stranded fd DNA complex from the crude extracts of fd-infected E. coli also bound to the column, as did isolated gene 5 protein; whereas fd single stranded DNA alone did not. These results may be relevant for the illucidation of the molecular events occurring in the early stages of fd phage assembly.

Comparison of glycerophosphate acyltransferases from Euglena chloroplasts and microsomes.

The acylation of sn-glycerol 3-phosphate is a common reaction in the pathways leading to the biosynthesis of glycerol-derived phospholipids, galactolipids, and sulfolipids. Enzymes catalyzing this reaction have been solubilized from Euglena chloroplasts, microsomes, and mitochondria (B. A. Boehler and M. L. Ernst-Fonberg (1976) Arch. Biochem. Biophys. 175, 229-235; L. V. Grobovsky, S. Hershenson, and M. L. Ernst-Fonberg (1979) FEBS Lett. 102, 261-264). Some characteristics of the reactions catalyzed by the acyl-CoA:sn-glycerol-3-phosphate O-acyltransferases (EC 2.3.1.15) solubilized from chloroplasts and microsomes of Euglena have been compared. Although the two enzymes have some common features, including stimulation by bovine serum albumin and phosphatidyl choline and sensitivity to sulfhydryl-binding reagents, they differ in their stabilities and responses to salt and glycerol. They exhibit different acyl-CoA substrate dependency curves. The proportions of monoacyl sn-glycerol-3-phosphate acyltransferase activity differ in the two solubilized enzyme preparations, and different products are produced by each of the glycerophosphate acyltransferases solubilized from chloroplasts and microsomes, respectively. Neither glycerophosphate acyltransferase will use palmitoyl- or myristoyl-acyl carrier protein (ACP) as a substrate whereas both use the corresponding CoA esters. Neither is inhibited by ACP, but the enzyme from microsomes is inhibited by CoA.

Receptor for bacteriophage lambda of Escherichia coli forms larger pores in black lipid membranes than the matrix protein (porin).

The receptor for phage lambda in Escherichia coli was isolated by cholate extraction and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein bands corresponding to the monomer and the dimer were eluted from the gel and tested for their activity to inactivate phage lambda and to form pores in black lipid membranes. It was found that only the dimer inactivated phage lambda, whereas both the monomer and the dimer were active in forming pores. The pore characteristics were similar to those exhibited by the matrix protein (porin) (R. Benz, K. Janko, W. Boos, and P. Läuger, Biochim. Biophys. Acta 511:305--319, 1978). In comparison, the lambda receptor showed a somewhat higher degree of cation specificity, and its pore size was larger. Assuming that the thickness of the outer membrane is 7.5 nm and that the pore is an ideal hydrophilic channel, the pore diameter in vivo was estimated to be 1.6 nm for the lambda receptor and 1.2 nm for the matrix protein.

Porin activity in the osmotic shock fluid of Escherichia coli.

Osmotic shock fluid of Escherichia coli exhibited pore-forming activity. This activity could be followed by an in vitro assay based on the conductivity increase for ions due to the presence of pores in black lipid membranes. The histogram (the distribution of conductivity increments in a single pore experiment) obtained with osmotic shock fluid from E. coli was identical to the histogram obtained by detergent-solubilized porin isolated from the outer membrane. The osmotic shock fluid from porin-negative mutants also exhibited pore activity, although the histogram and ion specificity were different from those of porin. Antibodies raised against detergent-solubilized porin were able to form precipitin lines by the Ouchterlony immunodiffusion technique when shock fluids, but not detergent-solubilized porin, were used. These antibodies prevented the formation of pores when shock fluids contained porin but not when shock fluids obtained from porin-negative mutants were used. Macroscopic membrane conductivity of shock fluids due to porin exhibited a concentration dependence, in contrast to detergent-solubilized porin. These results indicate that the hydrodynamic properties of periplasmic or "soluble" porin are different from those of the detergent-solubilized porin of the outer membrane. Periplasmic porin comprises about 0.7% of total protein in the osmotic shock fluid.