The organization and interaction of monoxygenase enzymes in the microsomal membrane.

Recently, a great number of investigations have been made on the organization and interactions of the monoxygenase enzymes in the microsomal membrane. Different organizational models have been proposed. The report reviews the general distributions and properties of membrane proteins and phospholipids, the incorporation of solubilized cytochrome P-450 into the membrane, the effect of temperature on the monoxygenase activities, and the rate-limiting steps of drug oxidations. It is believed that cytochrome P-450 and NADPH-cytochrome P-450 reductase are neither rigidly associated in molecular complexes nor randomly distributed in the membrane. Although these proteins do have lateral mobilities, the rate and the extent of the mobility remains to be determined.     

Newly made enzymes determine ongoing cell wall synthesis and the antibacterial effects of cell wall synthesis inhibitors.

Cell wall synthesis can continue with less than the total complement of cell wall synthetic enzymes present in normal growing cells. A method was developed to investigate whether there exists an excess of cell wall-synthesizing enzymes (penicillin-binding proteins [PBPs]) which all remain functional or whether a mixed population of functional and nonfunctional enzymes characterize normal cells. Surprisingly, cells in which less than 10% of the PBPs were functional could grow at a normal rate, as evidenced by increases in viable counts, culture turbidity, and rates of peptidoglycan, protein, and RNA synthesis. This subset of functional enzymes was biosynthetically new. Penicillin-induced lysis occurred contingent on the acylation of this same small fraction of PBPs, the copy number and affinities of which were below the level of detection by current fluorographic assay techniques. We propose that PBPs have a short functional half-life and that cell wall synthesis and bacterial lysis reflect the activity of newly synthesized PBPs.     

The synthesis and chemical properties of polyisoprenyl beta-D-mannopyranosyl phosphates.

2,3,4,6-Tetra-O-acetyl-beta-D-mannopyranosyl phosphate, free of the alpha anomer, was coupled with citronellol and dolichol in the presence of triisopropylbenzenesulfonyl chloride to give, after chromatographic purification and deacetylation, the respective polyisoprenyl beta-D-mannopyranosyl phosphates. These compounds were compared with the previously synthesized alpha anomers by means of their chromatographic properties, spectra, optical rotations, and hydrolysis reactions when treated with acid and alkali. To characterize the compounds resulting from these treatments, and to determine the mechanism of the alkaline hydrolysis, beta-D-mannopyranosyl phosphate was converted into beta-D-mannonpyranose 1,2-phosphate, and hence into D-mannose 2-phosphate, obtained as a mixture of alpha and beta anomers, characterized by infrared and nuclear magnetic resonance spectra and elemental analysis. Beta-D-Mannopyranosyl phosphate was readily separated by thin layer chromatography from the corresponding alpha anomer.     

Vitamin A and isoprenoid synthesis in the rat

1. Vitamin A-deficient rats were compared with similar animals given small amounts of vitamin A sufficient for adequate growth and with animals given large amounts of vitamin A. The effects of pair-feeding and feeding ad libitum were compared. 2. Ubiquinone and cholesterol concentrations in liver were measured at various stages of the deficiency, and the uptake of radioactive mevalonate and acetate into isoprenoid compounds was studied. 3. Ubiquinone concentrations in liver increased markedly in deficient rats compared with adequate controls, and heavy vitamin A supplementation had a further effect in depressing ubiquinone concentrations. These effects were unrelated to food intake or to the size of the organs. 4. Radioactive uptake into ubiquinone was often greater in deficient livers, especially during the early stages of the experiments, but the effect was not consistent. 5. Cholesterol concentrations were usually higher in deficient livers and these were more affected by the feeding regimen. 6. No consistent effect of vitamin A deficiency or of vitamin A dosage on the incorporation of mevalonate into cholesterol or squalene was found. 7. No evidence has been found for a specific effect of vitamin A on isoprenoid synthesis at the metabolic level.     

Outer membrane of gram-negative bacteria. XII. Molecular-sieving function of cell wall.

The permeability function the cell wall of gram-negative bacteria such as Salmoenlla was investigated by producing cells with an expanded periplasmic volume, and incubating them with radioactive non-utilizable oligo- and polysaccharides or polyethylene glycols. To quantitative the extent of penetration of these hydrophilic compounds into the periplasm, the radioactivity of the cell pellet was determined after centrifugation. We found that only di- and trisaccharides could fully diffuse into the periplasm, whereas higher-molecular-weight saccharides were nonpenetrable. In addition, low-molecular-weight polyethylene glycols rapidly diffused across the cell wall. Kinetics experiments also showed that both sucrose and raffinose in the periplasm exchanged rapidly with sugars in the medium, even at 0 degrees C. These results suggest that the cell wall acts as a molecular sieve, with an exclusion limit near 550 to 650 daltons for saccharides. We also suggest that the diffusion of these hydrophilic compounds most likely occurs through water-filled pores present in the cell wall of gram-negative bacteria.     

Molecular organization of peroxisomal enzymes: protein-protein interactions in the membrane and in the matrix

The beta-oxidation of fatty acids in peroxisomes produces hydrogen peroxide (H2O2), a toxic metabolite, as a bi-product. Fatty acids beta-oxidation activity is deficient in X-linked adrenoleukodystrophy (X-ALD) because of mutation in ALD-gene resulting in loss of very long chain acyl-CoA synthetase (VLCS) activity. It is also affected in disease with catalase negative peroxisomes as a result of inactivation by H2O2. Therefore, the following studies were undertaken to delineate the molecular interactions between both the ALD-gene product (adrenoleukodystrophy protein, ALDP) and VLCS as well as H2O2 degrading enzyme catalase and proteins of peroxisomal beta-oxidation. Studies using a yeast two hybrid system and surface plasmon resonance techniques indicate that ALDP, a peroxisomal membrane protein, physically interacts with VLCS. Loss of these interactions in X-ALD cells may result in a deficiency in VLCS activity. The yeast two-hybrid system studies also indicated that catalase physically interacts with L-bifunctional enzyme (L-BFE). Interactions between catalase and L-BFE were further supported by affinity purification, using a catalase-linked resin. The affinity bound 74-kDa protein, was identified as L-BFE by Western blot with specific antibodies and by proteomic analysis. Additional support for their interaction comes from immunoprecipitation of L-BFE with antibodies against catalase as a catalase- L-BFE complex. siRNA for L-BFE decreased the specific activity and protein levels of catalase without changing its subcellular distribution. These observations indicate that L-BFE might help in oligomerization and possibly in the localization of catalase at the site of H2O2 production in the peroxisomal beta-oxidation pathway.     

The mechanism of sugar-dependent repression of synthesis of catabolic enzymes in escherichia coli

Previous studies have indicated that the Escherichia coli adenylate cyclase (AC) activity is controlled by an interaction with the phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS). A model for the regulation of AC involving the phosphorylation state of the PTS is described. Kinetic studies support the concept that the velocity of AC is determined by the opposing contributions of PEP-dependent phosphorylation (V₁) and sugar-dependent dephosphorylation (V₂) of the PTS proteins according to the expression % V_AC = 100/[1 + (Max V₂/Max V₁)]. Physiological parameters influencing the rate of the PTS are discussed in the framework of their effects on cAMP metabolism. Factors that increase cellular concentration of PEP (and stimulate V₁) appear to enhance AC activity while increases in extracellular sugar concentration (which stimulate V₂) or internal levels of pyruvate (which inhibit V₁) inhibit the activity of this enzyme.