Comparison of two cytochromes P-450 from Candida maltosa: primary structures, substrate specificities and effects of their expression in Saccharomyces cerevisiae on the proliferation of the endoplasmic reticulum

cDNAs were cloned, sequenced and expressed which encode two different cytochrome P-450 forms of the alkane-assimilating yeast Candida maltosa, designated as P-450Cm1 and P-450Cm2. The amino acid sequences deduced were about 55% identical. Expression in Saccharomyces cerevisiae resulted in the formation of intact microsomal P-450 systems catalyzing the hydroxylation of n-hexadecane and lauric acid with significantly different substrate preferences. A massive proliferation of the endoplasmic reticulum was observed in the S. cerevisiae cells which produced P-450. Depending on the P-450 form expressed, distinctly organized stacks of paired membranes appeared and occupied considerable areas of the cytoplasm. As shown by immunoelectron microscopy for P-450Cm1, the protein expressed was highly concentrated within these newly formed membrane structures.     

Involvement of various organs in the initial plasma clearance of differently glycosylated rat liver secretory proteins

The initial plasma clearance and organ distribution of alpha 1-acid glycoprotein and alpha 2-macroglobulin carrying different types of oligosaccharide, side chains was studied in rats. The differently glycosylated proteins were synthesized by rat hepatocytes in culture in the presence of tunicamycin (unglycosylated form), swainsonine (hybrid type), or 1-deoxymannojirimycin (high-mannose type). Deglycosylated glycoproteins (Asn-GlcNAc) were obtained by endoglucosaminidase H treatment of high-mannose-type glycoproteins. Ten minutes after intravenous injection 3% of complex type, 26% of hybrid type, 84% of high-mannose type. 64% of unglycosylated and 80% of deglycosylated alpha 1-acid glycoprotein disappeared from the plasma. The respective values for alpha 2-macroglobulin were 26%, 42%, 59% and 67%. When the clearance of total hepatic secretory proteins was examined, major differences between glycosylated and unglycosylated (glyco)proteins were found, particularly in the case of low-molecular-mass polypeptides. Whereas complex-type alpha 1-acid glycoprotein and alpha 2-macroglobulin showed no accumulation in various organs, hybrid-type alpha 1-acid glycoprotein and alpha 2-macroglobulin were present in spleen and liver. High-mannose-type alpha 1-acid glycoprotein and alpha 2-macroglobulin also accumulated mainly in spleen and liver. Spleen had the highest specific activity; liver, due to its larger organ mass, represented the major organ for the uptake of high-mannose-type glycoproteins. Competition experiments with mannan and GlcNAc-bovine-serum-albumin showed a mannose/GlcNAc receptor-mediated removal. Whereas unglycosylated alpha 1-acid glycoprotein was taken up by the kidney, unglycosylated alpha 2-macroglobulin was found in the spleen. Deglycosylated glycoproteins (Asn-GlcNAc) were removed from the plasma via two different mechanisms: firstly, clearance by the kidney similar to the unglycosylated glycoproteins; secondly, clearance by a mannose/GlcNAc receptor-mediated uptake mainly into the spleen. We conclude that N-linked oligosaccharide side chains are important for the plasma survival of hepatic secretory glycoproteins and that unphysiologically glycosylated forms are cleared by different mechanisms.     

Inhibition of N-acetylneuraminate lyase by N-acetyl-4-oxo-D-neuraminic acid

We show that the 4-oxo analogue of N-acetyl-D-neuraminic acid strongly inhibits N-acetylneuraminate lyase (NeuAc aldolase, EC 4.1.3.3) from Clostridum perfringens (Ki = 0.025 mM) and Escherichia coli (Ki = 0.15 mM). In each case the inhibition was competitive. N-Acetyl-D-neuraminic acid; N-Acetylneuraminate lyase; N-Acetyl-D-neuraminic acid analog; 5-Acetamido-3,5-dideoxy-beta-D-manno-non-2,4-diulosonic acid; 2-Deoxy-2,3-didehydro-N-acetyl-4-oxo-neuraminic acid; Competitive inhibitor.     

Immunochemical evidence for the presence in human plasma of lipoproteins with apolipoprotein A-II as the major protein constituent

Apolipoprotein-A-containing lipoproteins have been studied by means of crossed immunoelectrophoresis with intermediate gels. The experiments confirmed the presence in human plasma of lipoprotein particles with both apoA-I and apoA-II (LpA) and of those with apoA-I but no apoA-II (LpAI). Furthermore, they obtained evidence for the occurrence in human plasma of small amounts of lipoproteins containing apoA-II but not apoA-I, apoB, apoC-II, apoC-III or apoE.     

Lipid interconversions in aging Mycoplasma capricolum cultures.

During the progression of Mycoplasma capricolum cultures from the early exponential to the stationary phase of growth, a decrease in the phospholipid-to-protein ratio and increases in both the unsaturated-to-saturated fatty acid ratio and the diphosphatidylglycerol (DPG)-to-phosphatidylglycerol (PG) ratio were found. The freedom of motion of spin-labeled fatty acids incorporated into the membrane remained unchanged throughout the growth cycle. The increase in DPG was almost stoichiometric with the decrease in PG. Furthermore, exogenous PG added to the medium was incorporated by the cells and partially converted to DPG. The DPG that was accumulated upon aging was always more unsaturated than the PG. This accumulation was enhanced in palmitic acid-poor media, but was inhibited even in aged cells when the cells were grown in palmitic acid-rich media, suggesting that the accumulation of DPG upon aging was associated with changes in the fatty acid composition of membrane lipids rather than with the transition of the cells from the exponential- to stationary-growth phase.     

Enhancement of translational efficiency by the Escherichia coli atpE translational initiation region: its fusion with two human genes

The cDNA sequences encoding mature human interleukin 2 (IL2) and beta-interferon (INF beta), respectively, were fused with various translational initiation regions and inserted into two different types of expression vector. The relative levels of expression of the two genes and the functional stability of their respective mRNAs were examined in vivo in Escherichia coli hosts. The addition of the 30-bp sequence, found immediately upstream of the E. coli atpE gene Shine-Dalgarno (SD) sequence, to the translational initiation regions of IL2 and INF beta increased the expression of both these genes by a factor of 6-10. Thus this sequence, which naturally acts within the E. coli atp operon to enhance the translational initiation frequency of the atpE gene, can increase the expression of other genes in E. coli. It may exemplify a specific type of recognition signal for the E. coli translational apparatus.     

Phase separation, ion permeability, and the isolation of membranes from osmotically stable mycoplasmas

The osmotic stability of M. gallisepticum was found to be a consequence of the synthesis of disaturated phosphatidylcholine incorporated into the cell membrane. The disaturated lipid induces the formation of segregated lipid domains, thus providing the sites for increased permeation of ions. Such permeation reduces the internal pressure so as to minimize cell swelling and subsequent lysis in a hypotonic medium. Purified membranes of M. gallisepticum can be prepared from cells suspended in an iso-osmotic NaCl solution containing either dicyclohexylcarbodiimide (DCCD), which blocks ATPase activity, or a mild alkaline buffer. Both conditions seem to interfere with cell volume regulation. These procedures can be used also to isolate membranes of other osmotically stable mycoplasmas.