Observation of a chloride-dependent intermediate during catalysis by angiotensin converting enzyme using radiationless energy transfer

Stopped-flow radiationless energy-transfer kinetics have been used to examine the effects of chloride on the hydrolysis of Dns-Lys-Phe-Ala-Arg by angiotensin converting enzyme. The kinetic constants for hydrolysis at pH 7.5 and 22 degrees C in the presence of 300 mM sodium chloride were KM = 28 microM and kcat = 110 s-1, and in its absence, KM = 240 microM and kcat = 68 s-1. The apparent binding constant for chloride was 4 mM, and the extent of chloride activation in terms of kcat/KM was 14-fold. The effects of chloride on the pre-steady-state were examined at 2 degrees C. In the presence of chloride, two distinct enzyme-substrate complexes were observed, suggesting multiple steps in substrate binding. The initial complex was formed during the mixing period (kobsd greater than 200 s-1) while the second complex was formed much more slowly (kobsd = 40 s-1 when [S] = 5 microM and [NaCl] = 150 mM). Strikingly, in the absence of chloride, only a single, rapidly formed enzyme-substrate complex was observed. These results are consistent with a nonessential activator kinetic mechanism in which the slow step reflects conversion of an initially formed complex, (E X Cl- X S)1, to a more tightly bound complex, (E X Cl- X S)2.     

Regulation of rat liver cell 3-hydroxy-3-methylglutaryl coenzyme A reductase by methoxypolyoxyethylated cholesterol

A water-soluble derivative of cholesterol, methoxypolyoxyethylated (MPOE) cholesterol, has been synthesized and used to study the regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key regulatory enzyme in cholesterol biosynthesis. MPOE cholesterol causes a specific, rapid and linear decline in HMG-CoA reductase in cultured rat liver cells. MPOE cholesterol is not a direct allosteric inhibitor of HMG-CoA reductase, does not appear to regulate HMG-CoA reductase through changes in membrane environment, and does not change the phosphorylation state and level of activation of rat liver cell HMG-CoA reductase. In order to confirm our data, which were consistent with a model in which MPOE cholesterol regulates the amount of HMG-CoA reductase and not its activity, we made direct measurements of reductase mRNA levels. The decline in HMG-CoA reductase in MPOE cholesterol-treated rat liver cells is preceded by the rapid disappearance of HMG-CoA reductase mRNA. As a water-soluble cholesterol derivative, MPOE cholesterol represents a useful model compound for studies on the regulation of the level of HMG-CoA reductase and its cognate mRNA.     

A complex translocation at the murine kappa light-chain locus.

We have previously reported that a segment of DNA from a murine plasmacytoma comprises DNA from three chromosomes, the immunoglobulin kappa light-chain locus on chromosome 6, the S mu locus on chromosome 12, and a region on chromosome 15. We now report that the reciprocal product contains DNA from only the kappa locus and chromosome 15 and not from S mu. We conclude that a complex series of events, including both a transposition of DNA and a translocation between chromosomes, generated these imperfect reciprocal products.     

Distributions of two recently inserted long interspersed elements of the L1 repetitive family at the Alb and beta h3 loci in wild mice populations

The presence of the L1 sequences, L1Md4 next to the pseudogene beta h3 and I12 found in the twelfth intron of the albumin gene, in certain strains of laboratory mice but not of others has led to the suggestion that these sequences were recent insertions into the Mus mus domesticus genome. To be sure that they are really recent insertions and not relics of an ancestral chromosome, we investigated the presence or absence of these sequences in populations of wild mice belonging to the semispecies M. m. domesticus and M. m. musculus as well as in other species of the genus Mus and in related murids. The sequence I12 in the albumin gene was found in 34% of the chromosomes of the wild mice belonging to M. m. domesticus and to a lesser extent (6%) in M. m. musculus. Of 114 M. m. domesticus chromosomes, L1Md4 was found in only nine, seven of which came from the same locality. Its presence was associated with the haplotype Hbbp, which is relatively rare in European populations of M. musculus. Since there was no evidence for the presence of these two L1 sequences in more distantly related species, we conclude that they are recent insertions in the M. musculus genome.      

Phospholipid biosynthesis and secretion by a cell line (A549) which resembles type II aleveolar epithelial cells.

The A549 cell line is a continuous cell line derived from a human adenocarcinoma of the lung. At low cell population density the cells contain relatively few lamellar bodies, but in mature cells in very confluent cultures lamellar bodies are abundant. The lamellar bodies from these cells are enriched for phosphatidylcholine and disaturated phosphatidylcholine. In mature cells, 45% of newly synthesized phosphatidylcholine is disaturated. Stimulation with the calcium ionophore A23187 produces exocytosis of phosphatidylcholine (46% disaturated). The A549 cell synthesizes, stores in lamellar bodies, and secretes phosphatidylcholine, and thus has many important biological properties of the alveolar epithelial type II cell.     

A picosecond pulse train study of exciton dynamics in photosynthetic membranes.

The fluorescence decay time of spinach chloroplasts at 77 degrees K was determined at 735 nm (corresponding to the photosystem I emission) using a train of 10-ps laser pulses spaced 10 ns apart. The fluorescence lifetime is constant at congruent to 1.5 ns for up to the fourth pulse, but then decreases with increasing pulse number within the pulse train. This quenching is attributed to triplet excited states, and it is concluded that triplet excitons exhibit a time lag of about 50 ns in diffusing from light harvesting antenna pigments to photosystem I pigments. The diffusion coefficient of triplet excitons is a least 300--400 times slower than the diffusion coefficient of singlet excitons in chloroplast membranes.     

Total synthesis of ceramide trihexoside accumulating with Fabry's disease.

We report the synthesis of natural ceramide trihexoside, viz. gal (alpha-1 leads to 4) gal (beta-1 leads to 4) gluc (beta-1 leads to 1) ceramide (XIII). It involves the Koenigs-Knorr reaction of the bromide II with the aglucon XIX, and of the chloride VII with the D-enantiomer of the ceramide ester XII. A positional isomer of XIII was obtained as a by-product. A novel technique in the Koenings-Knorr reaction is described.     

5-Methylthioribose kinase. A new enzyme involved in the formation of methionine from 5-methylthioribose.

The presence of a previously unidentified enzyme, tentatively designated 5-methylthioribose kinase, has been demonstrated in cell-free extracts of Enterobacter aerogenes. The enzyme catalyzes the ATP-dependent phosphorylation of 5-methylthioribose. ADP is one of the products of the reaction and, based on functional group analyses, the other product is 5-methylthioribose 1-phosphate. A 40-fold purified enzyme preparation has been obtained from a cell-free extract of E. aerogenes. Activity of the partially purified enzyme is totally dependent on the presence of a divalent cation and a sulfhydryl reagent. The substrate specificity of the enzyme is quite narrow, and the Km values for ATP and 5-methylthioribose are 7.4 X 10(-5) M and 8.1 X 10(-6) M, respectively. These results suggest that 5-methylthioribose kinase may be a primary enzyme involved in the recycling of the methylthio group of 5-methylthioribose back into methionine.     

The magnetic susceptibility of cytochrome oxidase in the 4.2-1.5 K range.

Sixteen low temperature measurements on eight independent cytochrome oxidase samples from two separate laboratories have yielded magnetic susceptibility data compatible with a model of spin-coupled iron and copper ions, as presented in the preceding paper (Tweedle, M.F., Wilson, L.J., García-I?iguez, L., Babcock, G. T., and Palmer, G. (1978) J. Biol. Chem. 253, 8065-8071). The data in the 1.5-77 K range match those attained at higher temperatures and the predictions of the spin-coupled model. Measurements on reduced samples confirm the high spin nature of one iron atom. No obvious uncoupling of the antiferromagnetic Fe-Cu interaction is detected in partly reduced samples.