Sialyl-transferase mitochondriale

A sialyl transferase activity is found in purified mitochondria. It is not due to residual contamination and this enzymatic system is located in the outer mitochondrial membrane. This proves mitochondrial autonomy in regard to glycoconjugate sialylation.     

Dehalogenation of dichloromethane by cell extracts of hyphomicrobium DM2

A facultatively methylotrophic bacterium was isolated from enrichment cultures containing dichloromethane as the sole carbon source. It was identified as a Hyphomicrobium species. The organism grew exponentially in batch cultures with 10 mM dichloromethane at a specific growth rate of 0.07 h^-1. The release of Cl^- from dichloromethane and the disapperance of substrate paralleled growth. Resting dichloromethane-grown cells, in the presence of potassium sulphite as a trapping agent, converted cichloromethane methane quantitatively to formaldehyde. The conversion of dichloromethane to formaldehyde by cell extracts was stricly dependent on glutathione. Other thiols were inactive. Glutathione was not consumed in the course of the reaction. The specific activity of the enzymic dehalogenation of dichloromethane amounted to 3.8 mkat/kg protein in extracts of dichloromethane-grown cells and to less than 0.1 mkat/kg protein in extracts from cells grown on methanol.     

Determination of rat muscles androgen-receptor complexes with methyltrienolone

In the present study, “in vitro” evidences are shown for the existence of a highly active 3α-hydroxysteroid dehydrogenase in the crude cytosol of rat muscle homogenates; the use of 5α-dihydrotestosterone (DHT) is therefore compromised in receptor binding measurements because of its extensive metabolism. The synthetic anabolic androgen, methyltrienolone (MT) palliates this disadvantage of DHT. Both steroids, as well as testosterone, appear to be bound to an 8–8.5 S androgen receptor on sucrose density gradient. The androgen receptor in the vastus and the levator ani bulbocavernosus complex (LA/BC) shows similar association constants, but the number of binding sites in LA/BC is about 5 times higher than in vastus. Otherwise, the total number of muscle androgen receptors seems to be invariant in adult and aged rats. The binding to these macromolecules can thus be measured “in vitro” provided specific and sensitive methods are utilized.     

Computation of the fraction of induced cells in enzyme induction systems

A theoretical model is developed for continuous multistage enzyme production systems, which consist of a growth fermentor used for growing microorganisms rapidly without enzyme production and a subsequent system of induction reactors in which enzymes induction and production occurs. The model allows the computation of the fraction of induced cells residing in the induction reactor for organisms exhibiting a lag phase in enzyme induction. For this model a general analytical solution was obtained for the cumulative internal residence time distribution of a series of n well-stirred vessels with a recycle. The theoretical results are compared in a preliminary way with experimentally measured cellulase productivities of continuous multistage cellulose fermentations with Trichoderma viride QM 9414.     

Molecular packing of cholesterol in phospholipid vesicles as probed by dehydroergosterol

Ergosta-5,7,9,22-tetraen-3-β-ol (dehydroergosterol) was synthesized and employed as a probe of cholesterol behavior in phospholipid bilayers. Circular dichroism (CD) spectra were obtained. The CD of dehydroergosterol in sonicated egg phosphatidylcholine vesicles was dependent on cholesterol concentration, while in unsonicated egg phosphatidylcholine liposomes and in vesicles obtained by oxctylglucoside dialysis, the CD observed was independent of cholesterol content. The CD of dehydroergosterol in sonicated sphingomyelin vesicles exhibited a different dependence on cholesterol content than seen in sonicated egg phosphatidylcholine vesicles. These data are interpreted in terms of differences between the packing of cholesterol in systems of large and small radii of curvature and in different interactions between dehydroergosterol and phosphatidylcholine and sphingomyelin.     

Site-specific mutagenesis of an essential histidine residue in pancreatic cholesterol esterase

The histidine residue essential for the catalytic activity of pancreatic cholesterol esterase (carboxylester lipase) has been identified in this study using sequence comparison and site-specific mutagenesis techniques. In the first approach, comparison of the primary structure of rat pancreatic cholesterol esterase with that of acetylcholinesterase and cholinesterase revealed two conserved histidine residues located at positions 420 and 435. The sequence in the region around histidine 420 is quite different between the three enzymes. However, histidine 435 is located in a 22-amino acid domain that is 47% homologous with other serine esterases. Based on this sequence homology, it was hypothesized that histidine 435 is the histidine residue essential for catalytic activity of cholesterol esterase. The role of His435 in the catalytic activity of pancreatic cholesterol esterase was then studied by the site-specific mutagenesis technique. Substitution of the histidine in position 435 with glutamine, arginine, alanine, serine, or aspartic acid abolished the ability of cholesterol esterase to hydrolyze p-nitrophenyl butyrate and cholesterol [14C]oleate. In contrast, mutagenesis of the histidine residue at position 420 to glutamine had no effect on cholesterol esterase enzyme activity. The results of this study strongly suggested that histidine 435 may be a component of the catalytic triad of pancreatic cholesterol esterase.     

Comparison of charge movement components in intact and cut twitch fibers of the frog. Effects of stretch and temperature

Charge movements were measured in frog intact fibers with the three-microelectrode technique and in cut fibers with the double Vaseline gap technique. At 13-14 degrees C, the ON segments of charge movement records from both preparations showed an early I beta component and a late I gamma hump component. When an intact fiber was cooled to 4-7 degrees C, the time-to-peak of I gamma (tp,gamma) was prolonged, but I gamma still appeared as a hump. Q-V plots from intact fibers at 4-7 degrees C were fitted with a sum of two Boltzmann distribution functions (method 1). The more steeply voltage-dependent component, identified with Q gamma, accounted for 32.1% (SEM 2.2%) of the total charge. This fraction was larger than the 22.6% (SEM 1.5%) obtained by separating the ON currents with a sum of two kinetic functions (method 2). The total charge in cut fibers stretched to a sarcomere length of 3.5 microns at 13-14 degrees C was separated into Q beta and Q gamma by methods 1 and 2. The fraction of Q gamma in the total charge was 51.3% (SEM 1.7%) and 53.7% (SEM 1.8%), respectively, suggesting that cut fibers have a larger proportion of Q gamma:Q beta than intact fibers. When cut fibers were stretched to a sarcomere length of 4 microns, the proportion of Q gamma:Q beta was unchanged. Between 4 and 13 degrees C, the Q10 of l/tp,gamma in intact fibers was 2.33 (SEM 0.33) and that of 1/tau beta was less than 1.44 (SEM 0.04), implying that the kinetics of I gamma has a steeper temperature dependence than the kinetics of I beta. When cut fibers were cooled from 14 to 6 degrees C, I gamma in the ON segment generally became too broad to be manifested as a hump. In a cut fiber in which I gamma was manifested as a hump, the Q10 of l/tp,gamma was 2.08 and that of l/tau beta was less than 1.47. Separating the Q-V plots from cut fibers at different temperatures by method 1 showed that the proportion of Q gamma:Q beta was unaffected by temperature change. The appearance of I gamma humps at low temperatures in intact fibers but generally not in cut fibers suggests an intrinsic difference between the two fiber preparations.