Cloning and characterization of a sucrase from Leuconostoc mesenteroides

A sucrase gene from Leuconostoc mesenteroides was cloned and expressed in Escherichia coli. The cloned enzyme did not show dextransucrase or sucrose phosphorylase activity. HPLC and GC-MS analyses of the sucrase products indicated the presence of fructose and glucose in equimolar amounts. IPTG induction did not increase sucrase activity in E. coli indicating that the cloned gene may be transcribed from its own promoter. To our knowledge, this is the first sucrase cloned from L. mesenteroides that has invertase activity.     

Hydrogen production by rumen holotrich protozoa: effects of oxygen and implications for metabolic control by in situ conditions

Experiments with washed suspensions of holotrich protozoa (Isotricha spp. and Dasytricha ruminantium) showed that both organisms have an efficient O2-scavenging capability (apparent Km values 2.3 and 0.3 microM, respectively). Reversible inhibition of H2 production increased almost linearly with increasing O2 up to 1.5 microM; higher levels of O2 gave irreversible inhibition. In situ determinations of H2, CH4, O2 and CO2 in ovine rumen liquor, using a membrane inlet mass spectrometer probe, indicated that O2 was present before feeding at 1-1.5 microM and decreased to undetectable levels (less than 0.25 microM) within 25 min after feeding. A transient increase in O2 concentration after feeding occurred only in defaunated animals and resulted in suppression of CH4 and CO2 production. The presence of washed holotrich protozoa decreases the O2 sensitivity of CH4 production by suspensions of a cultured methanogenic bacterium Methanosarcina barkeri. It is concluded that holotrich protozoa play a role in ruminal O2 utilization as well as in the production of fermentation end products (especially short-chain volatile fatty acids) utilized by the ruminant and H2 utilized by methanogenic bacteria. These hydrogenosome-containing protozoa thus both control patterns of fermentation by influencing O2 levels, and are themselves regulated by the low ambient O2 concentrations they experience in the rumen.     

Inactivation of the postural asymmetry factor at the stage of compensation for the postural disorder induced by unilateral removal of the motor area

The peptide nature of the posture asymmetry factor (PAF) produced in the brain after unilateral removal of the motor region of the neocortex was established. The inactivation of PAF activity in the brain toward the end of the third week after the removal is due to PAF inactivation by the endogenous factor absent from the intact brain. The molecular weight of the inactivation factor exceeds 5.0 kD that makes it possible to separate it from the PAF by gel filtration on Sephadex G-25. The correlation was marked between the increasing activity of the PAF inactivation factor and the recovery of the initial symmetric functioning of the spinal centers during three weeks after unilateral damage to the CNS.     

Extracellular and cell-associated forms of beta-glucosidase in Thermomonospora curvata

The thermophilic actinomycete, Thermomonospora curvata , released 16-times the beta-glucosidase when grown on protein-extracted lucerne fibre compared with growth on cellobiose or purified cellulose. The intracellular and extracellular betaglucosidases had the same mol. wts (66 kD), but the extracellular enzyme had higher affinities for both p -nitrophenyl glucoside and cellobiose and was more resistant to thermal inactivation.     

Photoconversion of 7-dehydrocholesterol to vitamin D3 in synthetic phospholipid bilayers

The incorporation of 7-dehydrocholesterol into synthetic phospholipid bilayers altered the distribution of products after photolysis. In liposomes, the relative amounts of 7-dehydrocholesterol and lumisterol were elevated, and tachysterol was reduced from the levels observed in hexane solution. Z to E isomerization of the previtamin to tachysterol is favored in organic solvents. The inhibition of this process is evidence that an ordered lipid matrix places a new constraint on the conformation of the ring B fission product--one in which the configuration is favorable for a return to a cyclized diene. Further, rate enhancements of up to 15-fold were observed for the thermal isomerization of the previtamin to vitamin D3 in liposomes. The free energies of activation for the reaction at 25 degrees C were reduced by 1.3-1.5 kcal/mol in the bilayer environment compared to that of hexane. As this reaction involves the concerted transfer of a hydrogen via a cyclic intermediate, it provides additional evidence for membrane stabilization of an all-cis conformation of the previtamin. Photoproduct ratios were also studied for 7-dehydrocholesterol adsorbed to a variety of solid supports. That nonspecific interactions of 7-dehydrocholesterol with lipid can influence product formation may have important implications with respect to the mechanism of vitamin D3 biosynthesis.