Analysis of mutations in cyclodextrin glucanotransferase from Bacillus stearothermophilus which affect cyclization characteristics and thermostability.

Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) produces cyclodextrin from starch. The CGTase molecule is composed of four globular domains, A, B, C, and D. In order to gain better understanding of the amylolytic and cyclization mechanisms of CGTase, mutant CGTases were constructed from a CGTase gene (cgt1) of Bacillus stearothermophilus NO2. Cgt1-F191Y (Phe at position 191 was replaced by Tyr), Cgt1-F191Y-F255Y, Cgt1-W254V-F255I, Cgt1-W254V, and Cgt1-F255I were constructed for the analysis of the NH2-terminal region. It was revealed that amino acids surrounding a spiral amylose are important for cyclization characteristics and that hydrophobic amino acids just after the Glu catalytic site play an important role in the hydrolysis characteristics of the enzyme. Mutant CGTases Cgt1-T591F and Cgt1-W629F were also constructed to study the role of a second substrate-binding site in domain D, and it was suggested that substrate binding at both domains A and D stabilized the enzyme and optimized cyclodextrin production.     

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.     

Cyclic nucleotide alterations in mixed leukocyte reaction: a preliminary report

Endogenous cyclic adenosine and guanosine monophosphate (cAMP, cGMP) levels were studied in human peripheral blood lymphocytes during mixed leukocyte reactions (MLR). cAMP level was consistently elevated in one-way MLR, with good correlation to 3H-thymidine uptake in these reactions. In contrast, cGMP level was practically unchanged. Irradiation of reacting cell populations resulted in inhibition of cyclic nucleotide phosphodiesterase (PDE) activity. These results suggest that metabolic alterations in cAMP may be associated with immune reactions of cellular recognition.