Release of rhodanese from Pseudomonas aeruginosa by cold shock and its localization within the cell.

Whole cells of Pseudomonas aeruginosa possess rhodanese activity. The enzyme can be released by rapidly resuspending the cells in cold Tris--HCl buffer. Approximately 95% of the rhodanese activity is released by cold shock. Release of the enzyme can be inhibited either by preincubating the cells with Mg2+ or by incorporating Mg2+ into the shocking buffer. The effect of Mg2+ can be reversed by washing the cells twice with buffer prior to cold shock. While rhodanese can be released from P. aeruginosa by cold shock, lactic dehydrogenase, a cytoplasmic enzyme, remains within the cell. Diazo-7-amino-1,3-napthalenedisulfonic acid, a compound which does not penetrate the cytoplasmic membrane, completely inactivated rhodanese and alkaline phosphatase, a periplasmic enzyme, whereas lactic dehydrogenase retained its full activity. These data suggest that rhodanese in P. aeruginosa, like alkaline phosphatase, is located distal to the cytoplasmic membrane in the periplasmic space. Electron micrographs also show that portions of the lipopolysaccharide outer membrane are shed from the cell during cold shock, while cells preincubated with Mg2+ did not release segments of their outer membrane.     

Cyclic AMP Phosphodiesterase in Leydig Cell Preparations of the Rat Testis

Enzymatic digestion of the interstitial tissue of early juvenile and adult rat testes resulted in an enrichment of the Leydig cell population. The cells of the intertubular preparation from adult testes were separated by centrifugal elutriation, according to differences in sedimentation velocity, a counter-flow centrifugation technique leading to 70% Leydig cell purity. Using this approach, it was possible to demonstrate that Leydig cells from adult testes contain only low affinity isoenzymes of cyclic AMP phosphodiesterase (PDE; E.C.: 3.1.4.17), an intracellular regulator of cAMP. Starch gel electrophoresis showed that the isozyme of cAMP PDE of Leydig cells is masked in crude testis homogenates due to the relatively low level of these cells in the total population. In Leydig cells, there are two different electrophoretic forms expressed which resemble two of eleven different molecular forms of cAMP PDE demonstrated for comparison in 21 different organs of the adult rat.
An interstitial cell preparation from early juvenile testes, with a Leydig cell content of up to 20%, was also investigated electrophoretically with regard to molecular forms of cAMP PDE, the properties of which were characterized by kinetic analysis of cAMP hydrolysis. The results presented are discussed in relation to the onset of testosterone synthesis in Leydig cells of prepubertal rats leading to the initiation of male puberty.     

An analysis of the sequence dependence of the structure and energy of A- and B-DNA models using molecular mechannics

Molecular-mechanics calculations have been carried out on the base-paired hexanucleoside pentaphosphates d(TATATA)₂, d(ATATAT)₂, d(A₆)·d(T₆), d(CGCGCG)₂, d(GCGCGC)₂, and d(C₆)·d(G₆) in both A- and B-DNA geometries. The calculated relative energies of these polymers are consistent with the relative stabilities of the polymers found experimentally. In particular, the results of our calculations support the observation that the homopolymer d(A)_n·d(T)_n is more stable in a B-DNA conformation, while the homopolymer d(G)_n·d(C)_n is more stable in an A-DNA conformation. The molecular interactions responsible for these differential stabilities include both inter- and intrastrand base stacking, as well as base–phosphate interactions. While definitive experiments on the heteropolymer stabilities have not yet been carried out, the results of our calculations also suggest a greater stability of the purine-3′,5′-pyrimidine sequence over the pyrimidine-3′,5′-purine sequence in both the A- and B-conformations. The reason for this greater stability lies in the importance of the inherent directionality (5′ → 3′ vs 3′ → 5′) of phosphate–base and base–base interactions. The largest conformation change observed on energy refinement is sugar repuckering, which occurs mainly on pyrimidine-attched sugars and only in the B-DNA geometry. We suggest a molecular mechanism, specifically, differential base–sugar steric interactions involving neighboring sugars, to explain why this repuckering occurs more with d(A₆)·d(T₆) than with other isomers.