Jin-Soo Kim

Dissociation/association properties of a dodecameric cyclomaltodextrinase : Effects of pH and salt concentration on the oligomeric state

As an effort to elucidate the quaternary structure of cyclomaltodextrinase I‐5 (CDase I‐5) as a function of pH and salt concentration, the dissociation/association processes of the enzyme were investigated under various pH and salt conditions. Previous crystallographic analysis of CDase I‐5 indicated that it existed exclusively as a dodecamer at pH 7.0, forming an assembly of six 3D domain‐swapped dimeric subunits. In the present study, analytical ultracentrifugation analysis suggested that CDase I‐5 was present as a dimer in the pH range of 5.0–6.0, while the dodecameric form was predominant at pH values above 6.5. No dissociation of the dodecamer was observed at pH 7.0 and the above. Gel filtration chromatography showed that CDase I‐5 dissociated into dimers at a rate of 8.58 × 10−2 h−1 at pH 6.0. A mutant enzyme with three histidine residues (H49, H89, and H539) substituted with valines dissociated into dimers faster than the wild‐type enzyme at both pH 6.0 and 7.0. The tertiary structure indicated that the effect of pH on dissociation of the oligomer was mainly due to the protonation of H539. Unlike the pH‐dependent process, the dissociation of wild‐type CDase I‐5 proceeded very fast at pH 7.0 in the presence of 0.2–1.0 m of KCl. Stopped‐flow spectrophotometric analysis at various concentrations of KCl showed that the rate constants of dissociation (kd) from dodecamers into dimers were 5.96 s−1 and 7.99 s−1 in the presence of 0.2 m and 1.0 m of KCl, respectively.

Ribonucleases Endowed with Specific Toxicity for Spermatogenic Layers

Bovine seminal ribonuclease (BS-RNase) is a dimer in which the subunits are cross-linked by disulfide bonds between Cys31 of one subunit and Cys32 of the other. Dimeric BS-RNase is resistant to ribonuclease inhibitor (RI), a protein endogenous to mammalian cells, and is toxic to a variety of cell types. Monomeric BS-RNase (like its homolog, RNase A) is bound tightly by RI and is not cytotoxic. The three-dimensional structure of the RI·RNase A complex suggests that carboxymethylation of C32S BS-RNase (to give MCM31) or C31S BS-RNase (MCM32) could diminish affinity for RI. We find that MCM31 and MCM32 are not only resistant to RI, but are also aspermatogenic to mice. In contrast to the aspermatogenic activity of dimeric BS-RNase, that of MCM31 and MCM32 is directed only at spermatogenic layers. Intratesticular injection of MCM31 or MCM32 affects neither the diameter of seminiferous tubules nor the weight of testes. Also in contrast to wild-type BS-RNase, MCM31 and MCM32 are not toxic to other cell types. Direct immunofluorescence reveals that MCM31 and MCM32 bind only to spermatogonia and primary spermatocytes. This cell specificity makes MCM31 and MCM32 of potential use in seminoma therapy and contraception.