Kinetic and circular dichroism studies of enzymes adsorbed on ultrafine silica particles

Negatively charged ultrafine silica particles (average diameter 20 nm) were used as support materials for adsorption immobilization of porcine trypsin, horseradish peroxidase, and bovine catalase under various conditions, and the changes in the enzyme activities and the circular dichroism (CD) spectra of these enzymes upon adsorption were measured. Since the light scattering intensity of the ultrafine particles was very low, the activities and the CD spectra of the enzymes adsorbed on the particle surfaces could be measured. The enzymes adsorbed at pH around and above their isoelectric points (pI) showed high activities. On the other hand, the enzymes adsorbed at pHs below their pI had significantly diminished activities and showed large CD spectral changes upon adsorption. The extent of CD spectral changes in the enzymes upon adsorption correlated very closely with that of the activity reduction. Therefore, the conformational changes in enzymes upon adsorption are one of the important factors that reduce the activities of adsorbed enzymes. These results demonstrate that the ultrafine particles are not only a novel support for enzyme immobilization but also are helpful for the molecular understanding of the immobilized enzymes. Correspondence to: A. Kondo     

Three-dimensional study of the cytoskeleton in macrophages and multinucleate giant cells by quick-freezing and deep-etching method.

The three-dimensional ultrastructure of multinucleate giant cells in subcutaneous granulomas was compared with those of peritoneal macrophages using a quick-freezing and deep-etching method. Subcutaneous granulomas were induced by implanting plastic coverslips in the dorsal subcutaneous tissue of rats. The quick-freezing and deep-etching replicas were prepared from the cells attached to the coverslips. Dense networks of actin filaments were distributed along all peripheral aspects (beneath the plasma membrane, and on free and coverslip-attached surfaces) of the multinucleate giant cells. On the coverslip-attached surface, numerous clathrin-coated pits and vesicles occurred between the actin filaments. In these cells, intermediate filaments, but not actin filaments, were the predominant cytoskeletal components in perinuclear regions and were attached to the cell nucleus, mitochondria and other vesicular cell organelles. A similar distribution of cytoskeletal components was observed in the mononuclear macrophages of the granulomas and the peritoneal macrophages. These results show that the cytoskeletal organization varies in different regions of the cytoplasm of multinucleate giant cells, while the characteristic cytoskeletal arrangement, resembling that of mononuclear macrophages, is maintained.     

A strong,preferential response of mice to polymorphic antigenic determinants of the chicken MHC,analyzed with mouse hybridoma (monoclonal) antibodies

CBA/J mice were immunized with B²/B² chicken RBC's (theB locus is the major histocompatibility complex (MHC) of the chicken). Spleen cells from these mice gave a much higher plaque-forming cell response when tested with RBC's bearing B² alloantigens, than with RBC's bearing any other MHC alloantigens. Similarly, immunization with chicken RBC's of other genotypes also produced responses that were highest when tested on the genotype used for immunization. Spleen cells from mice immunized 3 days previously with B²/B² RBC's were fused with mouse myeloma cells and hybrid clones which secreted anti-B² antibodies were selected. These monoclonal antibodies could be divided into three groups: (1) those which react with all genotypes of RBC's (panreactive); (2) those which react with only certain genotypes of RBC's and detect “public” MHC antigens; and (3) those which react with only B²/B² RBC's and detect “private” MHC antigens. Monoclonal antibodies which detect a private B² alloantigen were shown to be excellent typing reagents, as all birds of an outbred population which possessed the B² allele were easily detected using a simple one-step direct agglutination assay. No “false positives” were seen. The high and preferential response of the mouse to chicken MHC alloantigens suggested that mice might possess preexisting immunity to these antigens. In agreement with this hypothesis, normal mouse serum was found to have high titres of “natural” antibody against chicken MHC antigens.     

The rpoD gene functions as a multicopy suppressor for mutations in the chaperones, CbpA, DnaJ and DnaK, in Escherichia coli

Abstract The CbpA protein is an analog of the DnaJ molecular chaperone of Escherichia coli . The dnaJ ⁻ cbpA ⁻ double-null mutant exhibits severe defects in cell growth, namely, a very narrow temperature range for growth. To gain insight into the functions of CbpA as well as DnaJ, we isolated a multicopy suppressor gene that permits this dnaJ ⁻ cbpA ⁻~ mutant to grow normally at low temperatures. The suppressor gene was identified as rpoD , the gene that encodes the major σ ⁷⁰. The biological implications of this finding are examined and discussed.