(1) The cellular localization and tissue distribution of a novel magnesium-dependent, calcium-inhibitable protein phosphatase (MCPP) from bovine brain has been examined. The amino acid sequence of MCPP deduced from its cDNA indicates nuclear entry and localization motifs. Using a specific assay for MCPP, we found enzymatic activity in all cytosolic, microsomal, and nuclear fractions separated by differential centrifugation, albeit the percent of nuclear activity in various tissues differs. With rat tissues, the highest activities were seen in thymus and testis. Using an antibody against a unique region in MCPP, we found MCPP concentrated in the nucleus of Vero and HeLa cells by immunofluorescence techniques. Our observations suggest trafficking of MCPP among the cytosol, the endoplasmin reticulum, and the nucleus. The cDNA for a leukemogenic, nuclear protein, SET, that copurifies with MCPP has been obtained. The deduced amino acid sequence revealed that the bovine homolog is different from the human SET only in five amino acid residues. SET could be precipitated by anti-MCPP antibody in the presence of MCPP. It also served as substrate for MCPP. The findings are indicative of MCPP-SET interactions and further support a nuclear function for MCPP, likely in the regulation of cell cycle or differentiation. (2) The N-terminal fusion peptide of the gp41 envelope glycoprotein from human immunodeficiency virus (HIV) has been used as a mechanistic probe to identify the presence of receptors for gp41. It was found that the presence of a number of chemokine receptors and """"""""orphan chemokine receptors"""""""" (receptor without known chemokine ligand) made entry of the fusion peptide into various cells possible. When the chemokine receptors were transiently expressed in Vero cells, which could not be infected by HIV, the cells became penetrable to the fusion peptide. Also, when the p21 peptide of gp41, a region binding to gp41 receptor, was made into an affinity column, it was able to retain a protein in the cell lysate with a 40K molecular weight similar to that of chemokine receptors. Our results are consistent with the notion that the chemokine receptors are not merely co-receptors for interaction between the gp120 envelope protein of HIV and the CD4 receptor on the cell surface. Rather, they also serve as receptors for gp41.
| Huang, Charles Y; Zhou, Rixin; Yang, David C H et al. (2003) Application of the continuous variation method to cooperative interactions: mechanism of Fe(II)-ferrozine chelation and conditions leading to anomalous binding ratios. Biophys Chem 100:143-9 |
