Purification of active matrix metalloproteinase catalytic domains and its use for screening of specific stromelysin-3 inhibitors.

The matrix metalloproteinase (MMP) stromelysin-3 (ST3) has been shown to be involved in malignant tumor progression and therefore represents an attractive therapeutical target. In order to screen for ST3 synthetic inhibitors, we have produced and purified the catalytic domain of ST3, matrilysin, stromelysin-2, and membrane type-1 MMP from inclusion bodies in a bacterial system. Our strategy allowed the purification of MMPs directly in the active form, thereby avoiding in vitro activation. A total of 140,000 synthetic compounds from the Bristol-Myers Pharmaceutical Research Institute chemical deck were tested, using a substrate-based colorimetric enzymatic assay, in which ST3 activity was evaluated through its ability to cleave and inactivate alpha-1 proteinase inhibitor. One ST3 inhibitor belonging to the cephalosporin family of antibiotics was thereby identified.

Purification, characterization, and expression of rat epididymal beta-D-galactosidase.

The purpose of the present study is to purify, kinetically characterize and measure the amount of soluble acid beta-D-galactosidase (EC 3.2.1.23) in different anatomical regions (caput, corpus, and cauda) of the adult rat epididymis. Based upon SDS-PAGE analysis, the subunit molecular mass of the caput and cauda enzyme is approximately 85,000 daltons while the corpus enzyme is approximately 50,000 daltons. The apparent Km and Vmax values are 67, 24, and 59 microM and 5.0, 1.88 and 6.3 microM/min./-mg protein for the enzyme purified from the caput, corpus, and cauda regions of the epididymis, respectively. However, no regional differences in the amount of soluble enzyme protein are observed. These data demonstrates regional differences in the activity of epididymal acid beta-D-galactosidase and suggest that the observed regional differences in enzyme activity may be due to posttranslational modifications.

Quantification and localization of N-acetyl-beta-D-hexosaminidase in the adult rat testis and epididymis.

An affinity-purified polyclonal antibody against N-acetyl-beta-D-hexosaminidase (EC 3.2.1.52) from adult rat epididymis was used to develop an ELISA, to localize tissue antigen by immunocytochemistry, and to identify isoforms of the enzyme by Western blot analysis. While peak activity level was measured in the corpus region of the epididymis, the quantity of soluble enzyme protein was highest in the caput region. Luminal caput sperm were intensely immunopositive, whereas epithelial cells were weakly stained. The enzyme was localized to epithelial and sperm cells of the corpus and caudal regions. In the testis, the enzyme was restricted primarily to lymphatic spaces. Testicular cells were unreactive, but spermatids were weakly stained. Western blot analysis revealed three prominent immune-reactive protein variants of approximately 63 kDa, approximately 55 kDa, and approximately 31 kDa. The approximately 31-kDa immune-reactive protein variant was reduced by > 84% in the caput epididymis compared to the cauda and corpus regions. These data suggest that regional differences in N-acetyl-beta-D-hexosaminidase activity in the rat epididymis may be due to differences in cellular synthesis and/or posttranslational processing of the enzyme.

Purification and characterization of N-acetyl-beta-D-hexosaminidase in different anatomical regions of the adult rat epididymis.

The purpose of the present study was to purify and kinetically characterize N-acetyl-beta-D-hexosaminidases A and B (EC 3.2.1.52) from the caput, corpus and caudal regions of the adult rat epididymis. The molecular mass of the purified native enzyme was approximately 250,000 and approximately 223,000 daltons for the A and B isozymes, with a subunit molecular mass of approximately 63,000 and approximately 56,000 daltons, as determined by size exclusion chromatography and gel electrophoresis under reducing conditions. The apparent Michaelis-Menten constant and maximum velocity values were 0.60, 1.55 and 0.68 mM and 0.54, 3.20 and 2.30 microM/min./mg protein for the enzyme purified from the caput, corpus and caudal regions, respectively. These values were determined by using p-nitrophenyl-N-acetyl-beta-D-glucosaminide as the substrate. These data suggest that the enzyme may be more active in the corpus region of the epididymis than in the caput and caudal regions.

Identification of novel RNA-binding proteins that interact in the coding region of protein D sense RNA in vitro.

Ultraviolet (UV)-cross-linking and sodium dodecyl sulfate(SDS) polyacrylamide gel electrophoretic (PAGE) analysis were used to identify proteins of nuclear and cytosolic (S100) origin that specifically bind to an in vitro transcribed mRNA sequence for protein D. The coding region of the protein D cDNA was subcloned, in vitro transcribed to [32P]RNA, and incubated with nuclear and cytosolic extracts of enzymatically dispersed epididymal cells. As revealed by UV-cross-linking and SDS-PAGE analysis, two proteins exhibiting a molecular weight mass of approximately 2.5 and approximately 35 Kd that specifically recognize and bind to the in vitro transcribed mRNA sequence for protein D. Our findings suggest that the regulation of protein D gene expression in the rat epididymis may involve novel RNA-binding proteins.