The high molecular weight urinary matrix metalloproteinase (MMP) activity is a complex of gelatinase B/MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL). Modulation of MMP-9 activity by NGAL

Detection of matrix metalloproteinase (MMP) activities in the urine from patients with a variety of cancers has been closely correlated to disease status. Among these activities, the presence of a group of high molecular weight (HMW) MMPs independently serves as a multivariate predictor of the metastatic phenotype (). The identity of these HMW MMP activities has remained unknown despite their novelty and their potentially important applications in non-invasive cancer diagnosis and/or prognosis. Here, we report the identification of one of these HMW urinary MMPs of approximately 125-kDa as being a complex of gelatinase B (MMP-9) and neutrophil gelatinase-associated lipocalin (NGAL). Multiple biochemical approaches verified this identity. Analysis using substrate gel electrophoresis demonstrated that the 125-kDa urinary MMP activity co-migrates with purified human neutrophil MMP-9 x NGAL complex. The 125-kDa urinary MMP-9 x NGAL complex was recognized by a purified antibody against human NGAL as well as by a monospecific anti-human MMP-9 antibody. Furthermore, these same two antibodies were independently capable of specifically immunoprecipitating the 125-kDa urinary MMP activity in a dose-dependent manner. In addition, the complex of MMP-9 x NGAL could be reconstituted in vitro by mixing MMP-9 and NGAL in gelatinase buffers with pH values in the range of urine and in normal urine as well. Finally, the biochemical consequences of the NGAL and MMP-9 interaction were investigated both in vitro using recombinant human NGAL and MMP-9 and in cell culture by overexpressing NGAL in human breast carcinoma cells. Our data demonstrate that NGAL is capable of protecting MMP-9 from degradation in a dose-dependent manner and thereby preserving MMP-9 enzymatic activity. In summary, this study identifies the 125-kDa urinary gelatinase as being a complex of MMP-9 and NGAL and provides evidence that NGAL modulates MMP-9 activity by protecting it from degradation.     

Schizosaccharomyces pombe Aps1, a diadenosine 5',5' "-P1, P6-hexaphosphate hydrolase that is a member of the nudix (MutT) family of hydrolases: cloning of the gene and characterization of the purified enzyme

The fission yeast Schizosaccharomyces pombe contains a gene on chromosome I that encodes a hypothetical nudix hydrolase, YA9E. The gene, designated aps1, has been cloned and the protein has been purified from Escherichia coli with a yield of 10 mg of Aps1/L of culture. Aps1, composed of 210 amino acids with a calculated molecular mass of 23 724 Da, behaves as a monomer with a sedimentation coefficient of 1.92 S as determined by analytical ultracentrifugation. The effective hydrodynamic radius is about 29 A as determined by both analytical ultracentrifugation and gel-filtration chromatography. Aps1, whose expression was detected in S. pombe by Western blotting, is an enzyme that catalyzes the hydrolysis of dinucleoside oligophosphates, with Ap6A and Ap5A being the preferred substrates. The major reaction products are ADP and p4A from Ap6A and ADP and ATP from Ap5A. Values of Km for Ap6A and Ap5A are 19 microM and 22 microM, respectively, and the corresponding values of kcat are 2.0 s-1 and 1.7 s-1, respectively. The enzyme has limited activity on Ap4A and negligible activity on Ap3A, ADP-ribose, and NADH. Aps1 catalyzes the hydrolysis of mononucleotides with decreasing activity in order from p5A to AMP. Optimal activity with Ap6A as substrate is observed at pH 7.6 and in the presence of 0.1-1 mM MnCl2. Aps1 is the first nudix hydrolase isolated from S. pombe, and it is the first enzyme identified with this specific substrate specificity and reaction products.