Inhibition of sorbitol dehydrogenase by nucleosides and nucleotides.

Sorbitol dehydrogenase inhibitors have been found to prevent, or alleviate, various secondary complications of diabetes mellitus. In the present study, the effects of nucleosides and nucleotides on the rate of sorbitol oxidation catalyzed by the sheep liver enzyme were studied by steady-state kinetics at pH 7.4. Various such compounds, including ATP and the 2'-deoxy-analogues of ATP, ADP and AMP, reversibly inhibit enzyme activity by formation of enzyme-coenzyme-inhibitor ternary complexes. In each case, no deviations from linearity were seen in the double-reciprocal plots using sorbitol or NAD(+) as the varied substrate and there was a linear relationship between inhibitor concentration and the observed inhibitory effects. Sorbitol was docked into a model of the sheep SDH-NAD(+) complex based upon the structure of the human SDH-NAD(+) holoenzyme. The resulting structure of the ternary complex of sheep SDH, NAD(+) and sorbitol (PMDB ID code PM 0078068) shows that the reactive C-2 hydroxyl group of sorbitol is oriented toward the 4'-position of the nicotinamide moiety of the coenzyme, and that the adjacent primary hydroxyl group of sorbitol interacts with the catalytic zinc. The results indicate that the ribose moiety of the inhibitor structures is an important determinant for the observed effects. Specifically, the 2'-position of the ribose ring exerts an effect with respect to inhibitor potency.

Pancreatic trypsin activates human promatrix metalloproteinase-2.

In contrast to the prevalent view in the literature hitherto, the present study shows that pancreatic trypsin can activate human promatrix metalloproteinase-2 (proMMP-2). It is shown that trypsin's ability to activate proMMP-2 is dependent on various environmental factors such as the level of exogenously added Ca(2+) and Brij-35, temperature, as well as trypsin concentration. The activation occurred as a sequential processing of the proenzyme, initially generating an active 62kDa species. This was followed by successive truncation of the C-terminal domain, giving rise to active species of 56kDa, 52kDa and 50kDa. Tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) prevented the trypsin-mediated C-terminal truncation, without affecting the generation of the 62kDa species, while the presence of EDTA increased the rate of the trypsin-mediated activation of proMMP-2. MALDI-TOF MS analysis of the 50kDa form indicated that trypsin generated active forms with either Lys87 or Trp90 as the N-terminal residue and Arg538 as a C-terminal residue. The trypsin-activated MMP-2 was active in solution against both synthetic and physiologic substrates, and the steady-state kinetic coefficients k(cat), K(m) and k(cat)/K(m) were determined for the enzyme activated either by APMA, membrane-type 1 matrix metalloproteinase (MT1-MMP) or trypsin. The trypsin-activated MMP-2 exhibited slightly lower k(cat) and k(cat)/K(m) values as well as a slightly higher K(i) value against TIMP-1 compared to the enzyme activated by APMA or MT1-MMP. Docking studies of TIMP-1 revealed that the slightly weaker binding of the inhibitor to the trypsin-activated MMP-2 could be attributed to its shorter N terminus (Lys87/Trp90 versus Tyr81), as Phe83 and Arg86 interacted directly with the inhibitor. Our results suggest that the trypsin-activated MMP-2 possesses the catalytic and regulatory potential to be of significance in vivo.

Colchicine induces membrane-associated activation of matrix metalloproteinase-2 in osteosarcoma cells in an S100A4-independent manner.

Like the metastasis-associated protein S100A4, matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are important in physiological and pathological conditions. Previously, we showed that S100A4 is involved in the regulation of MMPs and TIMPs, and in the present work we have investigated whether the anti-inflammatory and microtubule-disrupting drug colchicine has an effect on the expression of these proteins in osteosarcoma cell lines (OHS) with high and low levels of S100A4. Colchicine treatment of the various OHS cells resulted in an increased expression of MT1-MMP and TIMP-2 mRNA, and a corresponding increase of these two proteins in isolated cell membranes. Colchicine-treated cells produced more of the activated form of MMP-2 than control cells. However, the drug did not affect the amount of MMP-2 and TIMP-1 mRNA or protein, and it reduced the S100A4 mRNA expression. Isolated cell membranes from the colchicine-treated cells were more effective in activating exogenous proMMP-2 than membranes from control cells, and inhibitory studies indicated that it was the colchicine-induced increase in MT1-MMP that caused the increased activation of endogenous MMP-2. A peptide inhibitor of nuclear factor kappaB nuclear translocation, SN50, blocked the colchicine-induced activation of proMMP-2 and reduced the synthesis of MMP-2 in colchicine-treated cells, but not in control cells. It can be concluded that colchicine modulates the expression of MT1-MMP and TIMP-2 and hence the activation of proMMP-2 independently of the S100A4 level in osteosarcoma cells.

S100A4 regulates membrane induced activation of matrix metalloproteinase-2 in osteosarcoma cells.

To study the role of the metastasis associated protein S100A4, an osteosarcoma cell line (OHS) with a high level of this protein was transfected with a vector containing a ribozyme that degrades S100A4 mRNA and, as controls, OHS cells were transfected with the vector alone. We have followed up our previous investigation (Bjørnland et al. 1999) by a detailed investigation of these cell lines' synthesis of MMP and TIMP proteins at different cell densities. It is shown that the cell lines with a low S100A4 level produced a reduced amount of immunoreactive MMP-2 at cellular subconfluence, while at confluence there was no difference compared to the control cells. The cell lines with a reduced S100A4 level produced less of the activated form of MMP-2 (62-kDa) and less TIMP-1 than the corresponding control cells, independent of cell density. Isolated cell membranes from cell lines with a reduced S100A4 level contained less MT1-MMP, MMP-2 and TIMP-2 compared to the control cells. Activation of exogenously added proMMP-2 was less effective with the former membrane preparations. It appeared that the mechanism behind the S100A4 dependent activation of proMMP-2 varied with cell density, as SN50, a peptide inhibitor of NF-kappaB nuclear translocation reduced the activation of MMP-2 at low cell density, but had no effect at high cell density. Thus, one of the mechanisms by which S100A4 may exert its effect on metastasis of some tumors is by regulating the MMP-2 activity.