Intact triple helical collagen molecules are highly resistant to proteolytic enzymes, whereas degraded (unwound) collagen is easily digested. This fact was exploited to develop a simpli fied method for the quantification of the amount of degraded collagen in the collagen net work of connective tissues. Essentially, the method involves extraction of proteoglycans with 4 M guanidinium chloride, selective digestion of degraded collagen by a-chymotrypsin, hydrolysis in 6 M HC1 of the released fragments as well as the residual tissue, and then mea surement of the amount of hydroxyproline in both pools* Since the digestion of degraded collagen by a-chymotrypsin and measurement of hydroxyproline is not restricted to a sper cific collagen type, this technique can be applied to a wide variety of connective tissues. The method was validated with articular cartilage. Levels of in situ degraded collagen were about four-fold higher in degenerated (fibrillated) cartilage than in its healthy counterpart derived from the same donor. More detailed investi gations revealed that the collagen damage in degenerated cartilage is more extensive at the cartilage surface than in the region adjacent to bone. This was not the case in healthy carti lage; identical low values were obtained at the surface and close to the bone. An impaired collagen network has been hypothesized to be the reason for the swelling of cartilage in os-4 ■* teoarthritis (OA). The present paper presents the first experimental evidence to support this hypothesis: more damage to the collagen network (i.e., more degraded collagen molecules within fibrils) is linearly related to more extensive swelling of the OA tissue in hypotonic saline.
Molecular modeling techniques were used to derive a predictive model for substrates of cytochrome P450 2D6, an isozyme known to metabolize only compounds with one or more basic nitrogen atoms. Sixteen substrates, accounting for 23 metabolic reactions, with a distance of either 5 A ("5-A substrates", e.g., debrisoquine) or 7 A ("7-A substrates", e.g., dextromethorphan) between oxidation site and basic nitrogen atom were fitted into one model by postulating an interaction of the basic nitrogen atom with a negatively charged carboxylate group on the protein. This acidic residue anchors and neutralizes the positively charged basic nitrogen atom of the substrates. In case of "5-A substrates" this interaction probably occurs with the carboxylic oxygen atom nearest to the oxidation site, whereas in the case of "7-A substrates" this interaction takes place at the other oxygen atom. Furthermore, all substrates exhibit a coplanar conformation near the oxidation site and have negative molecular electrostatic potentials (MEPs) in a part of this planar domain approximately 3 A away from the oxidation site. No common features were found in the neighbourhood of the basic nitrogen atom of the substrates studied so that this region of the active site can accommodate a variety of N-substituents. Therefore, the substrate specificity of P450 2D6 most likely is determined by the distance between oxidation site and basic nitrogen atom, by steric constraints near the oxidation site, and by the degree of complementarity between the MEPs of substrate and protein in the planar region adjacent to the oxidation site.(ABSTRACT TRUNCATED AT 250 WORDS)
Matrix metalloproteinases (MMPs) are involved in physiological tissue remodeling and pathological conditions like tumour metastasis and joint destruction. Until now, no convenient and sensitive MMP-activity assay in crude media like synodal fluid has been available. Therefore, the highly soluble fluorogenic substrate TNO211 (Dabcyl-Gaba-Pro-Gin-Gly-LeuGIu(EDANS)-AIa-Lys-NH2), containing the MMP cleavable Gly-Leu bond and EDANS/Dabcyl as fluorophore/quencer combination, was synthesized and characterized as an MMP specific substrate. We show that the fluorogenic assay using ] NO211 is sensitive and can detect MMP activity in culture medium from endothelial cells and untreated synovial fluid (SF) from RA and OA patients, and control subjects. MMP activity in SF significantly increased in the order C < OA < RA, thus the frequent use of OA samples as control in studies on RA is debatable.A ey words: Matrix metalloproteinase; Synovial fluid; I luorescence quenching
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