(Mea-Pro-Leu-Gl~Leu-DpaAla-Arg-NIH?) has been synthasiscd as a lluorogcnic substrate for the matrix mctalloprotcinascs. The highly fluorescent 7.methoxycoumarin group is eflicicntly quenched by energy transfer to the 2,4-dinitrophcnyl group. The punctuated mctalloprotcinase (PUMP, EC 3.42423) cleaves the substrute al the Gly-Leu bond wilh a 1904old increase in fluorrsccncc (A,, 328 nm. A,,, 393 nm). In assays or the buman malrix mclalloprolcinases.Mea-Pro-Lcu.Gly-Lcu.Dpa-Ala_Ar&-NH, is about SO LO 100 timcs more srnsitive than dinitrophcnyl-Pro-leu-Gly-lxu-Trp-Ala-o-Ark-NH2 and continuous assays can be made at enzyme concentrations compardblc IO those used with mucromolccular substrates. Specilicity constiants (k,,lK,) are reported for both synthetic substrates with PUMP. collagenuse. stromelysin and 72 kDa gclatinnsc.

The activation of leukocyte integrins through diverse receptors results in transformation of the integrin from a bent, resting form to an extended conformation, which has at least two states of ligand-binding activity. This highly regulated activation process is essential for T cell migration and the formation of an immunological synapse. The signalling events that drive integrin activation are complex. Some key players have been well-characterized, but other aspects of the signalling mechanisms involved are still unclear. This Review focuses on the integrin lymphocyte function-associated antigen 1 (LFA1; also known as αLβ2 integrin), which is expressed by T cells, and explores how disparate signalling pathways synergize to regulate LFA1 activity.

The cloning and expression of the full-length tissue inhibitor of metalloproteinase 2 (TIMP-2), delta 187-194TIMP-2, and delta 128-194TIMP-2 and the purification of these inhibitors and a cleaved version of TIMP-2 lacking nine C-terminal amino acids (delta 186-194TIMP-2) are described. The mechanism of inhibition of gelatinase A by the TIMPs was investigated by comparing the kinetics of association of TIMP-1, TIMP-2, the C-terminal deletions, and the mutants of both TIMPs which consisted of the N-terminal domain only. The full-length TIMPs inhibited gelatinase A rapidly with association constants of 3.2 x 10(6) M-1 s-1 for TIMP-1 and 2.1 x 10(7) M-1 s-1 for TIMP-2 at I = 0.2. The C-terminal peptide of TIMP-2 is proposed to exist as an exposed "tail" responsible for binding to progelatinase A and for increasing the rate of inhibition of active gelatinase A through electrostatic interactions with the C-terminal domain of the enzyme. The C-terminal domains of both TIMP-1 and TIMP-2 participate in low-affinity interactions with the C-terminal domain of gelatinase A which increase the rate of association by a factor of about 100 in both cases.

Recombinant 72 kDa gelatinase A and a truncated form lacking the C-terminal domain were shown to be activated by organomercurials and to possess similar activities towards a number of substrates. The truncated proenzyme differed from the full-length gelatinase in that it could not be activated by a membrane activator and did not bind tissue inhibitor of metalloproteinase (TIMP)-2. Kinetic studies also showed that the inhibition of the activated truncated enzyme, by both TIMP-1 and TIMP-2, was considerably decreased compared with the full-length enzyme. We conclude that the C-terminal domain plays an important role in the regulation of gelatinase A by a potential physiological activator and inhibitors.

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