Activation of the protease from human adenovirus type 2 is accompanied by a conformational change that is dependent on cysteine-104.

Adenovirus codes for a protease the activity of which can be regulated in vitro by an 11 residue peptide (GVQSLKRRRCF) derived from another viral protein, pVI. Three cysteine residues, one in the activating peptide and two in the protease (C104 and C122), play a central role in both activation and catalysis. Expression of protease mutants in insect cells has shown that C104 is not essential for proteolytic activity. GVQSLKRRRCF also caused a concentration-dependent increase in tryptophan fluorescence of protease expressed in Escherichia coli that paralleled the increase in proteolytic activity, indicating that activation was accompanied by a conformational change. Tryptophan fluorescence of C104S was not increased by the addition of GVQSLKRRRCF, nor was the fluorescence of wild-type protease increased by the addition of the peptide analogues where cysteine is replaced by aspartic acid or serine, suggesting that C104 is involved in activation and C122 in catalysis.

The protease of adenovirus serotype 2 requires cysteine residues for both activation and catalysis.

Sequence analysis and site-directed mutagenesis were used to study the mechanisms of activation and catalysis of the adenovirus type 2 (Ad2) protease. Primary structure alignments of proteases from 12 serotypes and previously elucidated inhibition profiles were used to target residues for mutagenesis. All conserved serine and cysteine residues were mutated separately and following expression in Escherichia coli their activity in a synthetic peptide assay was compared to that of wild-type recombinant protease. Mutants containing altered serine residues were active while mutations to cysteine-104 and cysteine-122 reduced activity by more than 95%. These results taken together with the known inhibition profile of the adenovirus protease confirm that it is a cysteine protease and suggest that one of these residues provides the active site nucleophile while the other is a part of the thiol-disulphide interchange mechanism previously reported to be involved in its activation.

Vanadate inhibits both intercellular adhesion and spreading on fibronectin of BHK21 cells and transformed derivatives.

Both intercellular adhesion and spreading on fibronectin of BHK21 hamster cells are inhibited by vanadate at concentrations that cause specific regulatory effects rather than general metabolic inhibition. Inhibition of aggregation of these cells in suspension (half-maximal in 10(-5) M vanadate) is rapid and reversible. The extent of inhibition, and its decline with culture age parallel inhibition by agents that depolymerize microtubules. Vanadate also reversibly inhibits spreading of both BHK cells and transformed derivatives on fibronectin. If 10(-4) M vanadate is added to BHK cells that have spread in its absence, they remain spread, but transformed derivatives are sensitive to rounding by vanadate at 10(-6) M. The mechanisms by which vanadate inhibits both intercellular adhesion and spreading are unknown, and may be different for the two phenomena. Possible sensitive targets include cytoplasmic dynein for the former, and protein tyrosyl phosphatase for the latter.