Practice question

Adherent mammalian cells deposit organized extracellular matrix and engage tissue culture surface through integrin mediated focal adhesions linking actin cytoskeleton to substrate, providing survival signaling through focal adhesion kinase FAK autophosphorylation tyrosine 397 recruiting Src, paxillin, vinculin, plus cadherin mediated cell-cell contacts dependent on calcium bridging extracellular domains EC1-EC2 homophilic binding. Subculture requires breaking these adhesions without irreversible damage to surface receptors. Trypsin serine protease belonging to PA clan S1 family purified from bovine or porcine pancreas as 223 amino acid 23.3 kDa polypeptide with six disulfide bonds stabilizing beta-barrel structure, active site His57 Asp102 Ser195 chymotrypsin numbering forming catalytic triad polarizing Ser195 hydroxyl increasing nucleophilicity for attack on peptide carbonyl producing tetrahedral oxyanion stabilized by backbone amides Gly193 Ser195 oxyanion hole, cleaving peptide bond on carboxyl side of basic residues arginine and lysine due to acidic Asp189 at bottom primary specificity pocket attracting positive side chain, characteristic tryptic digestion. Commercial working concentration 0.05 to 0.25 percent weight per volume often combined with chelator EDTA 0.