Trypsin action

The clones may be isolated either by surrounding each colony with a stainless steel cylinder and removing the cells by trypsin action or by initially seeding the cells into a multiwell plate and selecting those wells in which single colonies become established. If care is taken, single cells can be selected from a suspension and transferred to individual wells in a multiwell plate. 

It is a proteolytic enzyme, present in the intestine in its inactive form (zymogen), trypsinogen. Trypsinogen is converted into its active form, trypsin, by enteropeptidase, a specialized proteolytic enzyme secreted by intestinal cells. Some free trypsin formed also catalyses the conversion of trypsinogen into trypsin. Trypsin can also convert chymotrypsinogen and procarboxypeptidase into chymotrypsin and carboxypeptidase, respectively. Trypsin has different amino acid specificity when compared with other proteolytic enzymes. Trypsin hydrolyses those peptide bonds whose carboxyl groups are contributed by Lys or Arg residues and if the next residue is not proline. The number of smaller peptides resulting from trypsin action is equal to the total number of Arg and Lys residues in the protein plus one. 

For plating, wash the cells with PBS, aspirate the supernatant and add 2 ml trypsin-EDTA (0.25%) solution per 75-cm cultivation flask. Shake gently so that the solution can cover the entire cell are, and then remove all the trypsin with a Pasteur pipette and incubate the flask at 37°C for 2-3 min. Observe the cells under a microscope when the cells are detached, immediately add 10 ml of H441 culture medium to arrest the trypsin action. 

Figure 1. Effect of mild trypsin digestion on CPT I activity in permcaldized hepatocytes. Hepatocytes were preincubaled for 15 min in the absence (open circles) or in the presence (filled ciicles) of O.SpM OA. ( Ib were then permeabilized with digitonin and thorougiy washed with digitonin-free medium as described in ref. Per meabilized hepatocytes were subsequently resuspended at 1.5-2.0 mg piotdii mT and treated with varying con centrations of trypsin at 4 C. Trypsin action was stopped after 2 min and CPT I activity was subsequently determined in those permeabilized hepatocytes. Results correspond to 4 different cell preparations. Inset Mitochondria were isolated from permeabilized hepatocytes that had been treated without (lane a) or with (lane b) 17.5 pgmT trypsin and CPT I was detected by Western blotting. The arrow points to the 88 kDa band.

The initial rates of inactivation by trypsin of autophosphorylatico of the kinase and of LHC-II phosphorylation are faster than the release of the respective phosphorylated terminal peptides (fig.4B, cenpare with fig. 4A), and preceed the suppression by trypsin of the Mg ion stimulation of fluorescence. The observation that histone phosphorylation was also inhibited (fig.4B) rules out the possibility that inhibition of the other two activities was due to the digesticxi by trypsin of the substrates (mc-II and the kinase itself) rather than to inactivaticxi of the enzyme. These observations suggest that trypsin action on the enzyme may be dual a first hit would inactivate the enzyme by splitting at a site terminal with respect to the phosphorylated residue, and a second hit would hydrolyse off the phosphorylated peptide. 

As in the first exercise, trypsin action and measnrements of the fragment masses provide a handle on the states and information. 

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