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Protein Methylase II

(S-adenosylmethionine:protein Carboxyl O-methyltransferase; EC

Methylation of proteins is one of the key reactions in the post-translational modification of protein amino acid residues in the cell. It has been established that the basic and acidic amino acid residues of certain proteins are methylated in vivo.

Protein methylase II methylates the free carboxyl groups of dicarboxylic acid residues in a protein molecule. Existence of this enzyme, in mammalian tissues, was first reported by Liss and Edelstein (Liss, M. and Edelstein, M., Biochem. Biophys. Res. Commun., 26, 497, 1967). Protein methylase II from ox brain has been purified by Iqbal and Steenson (Iqbal, M. and Steenson, T., J. Neurochem., 27, 605, 1976).

The amount of enzyme which incorporates one picomole of methyl-14C into a substrate protein per minute at 37°C, pH 6.2.

Incorporation of S adenosyl-L-methyl-14C methionine into a protein is measured at pH 6.2. This pH is favorable for the assay of protein methylase II as interference from other protein methylases is minimal under these experimental conditions (Protein Methylation, Paik, W.K. and Kim, S., eds; Vol I, 113, 1980, Wiley, New York).

  1. 0.25 M Citric acid.
  2. 0.5 M Sodium phosphate (Na2HPO4).
  3. 0.02 M EDTA disodium salt, pH adjusted to 6.0 with 1 M NaOH.
  4. 2-Mercaptoethanol.
  5. Citrate-phosphate buffer cocktail. Prepared by mixing 6 ml of citric acid, 10 ml of sodium phosphate, 8 ml of EDTA and 0.08 ml of 2-mercaptoethanol.
  6. S-Adenosyl-L-(Methyl-14C) methionine (SAM), specific activity = 50 m Ci/mM diluted to yield a concentration of 0.1 mM and 100-150 dpm/picomole.
  7. Ovalbumin solution (10 mg/ml) in citrate-phosphate buffer cocktail.
  8. Protein methylase II (enzyme). Dilute in buffer cocktail to yield a final concentration of 20-30 µg protein per assay.
  9. 30% Trichloroacetic acid (TCA).
  10. 0.2 M Sodium phosphate buffer, pH 7.2.
  11. Chloroform:ether:ethanol mixture (1:2:2, v/v/v).
  12. Ethanol, 98%.
  13. Scintillation cocktail (available commercially).

  1. In a pyrex conical glass centifuge tube (kept in ice) pipette the following:
    Citrate-phosphate buffer cocktail 0.15 ml
    Ovalbumin solution 0.1 ml
    Enzyme (20-30 µg/0.1 ml) 0.1 ml
    Distilled water 0.15ml
  2. Incubate at 37°C for 3 minutes
  3. Initiate the reaction by adding 0.1 ml of (methyl 14C) SAM. Continue reaction for 10 minutes.
  4. Terminate the reaction by adding 0.5 ml of 30% TCA. The mixture is carefully overlayered with ethanol and centrifuged for 15 minutes in a table top clinical centrifuge.
  5. The supernatant is decanted and the precipitate is washed three times with 8 ml of TCA solution, once with chloroform: ether:ethanol, and once with ethanol.
  6. The precipitates in one set of tubes are transferred quantitatively into scintillation vials containing 10 ml of scintillation fluid and counted for radioactivity in a liquid scintillation counter. One ml of 0.2 M sodium phosphate buffer (pH 7.2) is added to each of the precipitates in a second set of tubes (serving as control), which are then placed in a boiling water bath for 5 minutes. This heat treatment decomposes the protein-methyl ester. After heating, the proteins are reprecipitated by addition of TCA, then washed once with TCA and once with ethanol. Finally the radioactivity in the precipitate is counted. The difference in the radioactivity between the heated and unheated tubes is taken as the radioactivity due to protein-methyl ester formed by the protein methylase II enzyme.

Activity (U/mg) = (CPM/Sample)(Enz. Diln.)
(CPM/picomole SAM)(mg Enz./ml)

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