Carboxyl methylation of nonhistone chromosomal proteins

Abstract

The in vitro methylation of nonhistone chromosomal proteins (NHCP) was investigated in nuclei isolated from the brain, liver, and thymus of 6-8-day-old rats. After the nuclei were incubated in the presence of 20 micron M S-adenosyl-L-[methyl-3H] methionine (1 Ci/mmol), the NHCP were separated from histones on hydroxylapatite and fractionated further on sodium dodecyl sulfate-acrylamide slab gel electrophoresis. After the gels were dried, autoradiography was used to detect [3H] methyl groups associated with these proteins. Four NHCP from the liver and thymus were methylated, while six methylated proteins were detected from the brain. None of the methylated proteins in these tissues corresponded with those from other organs, except for the component with a molecular weight of 66 000. It was evident that the methyl groups were esterified to the free carboxyl groups of NHCP since they are heat labile, yielding [3H]-methanol. The carboxyl-methylated NHCP from these organs were tightly bound to chromatin. Nucleoplasm and loosely associated NHCP were essentially devoid of methylated proteins. The carboxyl methylation of NHCP was verified in vivo. Six-day-old rats were given L-[methyl-3H]methionine (7 mCi/mmol) by intraperitoneal injection. The rats were killed at varying time periods and the NHCP isolated from gradient purified nuclei. Chromosomal nonhistone proteins, particularly from the liver, contained significant amounts of alkaline labile [(3)H] methyl groups.

Methyl-esterified proteins in a mammalian cell line

“…A low-pH gel system has been found necessary by some researchers for full recovery of base-labile methyl groups (Kloog et al, 1980; Freitag & Clarke, 1981; Billingsley et al, 1984). However, methyl esterifications of purified rat non-histone chromosomal proteins (Quick et al, 1981) as well as bovine photoreceptor rod outer segments (Swanson & Applebury, 1983) are stable during Laemmli gel electrophoresis. We find little difference between the two systems (Table I) and suspect that the SDS acts to protect the methyl esters from hydrolysis as suggested previously by others (Kleene et al, 1977;Galletti et al, 1978).…”

“…If methylation is being used to regulate the activity of these proteins, we would expect that both methylating and demethylating enzymes are present in the cells. At least one protein with methyl transferase activity has been identified in mammalian tissues (Paik & Kim, 1980a,b;Dilberto, 1982), and there are reports of methyl esterase activity as well (Venkatasubramanian et al, 1980;Quick et al, 1981). Demethylation was examined by using a pulse-chase experiment.…”

Purification and characterization of chemotactic methylesterase from Bacillus subtilis

“…Reversible methylation reactions have been found in a number of eukaryotic as well as prokaryotic systems (Goy et al, 1977;Goldman et al, 1982;Gagnon, 1979;Quick et al, 1981). Recently, methylesterase activity has been detected in a number of tissues (Gagnon, 1979).…”

“…Chemoattractants are found to stimulate demethylation of preformed methyl esters of proteins involved in rabbit neutrophil chemotaxis (Venkatasubramanian et al, 1980). Nonhistone chromosomal proteins are found to be methylated in a tissue-specific manner (Quick et al, 1981). Methylesterase activity has also been observed to be associated with chromatin (Quick et al, 1981).…”

Effect of 3-deazaadenosine on methionine metabolism in isolated perfused livers

“…The chromatin was dissolved in 0.5 M NaCI, 5.OM urea, and 1 lOmM sodium phosphate buffer (pH 6.8) and fractionated on a hydroxylapatite column as described previously (Quick et al 1981). Elution of the histones and nonhistone chromosomal proteins was followed spectrophotometrically at 280 nm, and radioactivity under the peaks was determined by the channels ratio technique using a Beckman scintillation spectrophotometer.…”