Increased lysine N-methylation of a 23-kDa protein during hepatic regeneration

The methylation of a 23-kDa nuclear protein increased after partial hepatectomy and methylation returned to basal levels after the initial stage of regeneration. The methylating enzyme was partially purified from rat liver by ammonium sulfate precipitation, DEAE-anion exchange chromatography and Butyl-Sepharose chromatography. The 23-kDa protein was purified from a nuclear fraction of liver tissue with SP-Sepharose. When the 23-kDa protein was methylated with the partially purified methyltransferase and analyzed on C18 high performance liquid chromatography (HPLC), the methylated acceptor amino acid was monomethyl lysine (MML). Previously, only arginine N-methylation of specific substrate proteins has been reported during liver regeneration. However, in this report, we found that lysine N-methylation increased during early hepatic regeneration, suggesting that lysine N-methylation of the 23-kDa nuclear protein may play a functional role in hepatic regeneration. The methyltransferase did not methylate other proteins such as histones, hnRNPA1, or cytochrome C, suggesting the enzyme is a 23-kDa nuclear protein- specific lysine N-methyltransferase.

The nuclear 16-kD protein methylation increases in the early period of liver regeneration in a hepatectomized rat

Methylation events play a critical role in various cellular processes including regulation of gene transcription and proliferation. We observed that methyltransferase activity underwent time-dependent changes in the cytosol of the rat hepatocytes upon partial hepatectomy. However, any change in the methylation of nuclear proteins is not clear during hepatocyte proliferation. The nuclear fraction possesses basal level of methyltransferase to catalyze methylation of several proteins ranging from 7 to 70 kD prior to any hepatecmony. The specific p16 (16 kD) band was transiently and heavily methylated post 1 day hepatectomy, and then became non- detectable, but not in the control liver. Methylation of p16 band was completely inhibited by exogenously added histones, particularly 2AS, 1, 2A and 2B subtypes. The methylated p16 protein remains stable in either acid or alkali- induced demethylation conditions, indicating that methylation is not likely to occur on isoaspartyl or C-terminal cysteinyl residues. Exogenous addition of non-hydrolyzable GTP caused a dose- dependent suppression of a p16 methylation suggesting that G-proteins might play a role as an endogenous methylation inhibitor in vivo. Taken together, we have identified the proliferation event associated-methylation of the nuclear p16 protein in the hepatocytes undergoing liver regeneration.

Increased methylation of the cytosolic 20-kD protein is accompanied by liver regeneration in a hepatectomized rat

Arginine methylation has been implicated in the signal transduction pathway leading to cell growth. Here we show that a regenerating rat liver following partial hepatectomy exhibited elevated methyltransferase activity as shown by increased methylation of a subset of endogenous proteins in vitro. The 20-kDa protein was shown to be a major cytosolic protein undergoing methylation in regenerating hepatocytes. Methylation of the 20-kDa protein peaked at 1 d following partial hepatectomy, which gradually declined to a basal level within the next 14 d. Likewise, methylation of exogenously added bulk histones followed the similar time kinetics as the 20-kDa protein, reflecting time-dependent changes in methyltransferase activity in regenerating hepatocytes. Presence of exogenously added bulk histone in the in vitro methylation assay resulted in dose-dependent inhibition of methylation of the 20-kDa protein. All the histone subtypes tested, histone 1, 2A, 2B, 3 or 4, were able to inhibit methylation of the 20-kDa protein while addition of cytochrome C, a-lactalbumin, carbonic anhydrase, bovine serum albumin, and g globulin minimally affected methylation of the 20-kDa protein. Since methylation of the 20-kDa protein preceded proliferation of hepatocytes upon partial hepatectomy, it is tempting to speculate that the methylated 20-kDa protein by activated histone-specific methyltransferase may be involved in an early signal critical for liver regeneration.

Arylamine Acetyltransferase Activity from a Regenerating Liver after Partial Hepatectomy and from a Cholestatic Liver after Common Bile Duct Ligation in Rats

A study was made of the change in arylamine acetyltransferase(AAT) activity in regenerating and/or cholestatic rat livers. Cytosolic, mitochondrial and microsomal AAT activities were determined over a period of 10 days in rat livers which were regenerating after 70%(median and left lateral lobes) partial hepatectomy and over a period of 42 days in rat livers with cholestasis induced by a common bile duct ligation. The values of Km and Vmax in these hepatic enzymes were measured. Both the cytosolic and the microsomal AAT activities in the regenerating rat livers showed significant increases from the first day to the third day after the partial hepatectomy. However, the mitochondrial AAT activity did not change. The cytosolic and the microsomal AAT activities in the cholestatic rat livers showed a significant increase on the first day and from the first day to the second day, respectively after the ligation; Both the cytosolic and the microsomal AAT activities showed significant decreases from the fourteenth day to the forty-second day after the ligation. However, the mitochondrial AAT activity did not change. The Vmax values of both the cytosolic and the microsomal AAT activity in the regenerating and/or cholestatic rat livers showed significant increases on the first day after the partial hepatectomy and/or the ligation. However, the Vmax values of both the cytosolic and the microsomal AAT activities in the cholestatic rat livers showed significant decreases on the twenty-eighth day after the ligation. On the other hand, the Km values of the above enzymes did not change. In view of the above results, the AAT activity in the regenerating rat liver appears to be due to the enzyme increasing its biosynthesis in the regenerating stage. The AAT activity in the cholestatic rat liver suggests that the enzymes is increasing its biosynthesis in the severe necrotizing stage, but decreasing its biosynthesis severe hepatic dysfunction stage.