Interaction between dietary methionine and methyl donor intake on rat liver betaine-homocysteine methyltransferase gene expression and organization of the human gene. (1/91)

We previously showed that rat liver betaine-homocysteine methyltransferase (BHMT) mRNA content and activity increased 4-fold when rats were fed a methionine-deficient diet containing adequate choline, compared with rats fed the same diet with control levels of methionine (Park, E. I., Renduchintala, M. S., and Garrow, T. A. (1997) J. Nutr. Biochem. 8, 541-545). A further 2-fold increase was observed in rats fed the methionine-deficient diet with supplemental betaine. The nutrition studies reported here were designed to determine whether other methyl donors would induce rat liver BHMT gene expression when added to a methionine-deficient diet and to define the relationship between the degree of methionine restriction and level of methyl donor intake on BHMT expression. Therefore, rats were fed amino acid-defined diets varying in methionine and methyl donor composition. The effect of diet on BHMT expression was evaluated using Northern, Western, and enzyme activity analyses. Similar to when betaine was added to a methionine-deficient diet, choline or sulfonium analogs of betaine induced BHMT expression. The diet-induced induction of hepatic BHMT activity was mediated by increases in the steady-state level of its mRNA and immunodetectable protein. Using methyl donor-free diets, we found that methionine restriction was required but alone not sufficient for the high induction of BHMT expression. Concomitant with methionine restriction, dietary methyl groups were required for high levels of BHMT induction, and a dose-dependent relationship was observed between methyl donor intake and BHMT induction. Furthermore, the severity of methionine restriction influenced the magnitude of BHMT induction. To study the molecular mechanisms that regulate the expression of BHMT, we have cloned the human BHMT gene. This gene spans about 20 kilobases of DNA and contains 8 exons and 7 introns. Using RNA isolated from human liver and hepatoma cells, a major transcriptional start site has been mapped using the 5' rapid amplification of cDNA ends technique, and this start site is 26 nucleotides downstream from a putative TATA box.  (+info)

Autolysosomal membrane-associated betaine homocysteine methyltransferase. Limited degradation fragment of a sequestered cytosolic enzyme monitoring autophagy. (2/91)

We compared the membrane proteins of autolysosomes isolated from leupeptin-administered rat liver with those of lysosomes. In addition to many polypeptides common to the two membranes, the autolysosomal membranes were found to be more enriched in endoplasmic reticulum lumenal proteins (protein-disulfide isomerase, calreticulin, ER60, BiP) and endosome/Golgi markers (cation-independent mannose 6-phosphate receptor, transferrin receptor, Golgi 58-kDa protein) than lysosomal membranes. The autolysosomal membrane proteins include three polypeptides (44, 35, and 32 kDa) whose amino-terminal sequences have not yet been reported. Combining immunoblotting and reverse transcriptase-polymerase chain reaction analyses, we identified the 44-kDa peptide as the intact subunit of betaine homocysteine methyltransferase and the 35- and 32-kDa peptides as two proteolytic fragments. Pronase digestion of autolysosomes revealed that the 44-kDa and 32-kDa peptides are present in the lumen, whereas the 35-kDa peptide is not. In primary hepatocyte cultures, the starvation-induced accumulation of the 32-kDa peptide occurs in the presence of E64d, showing that the 32-kDa peptide is formed from the sequestered 44-kDa peptide during autophagy. The accumulation is induced by rapamycin but completely inhibited by wortmannin, 3-methyladenine, and bafilomycin. Thus, detection of the 32-kDa peptide by immunoblotting can be used as a streamlined assay for monitoring autophagy.  (+info)

Apolipoprotein B mRNA and lipoprotein secretion are increased in McArdle RH-7777 cells by expression of betaine-homocysteine S-methyltransferase. (3/91)

The cDNA encoding rat betaine-homocysteine S-methyltransferase (BHMT) was isolated through production of monoclonal antibodies against protein fractions enriched with apolipoprotein B (apo B)-mRNA-editing complexes. BHMT mRNA was expressed predominantly in liver, and also in kidney, but not in small intestine. In stable McArdle RH-7777 (McA) cell lines expressing differing levels of BHMT, the editing efficiency of apo B mRNA was unchanged. Evaluation of apo B-mRNA expression revealed that steady-state levels were increased significantly and in parallel with BHMT protein expression. The highest levels of BHMT mRNA and BHMT enzyme activity expressed in stably transfected McA cells were comparable with those found in rat hepatocytes. In contrast to the changes in apo B-mRNA abundance, levels of other apolipoprotein-encoding mRNAs and several liver-specific and ubiquitously expressed mRNAs were unchanged by BHMT expression. In the cell line expressing the highest level of BHMT, apo B-containing lipoprotein secretion was increased, indicating utilization of increased endogenous message. Results suggest that apo B-mRNA abundance in McA cells is related to the expression of BHMT, an enzyme important in homocysteine metabolism.  (+info)

Leupeptin-induced appearance of partial fragment of betaine homocysteine methyltransferase during autophagic maturation in rat hepatocytes. (4/91)

A cytosolic enzyme, betaine homocysteine methyltransferase (BHMT), and its partial fragments were discovered as autolysosomal membrane proteins from rat liver in the presence of leupeptin [Ueno et al. (1999) J. Biol. Chem. 274, 15222-15229]. The present study was undertaken to further characterize the transport and processing of BHMT from cytosol to autolysosome and to test if the fragment can be used as an in vitro probe for the maturation step of macroautophagy. Upon subcellular fractionation, BHMT (p44) was found in all fractions, while its 32-kDa fragment (p32) was found only in the mitochondrial-lysosomal (ML) fraction. Incubation of isolated hepatocytes with leupeptin induced time-dependent accumulation of p32 in the ML fraction from 30 to 90 min after the start of incubation. However, chloroquine completely inhibited the appearance of p32, indicating that the processing from p44 to p32 is lysosomal. Incubation with Bafilomycin A(1), a vacuolar H(+)-ATPase inhibitor, together with leupeptin, led to linear accumulation of p44, but not of p32. The p44 accumulation rate was calculated to be 4.9%/h, which was comparable to autophagic sequestration rate. The distribution of p44 within the ML fraction turned out to be dual, i.e., the membrane-surface attached and luminal/sedimentable forms. Amino acids and 3-methyladenine, both of which specifically suppress macroautophagy, inhibited the accumulation of p32 as well as of p44. Finally, energy-dependent appearance of p32 was demonstrated during incubation of postnucler supernatant fractions, making it possible to establish an in vitro assay system. All the results strongly support the idea that BHMT is taken up and degraded to p32 through the macroautophagic pathway, and that p32 could be a novel probe for the maturation of macroautophagy.  (+info)

Dimethylglycine accumulates in uremia and predicts elevated plasma homocysteine concentrations. (5/91)

BACKGROUND: Hyperhomocysteinemia is a risk factor for atherosclerosis that is common in chronic renal failure (CRF), but its cause is unknown. Homocysteine metabolism is linked to betaine-homocysteine methyl transferase (BHMT), a zinc metalloenzyme that converts glycine betaine (GB) to N,N dimethylglycine (DMG). DMG is a known feedback inhibitor of BHMT. We postulated that DMG might accumulate in CRF and contribute to hyperhomocysteinemia by inhibiting BHMT activity. METHODS: Plasma and urine concentrations of GB and DMG were measured in 33 dialysis patients (15 continuous ambulatory peritoneal dialysis and 18 hemodialysis), 33 patients with CRF, and 33 age-matched controls. Concentrations of fasting plasma total homocysteine (tHcy), red cell and serum folate, vitamins B(6) and B(12), serum zinc, and routine biochemistry were also measured. Groups were compared, and determinants of plasma tHcy were identified by correlations and stepwise linear regression. RESULTS: Plasma DMG increased as renal function declined and was twofold to threefold elevated in dialysis patients. Plasma GB did not differ between groups. The fractional excretion of GB (FE(GB)) was increased tenfold, and FED(MG) was doubled in CRF patients compared with controls. Plasma tHcy correlated positively with plasma DMG, the plasma DMG:GB ratio, plasma creatinine, and FE(GB) and negatively with serum folate, zinc, and plasma GB. In the multiple regression model, only plasma creatinine, plasma DMG, or the DMG:GB ratio was independent predictors of tHcy. CONCLUSIONS: DMG accumulates in CRF and independently predicts plasma tHcy concentrations. These findings suggest that reduced BHMT activity is important in the pathogenesis of hyperhomocysteinemia in CRF.  (+info)

Selenium deficiency in Fisher-344 rats decreases plasma and tissue homocysteine concentrations and alters plasma homocysteine and cysteine redox status. (6/91)

The purpose of the present study was to determine the effect of graded amounts of dietary selenium on plasma and tissue parameters of methionine metabolism including homocysteine. Male weanling Fisher-344 rats (n = 7-8/group) were fed a selenium-deficient, torula yeast-based diet, supplemented with 0 (selenium deficient), 0.02, 0.05 or 0.1 microg (adequate) selenium (as selenite)/g diet. After 61 d, plasma total homocysteine and cysteine were decreased (P < 0.0001) and glutathione increased (P < 0.0001) by selenium deficiency. The concentrations of homocysteine in kidney and heart were decreased (P = 0.02) by selenium deficiency. The activities of liver betaine homocysteine methyltransferase, methionine synthase, S-adenosylmethionine synthase, cystathionine synthase and cystathionase were determined; selenium deficiency affected only betaine homocysteine methyltransferase, which was decreased (P < 0.0001). The ratios of plasma free reduced homocysteine (or cysteine) to free oxidized homocysteine (or cysteine) or to total homocysteine (or cysteine) were increased by selenium deficiency, suggesting that selenium status affects the normally tightly controlled redox status of these thiols. Most differences due to dietary selenium were between rats fed 0 or 0.02 microg selenium/g diet and those fed 0.05 or 0.1 microg selenium/g diet. The metabolic consequences of a marked decrease in plasma homocysteine and smaller but significant decreases in tissue homocysteine are not known.  (+info)

Methionine supply to growing steers affects hepatic activities of methionine synthase and betaine-homocysteine methyltransferase, but not cystathionine synthase. (7/91)

The effects of supplemental methionine (Met), supplied abomasally, on the activities of methionine synthase (MS), cystathionine synthase (CS) and betaine-homocysteine methyltransferase (BHMT) were studied in growing steers. Six Holstein steers (205 kg) were used in a replicated 3 x 3 Latin square experiment. Steers were fed 2.6 kg dry matter daily of a diet containing 83% soybean hulls and 8% wheat straw. Ruminal infusions of 180 g/d acetate, 180 g/d propionate, 45 g/d butyrate, and abomasal infusion of 300 g/d dextrose provided additional energy. An amino acid mixture (299 g/d) limiting in Met was infused into the abomasum to ensure that nonsulfur amino acids did not limit growth. Treatments were infused abomasally and included 0, 5 or 10 g/d L-Met. Retained N (20.5, 26.9 and 31.6 g/d for 0, 5 and 10 g/d L-Met, respectively) increased (P < 0.01) linearly with increased supplemental Met. Hepatic Met, vitamin B-12, S-adenosylmethionine and S-adenosylhomocysteine were not affected by Met supplementation. Hepatic folates tended (P = 0.07) to decrease linearly with Met supplementation. All three enzymes were detected in hepatic tissue of our steers. Hepatic CS activity was not affected by Met supplementation. Hepatic MS decreased (P < 0.01) linearly with increasing Met supply, and hepatic BHMT activity responded quadratically (P = 0.04), with 0 and 10 g/d Met being higher than the intermediate level. Data from this experiment indicate that sulfur amino acid metabolism may be regulated differently in cattle than in other tested species.  (+info)

Betaine-homocysteine methyltransferase: zinc in a distorted barrel. (8/91)

Betaine-homocysteine methyl transferase (BHMT) catalyzes the synthesis of methionine from betaine and homocysteine (Hcy), utilizing a zinc ion to activate Hcy. BHMT is a key liver enzyme that is important for homocysteine homeostasis. X-ray structures of human BHMT in its oxidized (Zn-free) and reduced (Zn-replete) forms, the latter in complex with the bisubstrate analog, S(delta-carboxybutyl)-L-homocysteine, were determined at resolutions of 2.15 A and 2.05 A. BHMT is a (beta/alpha)(8) barrel that is distorted to construct the substrate and metal binding sites. The zinc binding sequences G-V/L-N-C and G-G-C-C are at the C termini of strands beta6 and beta8. Oxidation to the Cys217-Cys299 disulfide and expulsion of Zn are accompanied by local rearrangements. The structures identify Hcy binding fingerprints and provide a prototype for the homocysteine S-methyltransferase family.  (+info)

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