Nicotinamide methylation tissue distribution,developmental and neoplastic changes

The distribution of cytosolic activity of nicotinamide:S-adenosylmethionine methyltransferase (nicotinamide methylase, EC 2.1.1.1) in normal tissues from adult rat and mouse and in tumors and the change in the enzyme activity during the the development of rat tissues were studied. (1) Rat liver exhibited the highest nicotinamide methylase activity among all adult tissues tested; other rat tissues, like adrenal, pancreas, kidney, brain and mouse tissues, had only less than 15% of the adult rat liver activity. (2) 3 days before birth, fetal liver showed a very low nicotinamide methylase activity (2% of adult rat liver), which, however, increased already 1 day before birth and reached the adult level on the day 28 after birth. (3) In a variety of hepatomas and ascites tumors, an inverse correlation, with some exceptions, between tumor growth rate and nicotinamide methylase activity could be seen. In all hepatomas, with the exception of Morris hepatoma 5123tc, nicotinamide methylase activity was significantly decreased in comparison to normal adult rat liver. The highly malignant Zajdela hepatoma, Yoshida sarcoma, sarcoma 180 and Ehrlich ascites tumor methylated nicotinamide only at a negligibly low rate. (4) Cultured RLC cells (an established rat liver cell line) from the stationary growth phase or G₁-arrested RLC cells had about half of the adult rat liver activity, yet the activity was 70% higher than that of the logarithmically growing RLC cells.     

The use of a tritium release assay to measure 6-N-trimethyl-L-lysine hydroxylase activity: synthesis of 6-N-[3-3H]trimethyl-DL-lysine

6-N-[3-³H]Trimethyl-dl-lysine was synthesized from 6-N-acetyl-l-lysine by the following chemical scheme: 6-N-acetyl-l-lysine → 2-keto-6-N-acetylcaproic acid → 2-[3-³H]keto-6-N-acetylcaproic acid → 2-[3-³H]keto-6-N-acetylcaproic acid oxime → 6-N-[3-³H]acetyl-dl-lysine → dl-[3-³H]lysine → 2-N-[3-³H]formyl-dl-lysine → 2-[3-³H]formyl-6-N-trimethyl-dl-lysine → 6-N-[3-³H]trimethyl-dl-lysine. Using a 70% ammonium sulfate fraction obtained from a high-speed rat kidney supernatant, the cosubstrate and cofactor requirements for 6-N-trimethyl-l-lysine hydroxylase activity as measured by tritium release from 6-N-[3-³H]trimethyl-dl-lysine were: α-ketoglutarate, ferrous ions, l-ascorbate, and oxygen, with added catalase showing a slight but distinct stimulatory effect. On incubation with the crude rat kidney preparation, the release of tritium from 6-N-[3-³H]trimethyl-dl-lysine was linear with both time of incubation and protein concentration. Hydroxylation of 6-N-trimethyl-l-lysine, as measured by tritium release from the labeled substrate, was examined in rat kidney, heart, liver, and skeletal muscle tissues, and found to be most active in the kidney.     

ϵ-N-Trimethyllysine desaminase de Neurospora crassa

?-N-Trimethyl-l-lysine is transformed into 2-keto-6-N-trimethylhexanoic acid by a soluble deaminase present in the mycelium of Neurospora crassa. The activity of the enzyme increases with the age of the mycelium with an optimum after 10 days of culture. ?-N-Trimethyllysine deaminase has been purified 200-fold by differential centrifugation and successive column chromatography on DEAE Sephadex and Ultrogel AcA-34. Some of its properties have been studied. This is the first enzyme reported to metabolise ?-N-trimethyllysine.     

Properties of rat 6-N-trimethyl-l-lysine hydroxylases: Similarities among the kidney,liver, heart,and skeletal muscle activities

Hydroxylation of 6-N-trimethyl-l-lysine(lys(Me₃)) to 3-hydroxy-6-N-trimethyl-l-lysine(3-HO-lys(Me₃)) by several rat tissues has been examined and compared. The kidney enzyme, which previously was shown to require molecular oxygen and α-ketoglutarate as cosubstrates, ferrous iron and ascorbate as cofactors, and to be stimulated by catalase, has a broad pH optimum ranging between 6.5 to 7.5 at 37 °C. As determined with crude tissue extracts from kidney, liver, heart, and skeletal muscle, similar apparent K_m values were obtained for substrate, cosubstrates, and cofactors. In view of similar kinetic parameters among the several lys(Me₃) hydroxylases examined in rat tissues, and the fact that the level of skeletal muscle lys(Me₃) hydroxylase activity is comparable to that of heart, liver, and kidney, because of its large total mass, skeletal muscle may contribute significantly to the biosynthesis of l-carnitine from lys(Me₃). The most effective inhibitors found, competitive with lys(Me₃), were 2-N-acetyl-6-N-trimethyl-l-lysine, 6-N-monomethyl-l-lysine, and 6-N-dimethyl-l-lysine. l-2-Amino-6-N-trimethylammonium-4-hexynoate, d-2-amino-6-N-trimethylammonium-4-hexynoate, and dl2-amino-6-N-trimethylammonium-cis-4-hexenoate, also inhibited hydroxylase activity but by a yet undetermined mechanism. Oxalacetate, succinate, and citrate inhibited the hydroxylation reaction by competing with α-ketoglutarate. The binding of ferrous iron to the enzyme was competitively inhibited by ions of “soft metals” (e.g., Cd²⁺, Zn²⁺) but not by those of “hard metals” (e.g., Ca²⁺, Mg²⁺). Preincubation of the crude kidney enzyme for 15 min at 37 °C with mercuriphenylsulfonate, N-ethylmaleimide, iodoacetate, or iodoacetamide resulted in considerable inhibition of 3-HO-lys(Me₃) formation. The degree of inhibition by N-ethylmaleimide could be reduced by including Zn (II) during preincubation of the enzyme. The effects of “soft” metals and sulfhydryl reagents on the enzyme suggest that sulfhydryl groups are required for ferrous iron binding in the active site.     

Analysis of unsaturated fatty acids, and their hydroperoxy and hydroxy derivatives by high-performance liquid chromatography.

A simple procedure for the detection of endo-β-N-acetylglucosaminidase H activity is described. The method utilizes N-[¹⁴C]methylribonuclease B as substrate. This is prepared from ribonuclease B by reductive alkylation of free amine groups in the protein with [¹⁴C]formaldehyde. Because the carbohydrate moiety of ribonuclease B has α-mannosyl residues at nonreducing terminal positions, the radioactive molecule binds to Sepharose-concanavalin A. Endo-β-N-acetylglucosaminidase action releases this mannose-containing oligosaccharide by splitting the di-N-acetylchitobiosyl residue that links it with the peptide and thereby renders the radioactive portion of the molecule unreactive with Sepharose-concanavalin A. This forms the basis of a convenient assay for screening column fractions during the purification of the endoglycosidase. Although protease or α-mannosidase activity might also be detected by the procedure, no difficulties were presented by these enzymes when the assay was used for the preparation of endo-β-N-acetylglucosaminidase H from Streptomyces plicatus.     

In vivo andin vitro methylation of lysine residues ofEuglena gracilis histone H1

We have earlier identified and purified two protein-lysine N-methyltransferases (Protein methylase III) fromEuglena gracilis [J. Biol. Chem.,260, 7114 (1985)]. The enzymes were highly specific toward histone H1 (lysine-rich), and the enzymatic products were identified as ε-N-mono-, di- and trimethyllysines. These earlier studies, however, were carried out with rat liver histone H1 as thein vitro substrate. Presently, histone H1 has been purified fromEuglena gracilis through Bio-Rex 70 and Bio-Gel P-100 column chromatography. TheEuglena histone H1 showed a single band on SDS-polyacrylamide gel electrophoresis and behaved like other histone H1 of higher animals, whereas it had a much higherR _f value than the other histones H1 in acid/urea gel electrophoresis. When theEuglena histone H1 was [methyl-³H]-labeledin vitro by a homologous enzyme (one of the twoEuglena protein methylase III) and analyzed on two-dimensional gel electrophoresis, three distinctive subtypes of histone H1 were shown to be radiolabeled, whereas five subtypes of rat liver histone H1 were found to be labeled. Finally, by the combined use of a strong cation exchange and reversed-phase Resolve C₁₈ columns on HPLC, we demonstrated thatEuglena histone H1 contains approximately 9 mol% of ε-N-methyllysines (1.40, 1.66, and 5.62 mol% for ε-N-mono-, di- and trimethyllysines, respectively). This is the first demonstration of the natural occurrence of ε-N-methyllysines in histone H1.     

Purification and characterization of the Novikoff hepatoma glycogen phosphorylase and its relations to a fetal form

The Novikoff hepatoma glycogen phosphorylase b has been purified over 300-fold, free of glycogen synthetase, some of its properties have been studied, and its relationship to fetal forms of rat muscle and liver phosphorylase has been established immunochemically. Its molecular weight is approximately 200,000, and, like the liver but unlike the muscle isozyme, it does not dimerize on conversion to the a form. However, it differs from the liver isozyme in being activated by AMP (K_a = 0.2 mM) and in not being activated by sulfate ion. Antibody to the adult rat muscle phosphorylase did not inhibit the activity of the tumor or liver isozyme. Although antibody to liver or hepatoma phosphorylase had no effect on adult muscle phosphorylase, each of these antibodies partially inhibited the other enzyme. These findings indicate the presence of some liver isozyme in the tumor, and this was confirmed by isoelectric focusing. Rat liver and muscle phosphorylase (and synthetase) were low during embryonal development but rose rapidly at or shortly after birth. Immunochemical studies revealed that both fetal liver and fetal muscle phosphorylases are immunologically identifiable with the tumor enzyme; and the fetal form is also present as a major form in rat kidney and brain.     

Identification of N5,N10-methylene tetrahydrofolate reductase as the enzyme involved in the 5-methyl tetrahydrofolate-dependent formation of a beta-carboline derivative of 5-hydroxytryptamine in human platelets.

An enzyme in human platelets or rat brain incubated with 5-methyl tetrahydrofolate (5MeH₄folate) yields formaldehyde (4, 13), which will combine with biogenic amines to form β-carbolines (5) or tetrahydroisoquinolines. This activity was purified 500-fold from human platelets which are the main storage site for 5-hydroxytryptamine in man. This enzyme was identical to N⁵, N¹⁰-methylene tetrahydrofolate (N⁵,N¹⁰-methylene H₄folate) reductase by the following criteria: (i) co-purification, (ii) heat denaturation, (iii) pH response, (iv) molecular weight, (5) cofactor requirements. A mechanism involving the enzymatic generation of formaldehyde followed by adduct formation with a biogenic amine is proposed.     

DNA methylases of Hemophilus influenzae Rd. I. Purification and properties

Hemophilus influenzae strain Rd DNA contains small amounts of 5-methylcytosine (0.012%) and significantly greater amounts of N-6-methyladenine (0.34%). Four DNA adenine methylases have been identified and purified from crude extracts of H. influenzae Rd by means of phosphocellulose chromatography. Each of the four enzymes requires (S-adenosyl-l-methionine as a methyl group donor and each differs in its ability to methylate various DNAs in vitro. DNA methylase I is related to the genetically described modification-restriction system in H. influenzae Rd, and is presumably the modification enzyme for that system. DNA methylase II introduces approximately 130 methyl groups into a phage T7 DNA molecule and protects T7 DNA from the H. influenzae Rd restriction enzyme, endonuclease R, described by Smith and Wilcox (1970). These findings indicate that DNA methylase II is the modification enzyme corresponding to endonuclease R. A third modification-restriction system, which does not affect T7 DNA, has been detected in H. influenzae Rd. DNA methylase III is apparently the modification enzyme for this system. The biological function of DNA methylase IV remains unknown.     

Mucin of the rat submaxillary gland: influence of the animal's age and sex on the composition of its glucidic fraction

Mucin was isolated from submaxillary glands of young (30 and 45 days) and adult (100 days) male and female rats; it was purified first by fractional precipitation with ethanol and then by zone electrophoresis at pH 6.0 on Pevikon. The mucin contains N-acetylhexosamines (N-acetylglucosamine and N-acetylgalactosamine, 3:2), galactose and sialic acid. The total sugar content of the glycoprotein of adult rats is about twice that of the young animals, while the galactose content remains almost unchanged. Incorporation of [¹⁴C]glucosamine into the rat submaxillary gland mucin starts to increase considerably at the moment of sexual maturity and reaches a constant level (90 days); the increase in biosynthesis velocity is higher in males than in females.     

A rapid method for the purification of S-adenosylmethionine: Protein-carboxyl O-methyltransferase by affinity chromatography

A simple method to purify S-adenosylmethionine: protein-carboxyl O-methyltransferase (protein methylase II, EC 2.1.1.24) from calf brain has been developed using affinity chromatography. The product of the reaction, S-adenosyl-l-homocysteine, which is a competitive inhibitor of the enzyme, was covalently linked to Sepharose beads. This gel proved to be an effective binder for protein methylase II at pH 6.2 and allowed for specific removal of the enzyme by the addition of the methyl donor substrate, S-adenosyl-l-methionine to the elution buffer. One step using this affinity chromatography column resulted in 377-fold purification of the enzyme and 71% recovery of the activity. Subsequent Sephadex G-100 chromatography enabled the enzyme to be purified 3000-fold from the calf brain whole homogenate. The purified enzyme showed a number of protein methylase II activity peaks following preparative gel electrophoresis with one major enzyme peak.     

Isolation and comparative studies of mitochondrial F1-ATPase from Morris hepatoma and rat liver.

A simple method of isolating mitochondrial ATPase from rat liver and Morris hepatoma cell lines by chloroform extraction and chromatography on DEAE-Sephadex is described. This method is suitable even when small amounts of starting material with relatively low specific ATPase activity (in the case of hepatoma mitochondria and submitochondrial particles) are available. The isolated enzyme from both rat liver and hepatomas had a high specific activity, was similarly activated by bicarbonate and 2,4-dinitrophenol, and had a typical five-band pattern in sodium dodecyl sulfate electrophoresis. Prior to DEAE-Sephadex chromatography, an additional protein band which migrates between the δ and ? subunits in the tumor F₁-ATPase preparation was observed. The purified enzymes were cold labile and restored oxidative phosphorylation function of F₁-ATPase depleted submitochondrial particles prepared from rat liver. The ATPase activity of the isolated enzymes was inhibited by mitochondrial ATPase inhibitor protein. The apparent stoichiometry of the inhibitor protein to the purified ATPase was extrapolated to be 2:1.     

METHYLASES OF MYELIN BASIC PROTEIN AND HISTONE IN RAT BRAIN

—The two enzymes methylating myelin basic protein and histone were purified 170- and 250-fold respectively from the cell sap fraction of rat brain. These enzymes methylated only arginine residues of the two proteins. The enzyme activities were present in all organs tested. Testis has the highest, brain a moderate and liver the lowest activity. Most of the activities were present in the cell sap fraction in brain, liver and testis. Methylation of myelin basic protein and histone was examined in both the cell sap and solubilized nuclear fraction of rat brain during life span after birth. The myelin basic protein methylating activity in the cell sap fraction increased during myelination. Histone methylase from the nuclear fraction was highest at birth and dropped rapidly thereafter. The other activities remained unchanged. The natural occurrence of N^G-mono- and N^G,N^G-dimethylarginine residues in histones prepared from rabbit liver was demonstrated.     

Purification and properties of protein methylase II

Protein methylase II (S-adenosyl-methionine:protein-carboxyl methyltransferase) from calf thymus was purified approximately 2400-fold with a yield of 7% by incorporating the pH 5.1 treatment and QAE (triethylaminoethyl)-Sephadex column chromatography to the published purification steps (Kim and Paik (1970) J. Biol. Chem., 245, 1806). The enzyme is found stable at pH 10.2, but loses 50% of its activity in 60 min at pH 5. The enzyme activity disappeared in 8 m urea 2.5 m guanidine hydrochloride at pH 8.0. However, about 80% of the activity returned upon dialysis of the mixture. The highly purified enzyme is stable for at least 2 yr in the presence of 50% glycerol at pH 8.0 or in the form of lyophilized powder. Protein methylase II from different tissues exhibits different pI values, determined by isoelectrofocusing; 4.85 with the enzyme preparation isolated from calf thymus, 5.8 from calf spleen, and 5.08 from rat testis. Reinvestigation of the methanol-forming enzyme system from calf posterior pituitary gland by Axelrod and Daly [Science 150, 892 (1965)] indicated that this enzyme is identical with protein methylase II.     

Partial purification and characterisation of S-adenosylmethionine:protein-histidine N-methyltransferase from rabbit skeletal muscle

A new class of protein methylase (S-adenosylmethionine:protein-histidine N-methyltransferase) which methylates histidine residues of protein substrates using S-adenosylmethionine as the methyl donor has been partially purified from rabbit skeletal muscle, 22-fold with a yield of 56%. The enzyme activity was monitored using denatured myofibrils from young rabbit skeletal muscle as the methyl acceptor protein substrate. The enzyme was localised in the myofibrillar fraction and myofibrils isolated in pure form represented the enzyme-substrate complex. The enzyme was solubilised in 0.275 M KCl. The methylase showed no requirement for any metal ion and has a pH optimum of 8.0. It was shown to require a reducing agent like mercaptoethanol for its activity. It was also shown that cardiac and skeletal muscle forms of actins obtained from different species serve as the efficient methyl acceptor protein substrates. Since the enzyme was found to methylate specifically the histidine residues of actin we propose to designate this new methylase as protein methylase IV, to distinguish it from the already known protein methylases I, II and III.     

S-adenosylmethionine: Protein-carboxyl methyltransferase from erythrocyte

Protein methylase II (S-adenosylmethionine:protein—carboxyl methyltrans-ferase), which modifies free carboxyl residues of protein, was purified from both rat and human blood, and properties of the enzymes were studied. The pH optima for the reaction were dependent on the substrate proteins used; pH 7.0 was found with endogenous substrate, 6.1 with plasma, 6.5 with γ-globulin, and 6.0 with fibrinogen. The molecular weight of the enzymes from both rat and human erythrocytes were identical (25,000 daltons) determined by Sephadex G-75 chromatography. Partially purified enzyme from rat erythrocytes showed three peaks on electrofocusing column at pH 4.9, 5.5 and 6.0. The K_m values of the enzymes from rat and human erythrocytes showed 3.1 × 10^?6m and 1.92 × 10^?6m at pH 6.0, 1.96 × 10^?6m and 1.78 × 10^?6m at pH 7.2, respectively, for S-adenosyl-l-methionine. It is also found that S-adenosyl-l-homocysteine is a competitive inhibitor for protein methylase II with K_i value of 1.6 × 10^?6m.     

Purification and Properties of β-N-Acetylhexosaminidase from Mucor fragilis Grown in Bovine Blood

Mucor fragilis grown on bovine blood powder as the sole carbon source abundantly produced β-N-acetylhexosaminidase. The enzyme activity was several times higher than that of a culture obtained with glucose medium. The enzyme had two different molecular weight forms. The high-molecular-weight form had somewhat higher β-N-acetylgalactosaminidase activity than the lower-molecular-weight enzyme which had β-N-acetylgalactosaminidase activity equivalent to about 40% of its β-N-acetylglucosaminidase activity. Bovine blood seemed to induce both enzymes, but N-acetylamino sugars specifically induced the low-molecular-weight form. N-Acetylgalactosamine had an especially marked effect on activity. The low-molecular-weight form of enzyme was purified from the culture filtrate by fractionation with ammonium sulfate and various column chromatographies. The purified enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The optimum pH was 4.0 to 5.0 for β-N-acetylglucosaminidase activity and 5.5 to 6.5 for β-N-acetylgalactosaminidase activity. The enzyme hydrolyzed natural substrates such as di-N-acetylchitobiose, tri-N-acetylchitotriose, and a glycopeptide obtained by modification of fetuin.     

A β-citryl-L-glutamate-hydrolysing enzyme in rat testes

An enzyme responsible for the deacylation of β-citryl-L-glutamate to citrate and glutamate has been characterized in rat testis. The enzyme required manganese ion for full activity and was strongly inhibited by nucleotides such as ATP or GTP. The activity was localized in the particulate fractions. The enzyme favored $\text{N-}\text{formyl-}\text{L}\text{-glutamate}\text{> β-}\text{citryl-}\text{L}\text{-glutamate}\text{> β-}\text{citryl-}\text{L}\text{-glutamine}$ in a decreasing order. The amidohydrolyase activity was highest in the testis and lung, a moderate activity was detected in heart, kidney and intestine, and low in brain, thymus, stomach, skeletal muscle, spleen and liver. These findings suggest that the amidohydrolase is different from any of amidohydrolases reported so far, amidohydrolase I (EC 3.5.1.14), II (EC 3.5.1.15), III, N-acetyl-lysine deacylase (EC 3.5.1.17) and N-acetyl-β-alanine deacetylase (EC 3.5.1.21), and various peptidases.     

NG-Methylarginines: Biosynthesis,biochemical function and metabolism

Summary N^G-Methylarginines (N^G-monomethylarginine, N^G, N^G-dimethylarginine and N^G, N^G-dimethylarginine) occur widely in nature in either proteinbound or in free states. They are posttranslationally synthesized by a group of enzymes called protein methylase I with S-adenosyl-L-methionine as the methyl donor. The enzymes are highly specific not only towards arginine residues but also towards the protein species. Since transmethylation reaction is energy-dependent in the form of S-adenosyl-L-methionine and is catalyzed a group of highly specific enzymes, it is quite logical to assume that the enzymatic methylation of protein-bound arginine residues play an important role in the regulation of the function and/or metabolism of the protein. When determined with histones asin vitro substrates, protein methylase I activity parallels closely the degree of cell proliferation, and the myelin basic protein (MBP)-specific protein methylase I activity decreases drastically in dysmyelinating mutant mouse brain during myelinating period, suggesting an important role played in the formation and/or maintenance of myelin. When the methylated proteins are degraded by intracellular proteolytic enzymes, free N^G-methylarginines are generated. Some of these free N^G-methylarginines, particularly N^G-monomethylarginine, are extensively metabolized by decarboxylation, hydrolysis, transfer of methylamidine and deimination reaction. Recent experiment demonstrates that some of the N^G-methylarginines may be involved in the neutralization of activity of nitric oxide (NO) which has attracted a great deal of attention as vascular smooth muscle relaxation factor.     

Comparative study of the tRNA-methylases of normal and tumor tissues. I. Spectrum of renal and carcinoma RA methylases

A comparative study of rat kidney and carcinoma RA tRNA-methylase activity has been carried out using partially purified enzyme preparations and total E. coli tRNA. Also the nuclease activity of the methylase preparations from kidney and carcinoma was compared. It was established that the methylase activity in carcinoma preparations is higher, whereas the nuclease activity is lower in comparison to the enzyme preparations from liver. No formation of some specific methylated compounds could be established in the case of carcinoma. It was established that the relative contribution of individual methylases to the elevated level of total tRNA-methylase activity in carcinoma is different. Maximal enhancement of activity was established for the methylase forming m5U, whereas the activity of the enzymes, transfering the methyl group to the fifth position of C is practically equal in kidney and carcinoma tissues. Experimental results and theoretical evaluation of the hypotheses suggested to explain the higher methylase activity in tumor tissues allowed to reject some of them.