Biodistribution of radiolabeled adenosylcobalamin in patients diagnosed with various malignancies.

OBJECTIVE: To study the biodistribution of a vitamin B12 analog, indium In 111-labeled diethylenetriaminepentaacetate adenosylcobalamin (In 111 DAC), in patients recently diagnosed as having primary or recurrent malignancy. PATIENTS AND METHODS: Thirty patients (14 women and 16 men) with radiographically or clinically diagnosed breast, lung, colon, sarcomatous, thyroid, or central nervous system malignancies were studied prior to definitive surgery or biopsy. A maximum of 650 microCi (2.2 microg) of In 111 DAC was administered intravenously. Vitamin B12 and folate levels were determined prior to injection. Serum clearance and urinary and stool excretion of the tracer were measured. Images were routinely obtained at 0.5, 3 to 5, and 20 to 24 hours after injection. Biodistribution of In 111 DAC was determined by computer analysis of regions of interest. RESULTS: Serum T1/2 clearance was 7 minutes. Average urinary and stool excretion of the injected dose over 24 hours was 26.1% and 0.4%, respectively. The greatest focal uptake of In 111 DAC occurred in the liver and spleen, followed by the nasal cavity and salivary and lacrimal glands. The average tumor uptake of the injected dose was 2% at 30 minutes and 1.5% at 24 hours. High-grade primary and metastatic breast, lung, colon, thyroid, and sarcomatous malignancies were all imaged at 3 to 5 hours after injection. Central nervous system tumors and advanced metastatic prostate cancer were best identified at 24 hours. Mammographically occult, palpable, and nonpalpable breast cancers were delineated by In 111 DAC. Low-grade malignancies as well as early skeletal metastatic disease were not effectively imaged by the vitamin B12 tracer. Patients with elevated baseline vitamin B12 or those concurrently taking corticosteroids appeared to have optimal visualization of their malignancies. CONCLUSION: Vitamin B12 may be a useful vehicle for delivering diagnostic and therapeutic agents to various malignancies. Further evaluation of cobalamin analogs and their interaction with transport proteins and cellular receptors within malignant tissue and infection is warranted.

Synthesis and characterization of nido-carborane-cobalamin conjugates.

Three vitamin B12 (cyanocobalamin) conjugates bearing one nido-carborane molecule or two nido-carborane molecules linked to the propionamide side chains via a four carbon linker have been synthesized. Reaction of o-carboranoylchloride with 1,4-diaminobutane in pyridine produced nido-carboranoyl(4-amidobutyl)amine, which was linked to the b- and d-monocarboxylic acids and the b,d-dicarboxylic acid of cyanocobalamin. Mass spectrometry analysis as well as 11B nuclear magnetic resonance demonstrated that during the reaction of o-carboranonylchloride with diaminobutane one of the boron atoms was eliminated. In vitro biological activity of the cyanocobalamin-nido-carborane conjugates was assessed by the unsaturated vitamin B12 binding capacity assay. When compared with 57Co cyanocobalamin, the biological activity of cyanocobalamin-b-nido-carborane, cyanocobalamin-d-nido-carborane, and cyanocobalamin-b-d-bis-nido-carborane conjugates were 92.93%, 35.75%, and 37.02%, respectively. These findings suggest that the 10B cobalamin conjugates might be useful agents in treating malignant tumors via neutron capture therapy.

Tumor imaging via indium 111-labeled DTPA-adenosylcobalamin.

Vitamin B12 is essential for life. Lack of it results in pernicious anemia and death. Conversely, the demand for vitamin B12 increases in rapidly dividing tumors. This is secondary to the direct involvement of vitamin B12 in mitochondrial metabolism as well as its indirect role in the production of thymidylate and S-adenosylmethionine. The latter 2 substances are needed for DNA synthesis and cellular methylation reactions, respectively. Novel radiolabeling of adenosylcobalamin has proven to be useful in the imaging of transplanted and spontaneous tumors in animals. Herein, we describe what we believe to be the first human to have imaging with conventional gamma cameras of vitamin B12 metabolism in a breast tumor.

Transcobalamin II receptor imaging via radiolabeled diethylene-triaminepentaacetate cobalamin analogs.

UNLABELLED: Rapidly dividing cells up-regulate the number of transcobalamin II receptors during DNA replication. We have developed diethylene-triaminepentaacetate (DTPA) cobalamin analogs for the purpose of imaging transcobalamin II receptors in malignant and nonmalignant tissue. METHODS: Methyl-, adenosyl- and cyanocobalamin-b-(4-aminobutyl)-amide-DTPA analogs were synthesized. In vitro binding of the analogs to the transcobalamin proteins was assessed by the unsaturated vitamin B12 binding capacity assay and compared to DTPA and cyanocobalamin. The biodistribution of the 111In-DTPA cobalamin analogs was measured at 24 hr after injection into sarcoma-bearing mice and non-tumor-bearing mice and pigs. RESULTS: Methyl-, adenosyl- and cyanocobalamin-b-(4-aminobutyl)-amide-DTPA analogs and DTPA were 94.0%, 90.4%, 66.4%, and 3.6%, respectively, as efficient in binding to the transcobalamin proteins when compared to cyanocobalamin. At 24 hr after administration, the cobalamin analogs had 5-17 times and 20-29 times, respectively, the amount of uptake within the resected tissue samples and transplanted sarcomas when compared to 111In-DTPA. CONCLUSION: The radiolabeled DTPA cobalamin analogs are biologically active. Preliminary animal studies suggest that the analogs could be effective in vivo transcobalamin II receptor imaging agents.

Mechanism-based inhibition of ribonucleoside diphosphate reductase from Corynebacterium nephridii by 2'-C-methyladenosine diphosphate.

The interaction of the adenosylcobalamin-dependent ribonucleoside diphosphate reductase of Corynebacterium nephridii with 2'-C-methyladenosine diphosphate (2'-C-methylADP) has been investigated in more detail [Ong, S. P., McFarlan, S. C., & Hogenkamp, H. P. C. (1993) Biochemistry 32, 11397-11404]. This nucleotide analog partitioned between normal reduction to 2'-deoxy-2'-C-methyladenosine diphosphate and decomposition to adenine, 2-methylene-3(2H)-4-methylfuranone, and presumably pyrophosphate. Reaction of the reduced enzyme with 2'-C-methylADP caused the development of a chromophore at 318 nm that is characteristic of the modification of the enzyme by the furanone [Harris, G., Ator, M., & Stubbe, J. (1984) Biochemistry 23, 5214-5225]. Incubation of [5'-3H2]-2'-C-methylADP with reduced reductase resulted in the covalent incorporation of the radiolabel into the protein and into aquocobalamin. A similar incubation of the enzyme, the labeled nucleotide analog, and dithiothreitol resulted in the formation of three radioactive hydrophilic compounds. Mass spectroscopic analysis of one of these compounds showed the presence of 2-methylene-3(2H)-4-methylfuranone. 2'-Deoxy-2'-C-methylADP is a very effective promoter of the tritium exchange reaction between [5'-3H2]adenosylcobalamin and the solvent, confirming that the exchange reaction is an integral part of the overall reduction. All these observations are consistent with the proposal that 2'-C-methylADP serves as a substrate and a mechanism-based inhibitor of the ribonucleotide reductase of C. nephridii, indicating that the enzyme is able to catalyze the conversion of the nucleotide analog to a 2'-deoxy-2'-C-methyl-3'-ketonucleotide that can collapse to the reactive 2-methylene-3(2H)-4-methylfuranone. Surprisingly, 2'-C-methylADP did not serve as either a substrate or an inhibitor of the ribonucleoside diphosphate reductase of Escherichia coli.

Purification and partial characterization of a putative thymidylate synthase from Methanobacterium thermoautotrophicum.

A protein catalyzing the tritium exchange of [5-3H]deoxyuridine monophosphate ([5-3H]dUMP) for solvent protons and the dehalogenation of 5-bromo-deoxyuridine monophosphate (Br-dUMP) has been isolated from the methanogenic archaea Methanobacterium thermoautotrophicum. These two activities are well-established side reactions of thymidylate synthase and do not require cofactors. Sodium dodecylsulfate/polyacrylamide gel electrophoresis of the purified enzyme showed a single band with a molecular mass of 27 kDa. The suggested molecular mass of the native protein calculated from sedimentation equilibrium experiments was 33.5 kDa, indicating that the enzyme is a monomer. The pH optima were 9.0 and 7.0 for the exchange reaction and the dehalogenation, respectively. The effects of temperature, salt, reducing agent and inhibitors were determined. The apparent Km for the tritium exchange from [5-3H]dUMP was 7 microM and for the dehalogenation of Br-dUMP was 14 microM. However, thus far, the conditions for dTMP synthesis from dUMP have not yet been established. Incubation of the enzyme with dUMP, tetrahydromethanopterin, a folate analog present in methanogens, and formaldehyde did not yield dTMP. The first 30 amino acids of the amino terminus have been sequenced. However, there is no similarity with any of the thymidylate synthases. Surprisingly, the protein from M. thermoautotrophicum appears to be related to chitin synthases from several organisms.

2'-C-methyladenosine and 2'-C-methyluridine 5'-diphosphates are mechanism-based inhibitors of ribonucleoside diphosphate reductase from Corynebacterium nephridii.

The interaction of the adenyosylcobalamin-dependent ribonucleoside diphosphate reductase of Cornyebacterium nephridii with 2'-C-methyladenosine 5'-diphosphate (2'-MeADP) and 2'-C-methyluridine 5'-diphosphate (2'-MeUDP) has been investigated. The nucleotide analogs are converted to adenine and uracil, respectively, suggesting that they may be mechanism-based inhibitors. In addition, both analogs generate nucleotides with properties expected for the 2'-deoxy-2'-C-methylnucleotides. The nucleoside obtained after enzymatic dephosphorylation of the product formed from 2'-MeADP has been identified as 2'-deoxy-2'-C-methyladenosine by 1H NMR and mass spectroscopies. Adenine is the major product derived from 2'-MeADP, indicating that the degradation pathway predominates. During the reaction, the carbon-cobalt bond of the coenzyme is cleaved irreversibly to yield 5'-deoxyadenosine and cob(II)alamin. 2'-MeADP is a potent competitive inhibitor of the reduction of the purine nucleotides ADP and GDP, while 2'-MeUDP competitively inhibits the reduction of the pyrimidine nucleotides UDP and CDP. 2'-MeADP is a very effective promoter of the tritium exchange reaction between [5'-3H2]adenosylcobalamin and the solvent, indicating that the exchange reaction is an integral part of the overall reduction. All these observations are consistent with the reaction mechanism proposed by Stubbe and co-workers [Harris, G., Ashley, G. W., Robins, M. J., Tolman, R. L., & Stubbe, J. (1987) Biochemistry 26, 1895-1902 (1987); Stubbe, J. (1990) J. Biol. Chem. 265, 5329-5332] in which they suggest that the partitioning between reduction and inactivation occurs at the level of the 2'-deoxy-3'-ketoribonucleotide intermediate.

Ribonucleotide diphosphate reductase from human metastatic melanoma.

Cell free extracts from metastases of human melanoma contain a highly active ribonucleoside diphosphate reductase (RR) which uses guanosine diphosphate (GDP) as substrate and deoxythymidine triphosphate (dTTP) as effector. No activity could be detected in these extracts when cytidine diphosphate (CDP) was used as the substrate with adenosine triphosphate (ATP) as effector. The activity of this RR required the presence of either magnesium or calcium: there was a time lag before cell extracts from melanotic melanoma metastases showed full activities, but extracts from amelanotic tumors showed normal kinetics in the presence of these divalent cations. By contrast to other RRs, the activities in cell-free extracts could not be inhibited by hydroxyurea (10(-2) M). Even though an activity related free radical could be detected by electron paramagnetic resonance spectroscopy at 77 degrees K, the signal could not be quenched by 10(-2) M of this free radical trap. However, after ammonium sulphate fractionation, enzyme activity from melanotic melanoma was inhibited by 66% in 1 h. In the presence of substrates, effector and cofactors, the radical signal at g = 2.009 was also quenched by 60%; in the absence of substrate, effectors and cofactors, this signal was unaffected. These results indicate that two different free radicals must be present on melanoma RR. One is present in the resting enzyme, and the other is used during catalytic activity. The thiolate-active site of RR from melanoma was inhibited by the new nitrosourea anti-tumour drug fotemustine (IC50 = 10(-4) M as determined from a dose-response study).(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of 3'-C-methyladenosine and 3'-C-methyluridine diphosphates and their interaction with the ribonucleoside diphosphate reductase from Corynebacterium nephridii.

Two nucleoside diphosphate analogs, 3'-C-methyl-ADP and 3'-C-methyl-UDP, have been tested as substrate and/or allosteric effectors using the adenosylcobalamin-dependent ribonucleoside diphosphate reductase of Corynebacterium nephridii. Neither analog was a substrate for the reductase. However, they did function as allosteric effectors and as inhibitors of the reduction of ADP and UDP, respectively. The nucleotide analogs did not stimulate the hydrogen exchange reaction between [5'-3H2]adenosylcobalamin and the solvent, indicating that the cleavage of the 3'-carbon-hydrogen bond is a prerequisite for the exchange reaction. A reinvestigation of the requirements for the exchange reaction revealed that the deoxyribonucleoside diphosphate products are very effective promoters of this reaction. Indeed, the deoxyribonucleoside diphosphates were found to be more effective in promoting the exchange reaction than the ribonucleoside diphosphate substrates. In contrast, the deoxyribonucleoside triphosphate effectors, dATP, dUTP, and dTTP, were only marginally effective as promoters of this reaction.

The purification, characterization, and primary structure of a small redox protein from Methanobacterium thermoautotrophicum, an archaebacterium.

A small redox-active protein has been purified to homogeneity from cell-free extracts of the strictly anaerobic thermophilic methanogen, Methanobacterium thermoautotrophicum (strain Marburg). The purification consisted of streptomycin sulfate and acid treatments and three chromatographic steps using Sephadex G-75, Mono Q HR 10/10, and Superose 12 HR 10/30 columns. When these procedures were carried out under strictly anaerobic conditions, approximately 3 mg of this protein could be isolated from 45 g of wet cell paste. Like the thioredoxins and glutaredoxins, it is a small acidic protein (pI = 4.2) consisting of 83 amino acids (M(r) = 9136). In the presence of dithiothreitol or dihydrolipoate, the protein serves as a hydrogen donor for the ribonucleotide reductase from Escherichia coli, and it catalyzes the reduction of insulin. However, it does not interact with the thioredoxin reductases from E. coli or Corynebacterium nephridii and does not function as a hydrogen donor for the ribonucleotide reductase of C. nephridii. The amino acid sequences determined by automated Edman degradation of the 14C-carboxymethylated protein and of peptides derived from trypsin and chymotrypsin digestions show a redox-active site -Cys-Pro-Tyr-Cys-, typical of the glutaredoxins. Its amino acid sequence shows moderate identity with the known glutaredoxins (E. coli, yeast, rabbit bone marrow, calf thymus, and pig liver) when the proteins are aligned at the active site. The secondary structure of the glutaredoxin-like protein predicted by the Chou-Fasman procedure shows that it is similar to the known glutaredoxins. However, surprisingly, the protein does not function as a glutathione-disulfide oxidoreductase in the presence of glutathione and glutathione reductase. This glutaredoxin-like protein may be a component of a ribonucleotide-reducing system distinct from the previously described systems utilizing thioredoxin or glutaredoxin.

A possible new class of ribonucleotide reductase from Methanobacterium thermoautotrophicum.

The ribonucleotide reductase from the strictly anaerobic methanogen Methanobacterium thermoautotrophicum has been partially purified by ion-exchange and gel-filtration chromatography. Its molecular weight is estimated to be 100,000 by the latter step. Unlike all previously studied ribonucleotide reductases, the enzyme does not employ dithiol compounds such as dithiothreitol as artificial electron donors in in vitro assays. Inhibition of the enzyme by S-adenosylmethionine, oxygen, and azide further distinguishes it from the Escherichia coli anaerobic enzyme, the iron- and manganese-containing, and the adenosylcobalamin-dependent enzymes. Our preliminary results suggest that this enzyme has an activation mechanism different from the known classes of ribonucleotide reductases.

Reductive formation of carbon monoxide from CCl4 and FREONs 11, 12, and 13 catalyzed by corrinoids.

In an earlier publication, we reported that corrinoids catalyze the sequential reduction of CCl4 to CHCl3, CH2Cl2, CH3Cl, and CH4 with titanium(III) citrate as electron donor [Krone, U. E., Thauer, R. K., & Hogenkamp, H. P. C. (1989) Biochemistry 28, 4908-4914]. However, the recovery of these products was less than 50%, indicating that other products were formed. We now report that, under the same experimental conditions, CCl4 is also converted to carbon monoxide. These studies were extended to include FREONs 11, 12, 13, and 14. Corrinoids were found to catalyze the reduction of CFCl3, CF2Cl2, and CF3Cl to CO and, in the case of CFCl3, to a lesser extent, to formate. CF4 was not reduced. The rate of CO and formate formation paralleled that of fluoride release. Both rates decreased in the series CFCl3, CF2Cl2, CCl4, and CF3Cl. The reduction of CFCl3 gave, in addition to CO and formate, CHFCl2, CH2FCl, CH3F, C2F2Cl2, and C2F2Cl4. The product pattern indicates that the corrinoid-mediated reduction of halogenated C1-hydrocarbons involves the intermediacy of dihalocarbenes, which may be a reason why these compounds are highly toxic for anaerobic bacteria.

Coenzyme F430 as a possible catalyst for the reductive dehalogenation of chlorinated C1 hydrocarbons in methanogenic bacteria.

Corrinoids, such as aquocobalamin, methylcobalamin, and (cyanoaquo)cobinamide, catalyze the reductive dehalogenation of CCl4 with titanium(III) citrate as the electron donor [Krone et al. (1989) Biochemistry 28, 4908-4914]. We report here that this reaction is also effectively mediated by the nickel-containing porphinoid, coenzyme F430, found in methanogenic bacteria. Chloroform, methylene chloride, methyl chloride, and methane were detected as intermediates and products. Ethane was formed in trace amounts, and several as yet unidentified nonvolatile compounds were also generated. The rate of dehalogenation decreased in the series of CCl4, CHCl3, and CH2Cl2. With coenzyme F430 as the catalyst, the reduction of CH3Cl to CH4 proceeded more than 50 times faster than with aquocobalamin. Cell suspensions of Methanosarcina barkeri were found to catalyze the reductive dehalogenation of CCl4 with CO as the electron donor (E'0 = -0.524 V). Methylene chloride was the main end product. The kinetics of CHCl3 and CH2Cl2 formation from CCl4 were similar to those with coenzyme F430 or aquocobalamin as catalysts and titanium(III) citrate as the reductant.

Purification, characterization and revised amino acid sequence of a second thioredoxin from Corynebacterium nephridii.

A second thioredoxin, distinct from the one reported by Meng and Hogenkamp in 1981 (J. Biol. Chem. 256, 9174-9182), has been purified to homogeneity from an Escherichia coli strain containing a plasmid encoding a Corynebacterium nephridii thioredoxin. Thioredoxin genes from C. nephridii were cloned into the plasmid pUC13 and transformants were identified by complementation of a thioredoxin negative (trxA-) E. coli strain. The abilities of the transformants to support the growth of several phages suggested that more than one thioredoxin had been expressed [Lim et al. (1987) J. Biol. Chem. 262, 12114-12119]. In this paper we present the purification and characterization of one of these thioredoxins. The new thioredoxin from C. nephridii, designated thioredoxin C-2, is a heat-stable protein containing three cysteine residues/molecule. It serves as a substrate for C. nephridii thioredoxin reductase and E. coli and Lactobacillus leichmannii ribonucleotide reductases. Thioredoxin C-2 catalyzes the reduction of insulin disulfides by dithiothreitol or by NADPH and thioredoxin reductase and is a hydrogen donor for the methionine sulfoxide reductase of E. coli. Spinach malate dehydrogenase (NADP+) and phosphoribulokinase are activated by this thioredoxin while glyceraldehyde-3-phosphate dehydrogenase (NADP+) is not. Like the thioredoxin first isolated from C. nephridii, this new thioredoxin is not a reducing substrate for the C. nephridii ribonucleotide reductase. The complete primary sequence of this second thioredoxin has been determined. The amino acid sequence shows a high degree of similarity with other thioredoxins. Surprisingly, in contrast to the other sequences, this new thioredoxin contains the tetrapeptide -Cys-Ala-Pro-Cys- at the active site. With the exception of the T4 thioredoxin, this is the first example of a thioredoxin that does not have the sequence -Cys-Gly-Pro-Cys-. Our results suggest that, like plant cells, bacterial cells may utilize more than one thioredoxin.

Inhibitory effect of adenosine dialdehyde on in situ murine neuroblastoma growth.

The effect of adenosine dialdehyde (AD) on in situ tumor growth and host survival was evaluated in the C1300 murine neuroblastoma tumor model prepared by implantation of murine neuroblastoma cells into A/J mice. AD was administered s.c. by one of the following treatment regimens: regimen A, single daily dose for 5 days; regimen B, minipump infusion for 7 days; regimen C, minipump infusion for 14 days; regimen D, minipump infusion for two 7-day periods interspersed by a 7-day drug free interval. AD doses of 1.5 to 2.5 mg/kg/day infused over a 7-day period (regimen B) significantly increased the mean life span of tumor bearing mice from 20.9 +/- 1.2 days (mean +/- 2 SEM) in diluent treated controls to 35.3 +/- 2.1 days in AD treated animals (mean increase +/- 2 SEM: 69 +/- 10%; P less than 0.0001). This treatment regimen also produced a 56 +/- 13% decrease in tumor diameter (P less than 0.0001). Administration of AD for two 7-day infusion periods, interspersed by a 7-day drug free interval (regimen D), increased mean life span 80% (controls, 21.3 +/- 4.4 days; AD treated 38.4 +/- 5.6 days; P less than 0.0005). Hematopoietic toxicity was not observed when doses between 2 and 3 mg/kg/day of AD were infused for 7 days (regimen B). These data suggest that steady state infusions of AD can significantly suppress murine neuroblastoma tumor growth with little systemic toxicity. In contrast, single daily injections of AD were ineffective and toxic to the tumor bearing host.

Cloning, expression, and nucleotide sequence of a gene encoding a second thioredoxin from Corynebacterium nephridii.

A gene encoding thioredoxin in Corynebacterium nephridii was cloned in Escherichia coli by complementation of a thioredoxin mutant. Transformants that appeared to complement were analyzed for the presence of thioredoxin by the coupled assay using methionine sulfoxide reductase. Of 18 transformants, four contained high levels of thioredoxin activity. Transformants containing plasmids pLCN2 and pLCN4 were unable to support replication of T7 phage, in spite of their thioredoxin activities, and were studied in more detail. The plasmid pLCN2 contains a 1.85-kilobase Sau3AI insert, whereas pLCN4 contains a 10-kilobase TaqI insert. These plasmids complement all phenotypes of a thioredoxin-deficient strain except for replication of T7 phage. The nucleotide sequence of a 620-base pair HinfI fragment encoding thioredoxin derived from either plasmid indicated that the protein derived from this DNA is different from the thioredoxin of C. nephridii previously reported (Meng, M., and Hogenkamp, H.P.C. (1981) J. Biol. Chem. 256, 9174-9182). The amino acid sequence predicted from the nucleotide sequence shows a high degree of homology with other procaryotic thioredoxins. However, the new thioredoxin contains the tetrapeptide -Cys-Ala-Pro-Cys- at the active site and a third half-cystine residue in the carboxyl-terminal domain of the protein. The molecular weight of this thioredoxin, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is smaller than that estimated from the DNA sequence, suggesting that processing may have occurred.

Cytotoxic action of adenosine nucleoside and dialdehyde analogues on murine neuroblastoma in tissue culture: structure-activity relationships.

A series of purine nucleoside and dialdehyde analogues was studied to determine their potency as inhibitors of C-1300 murine neuroblastoma cell growth in tissue culture. Tumor cells were incubated with each analogue for 72 h, and the number of viable cells was determined at 24, 48, and 72 h. Dose-response curves were generated (concentration range, 10(-8) to 10(-3) M), and the drug concentration producing 50% inhibition of cell growth was calculated for each analogue. The 50% inhibitory concentrations, in ascending order of potency, were as follows: adenosine (inactive); S-adenosylhomocysteine (inactive); methylfuryladenine (5.6 X 10(-1)M); adenosine 5'-carboxylic acid (2.0 X 10(-1)M); 5'-chloro, 5'-deoxy-ara-adenosine (3.0 X 10(-2)M); sinefungin (1.7 X 10(-3) M); 5'-deoxyadenosine (2.2 X 10(-4)M); 5'-chloro, 5'-deoxyadenosine (2.1 X 10(-4)M); 3',5'-dichloro, 2',3',5'-trideoxyadenosine (1.3 X 10(-4)M); 3-deazaadenosine (5.6 X 10(-5)M); and adenosine dialdehyde (1.5 X 10(-6)M). Oxidation of the pentose to a dialdehyde increased, whereas reduction of the dialdehyde or substitution of adenine with either hypoxanthine, guanine, uracil, or cytosine decreased the inhibitory potency. The analogues 4',5'-anhydroadenosine dialdehyde and 5'-deoxyadenosine dialdehyde, which cannot be phosphorylated at the 5' position, had 50% inhibitory concentrations of 9.1 X 10(-6) and 7.6 X 10(-6)M, respectively. These data suggest that the inhibitory action and potency of nucleoside dialdehydes on neuroblastoma growth are independent of their capacity to undergo a kinase-mediated phosphorylation at the 5' position.

Reaction of alkylcobalamins with thiols.

Carbon-13 NMR spectroscopy and phosphorus-31 NMR spectroscopy have been used to study the reaction of several alkylcobalamins with 2-mercaptoethanol. At alkaline pH, when the thiol is deprotonated, the alkyl-transfer reactions involve a nucleophilic attack of the thiolate anion on the Co-methylene carbon of the cobalamins, yielding alkyl thioethers and cob(II)alamin. In these nucleophilic displacement reactions cob(I)alamin is presumably formed as an intermediate. The higher alkylcobalamins react more slowly than methylcobalamin. The lower reactivity of ethyl- and propylcobalamin is probably the basis of the inhibition of the corrinoid-dependent methyl-transfer systems by propyl iodide. The transfer of the upper nucleoside ligand of adenosylcobalamin to 2-mercaptoethanol is a very slow process; S-adenosyl-mercaptoethanol and cob(II)alamin are the final products of the reaction. The dealkylation of (carboxymethyl)cobalamin is a much more facile reaction. At alkaline pH S-(carboxymethyl)mercaptoethanol and cob(II)alamin are produced, while at pH values below 8 the carbon-cobalt bond is cleaved reductively to acetate and cob(II)alamin. The reductive cleavage of the carbon-cobalt bond of (carboxymethyl)cobalamin by 2-mercaptoethanol is extremely fast when the cobalamin is in the "base-off" form. Because we have been unable to detect trans coordination of 2-mercaptoethanol, we favor a mechanism that involves a hydride attack on the Co-methylene carbon of (carboxymethyl)cobalamin rather than a trans attack of the thiol on the cobalt atom.

Effect of adenosine analogues on protein carboxylmethyltransferase, S-adenosylhomocysteine hydrolase, and ribonucleotide reductase activity in murine neuroblastoma cells.

The noncompetitive S-adenosylhomocysteine (AdoHcy) hydrolase antagonist adenosine dialdehyde (AD) has been shown to suppress the growth of cultured C-1300 murine neuroblastoma (MNB) cells. The enzymatic sites at which AD and other nucleoside analogues exert their cytotoxic effects have been postulated to include protein carboxylmethyltransferase (PCM), AdoHcy hydrolase, and ribonucleotide reductase. AD (10(-5) M) increased PCM activity 350% in suspensions prepared from disrupted cells after 72 h of drug exposure; in contrast, 3-deazaadenosine (10(-4) M) increased PCM activity 57%, whereas AdoHcy and sinefungin had no effect. When intact MNB cells were incubated with AD for varying time periods up to 72 h and then pulse labeled with the S-adenosylmethionine precursor L-[3H]-methionine, AD (10(-8) to 5 X 10(-6) M) produced a concentration-dependent inhibition of protein carboxylmethylation which persisted for up to 6 h. Following extended periods of AD treatment (48 to 72 h), AD (10(-6) to 10(-5) M) produced a 250% increment in protein carboxylmethylation, similar in magnitude to that observed in disrupted cell preparations. This increase in carboxylmethylation was observed at timepoints when AdoHcy hydrolase activity remained suppressed. The inhibitory effect of AD on AdoHcy hydrolase activity was maximal within 4 h and still apparent after 72 h of incubation. In contrast, AD treatment had no effect on the ribonucleotide reductase activity of MNB cells. These data suggest that the cytotoxic effect of AD on MNB cells results directly from its inhibition of AdoHcy hydrolase activity and indirectly through its suppression of methyltransferase enzyme systems. The potential linkage between the observed long-term elevations in PCM activity and AD-induced cytotoxicity remains to be defined.

Methyl transfer from methylcobalamin to thiols. A reinvestigation.

The methyl transfer from methylcobalamin to thiols has been reinvestigated. By use of methylcobalamin selectively enriched with 13C in the methyl moiety, the methyl transfer to thiols was followed by 13C NMR. The methyl transfer occurs in aqueous mildly alkaline (pH 8-12) solution, even in the complete absence of oxygen. 31P NMR and EPR studies demonstrate that cob(II)alamin is the final corrinoid product. However, the pH dependence of the methyl-transfer reaction from methylcobalamin to beta-mercaptoethanol is consistent only with a nucleophilic displacement of the methyl group by a thiolate anion, resulting in the heterolytic cleavage of the carbon-cobalt bond. Difference visible spectroscopic measurements of the reaction mixture suggest that cob(I)alamin is formed as an intermediate.