Critical evaluation of the selective tritiation method of determining C-terminal amino acids and its application to luteinizing hormone

Holcomb, George; James, Sally A.; Ward, Darrell N.

doi:10.1021/bi00844a007

Cited by 71 publications

(22 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Amino acids released from the C-terminus of peptides by the action of carboxypeptidase A and B or by carboxypeptidase C (16) were identified on the analyser. The C-terminal residue was in some cases confirmed by the tritiation method (17). Carboxymethylation of the protein (0.1 mM) was carried out with 200 mM l4C]iodoacetate in 0.2 M citrate-phosphate buffer, pH 6.3, at 00 over a period of 22-24 hr.…”

Section: Methodsmentioning

confidence: 99%

Amino-Acid Sequence of Dihydrofolate Reductase from a Methotrexate-resistant Mutant of Streptococcus faecium and Identification of Methionine Residues at the Inhibitor Binding Site

Gleisner

Peterson

Blakley

1974

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The amino-acid sequence of dihydrofolate reductase (7,8-dihydrofolate: NADP + oxidoreductase, EC 1.5.1.4) from S. faecium var Durans strain A is reported, and methionine residues 28 and 50 are shown to be protected by the inhibitor aminopterin from carboxymethylation by iodoacetate which occurs in absence of the inhibitor. Comparison of the sequence with that of the Escherichia coli reductase reveals two domains of considerable homology, one (the N-terminal region) presumably concerned with dihydrofolate and inhibitor binding and the other with dinucleotide binding. No significant sequence homology was found between larger dehydrogenases and the dihydrofolate reductases, which must, therefore, have evolved from a different ancestral protein.Dihydrofolate reductase (7,8-dihydrofolate: NADP+ oxidoreductase, EC 1.5.1.4) is a monomeric, NADPH-linked enzyme and, as isolated from various bacterial and mammalian sources, has a molecular weight in the range of 15,000 to 30,000 (1). The small size is advantageous for structural studies and these should elucidate the mode of binding of inhibitors to the catalytic site. This is of special interest because of the clinical use of such inhibitors; thus, methotrexate has been used in the treatment of neoplastic disease, trimnethoprim for bacterial infections, and pyrimethamine in the treatment of malaria. Comparative structural studies of this enzyme are also of interest because some inhibitors (such as trimethoprim) discriminate efficiently between the bacterial and mammalian reductases (2), and also because bacteria develop resistance which in some cases is due to selectively increased production of one isozyme which is not the predominant species in the sensitive parent strain (3).In this paper we report the complete primary sequence of one of the two dihydrofolate reductases present in a methotrexate-resistant mutant of Streptococcus faecium var Durans (4, 5). This mutant, strain A, was isolated (6) and kindly provided by Dr. D. J. Hutchison of the Sloan Kettering Institute. The enzyme investigated in this study was previously designated by us as the "mutant" enzyme, since it is the predominant form in the mutant organism (5), but it might more properly be called isozyme II since it has less mobility than isozyme I on polyacrylamide gel electrophoresis.Isozyme II, like mammalian dihydrofolate reductases, is capable of reducing folate as well as dihydrofolate although folate reduction is relatively slow. The enzyme predominant in the wild strain is also present in increased levels in the mutant and is designated "wild" enzyme or isozyme I. Like many other bacterial dihydrofolate reductases, it reduces dihydrofolate but not folate. Resistance of strain A to methotrexate is attributed to increased capacity of the organism to reduce the folate supplied in the synthetic medium because of the vastly increased level of isozyme II over that in the sensitive, wild strain.We also report on the position, in the primary sequence of isozyme II, of methionine residues. These re...

show abstract

Section: Methodsmentioning

confidence: 99%

Amino-Acid Sequence of Dihydrofolate Reductase from a Methotrexate-resistant Mutant of Streptococcus faecium and Identification of Methionine Residues at the Inhibitor Binding Site

Gleisner

Peterson

Blakley

1974

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…, The toxin was not stained on paper by ninhydrin, indicating the absence of free amino groups, nor was a free amino group available for dansylation, but the hydroxyl group of tyrosine did dansylate. Selective tritiation (Holcomb et al 1968) indicated the absence of the free carboxyl groups of alanine, methionine, or tyrosine (the only monocarboxylic amino acids present) whilst both dicarboxylic amino acids incorporated tritium indicating the presence of free alpha carboxyl groups in these amino acids.…”

Section: Purification and Chemical Composition Of The Toxinmentioning

confidence: 99%

Isolation, Characterization and Pathology of the Toxin From a Microcystis Aeruginosa (= Anacystis Cyanea) Bloom

Elleman¹,

Falconer²,

Jackson³

et al. 1978

Aust. Jnl. Of Bio. Sci.

128

View full text Add to dashboard Cite

The nature of the toxicity of a bloom of blue-green alga, M. aeruginosa (= Anacystis cyanea), that occurred in a man-made lake was investigated. Crude algal bloom extracts were toxic to laboratory mice when injected intraperitoneaIIy. The lethal dose (LDlOO) of these extracts was 15-30 mg of lyophilized algal bloom per kilogram body weight. The toxin was purified by a procedure that included ammonium sulphate fractionation, solvent extraction, acid precipitation, Sephadex G25 and DEAE-Sephadex chromatography, and high-voltage electrophoresis at pH 6·5. The preparation gave a single spot on high-voltage electrophoresis at pH 9·0, had no free amino group, and was characterized by a simple amino acid composition of equimolar quantities of L-methionine, L-tyrosine, D-alanine, D-glutamic acid, erythro fi-methyl aspartic acid and methylamine.The LDso for the purified toxin was estimated to be 0·056 mg/kg of mice, and the approximate LD100 is 0·070 mg/kg, based on the total material found from amino acid analysis.Parenteral administration of the purified toxin to mice produced extensive liver lobular haemorrhage and death within 1-3 h. Repeated inoculation of sublethal doses daily over some weeks produced progressive hepatocyte degeneration and necrosis and the development of fine hepatic fibrosis.

show abstract

“…The original analysis reported by Burley (1975) has one less residue of both isoleucine and tyrosine. Tyrosine was the only amino acid found at the C-terminus by selective tritiation (Holcomb et al 1968), and lysine the only N-terminal residue by dansylation of the protein (Gray 1967). Tryptic peptides were separated on G25 Sephadex (five coupled columns, each 120 by 0·9 cm) in pyridine-l M ammonia (70:30, v/v).…”

Section: Resultsmentioning

confidence: 99%

“…It was' degraded until the C-terminal Lys was reached. The positions of the two carboxyl groups were obtained from the thermolytic peptides Met-Leu-Glu-Lys (m = 0·0) and Leu-Ala-Glu-Gln (m = -0·49); the latter peptide was tritiated at the C-terminal residue by the method of Holcomb et al (1968), then digested for 1 h with carboxypeptidase A and the [3H]glutamine (at the origin) identified as the only radioactive spot after electrophoresis at pH 6·5. Table 4.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Primary Structure of Apovitellenin I From Hen Egg Yolk and its Comparison With Emu Apovitellenin I

Ta¹,

Inglis²

1976

Aust. Jnl. Of Bio. Sci.

View full text Add to dashboard Cite

The amino acid sequence of apovitellenin I from hen egg yolk has been determined using both automatic and manual procedures; it comprises 82 residues. Hen apovitellenin shows considerable homology with emu apovitellenin I which contains 84 residues. Besides two deletions in the sequence, the hen protein differs in 28 positions from the emu protein; 26 of these positions may have arisen from single base changes. The changes are largely conservative ones, which suggest that the structure and function have been preserved despite extensive mutation.

show abstract

Critical evaluation of the selective tritiation method of determining C-terminal amino acids and its application to luteinizing hormone

Cited by 71 publications

References 12 publications

Amino-Acid Sequence of Dihydrofolate Reductase from a Methotrexate-resistant Mutant of Streptococcus faecium and Identification of Methionine Residues at the Inhibitor Binding Site

Amino-Acid Sequence of Dihydrofolate Reductase from a Methotrexate-resistant Mutant of Streptococcus faecium and Identification of Methionine Residues at the Inhibitor Binding Site

Isolation, Characterization and Pathology of the Toxin From a Microcystis Aeruginosa (= Anacystis Cyanea) Bloom

Primary Structure of Apovitellenin I From Hen Egg Yolk and its Comparison With Emu Apovitellenin I

Contact Info

Product

Resources

About