John H. Mangum

Studies on a ferrous-ion-oxidizing bacterium. II. Cytochrome composition☆

Abstract 1. 1. Cells of an iron-oxidizing bacterium have been shown to contain both cytochrome c and cytochrome a 1 . No cytochrome b was detected in whole cells or cell-free extracts. 2. 2. The cytochrome c was partially purified and had absorption maxima at 552, 523, and 417 mμ. Pyridine and cyanide hemochromogens showed it to be a c type cytochrome. The E 0 ′ at pH 7.0 was 0.31 v., while at pH 2.9 the potential was 0.38 v. The cytochrome was not oxidized by the air, and its concentration in the cell was 88 mg./g. nitrogen. 3. 3. Both the cytochrome c and cytochrome a 1 were reduced when ferrous ion was added to a cell suspension. The relation of the cytochrome composition to the physiology of the organism is discussed, and it is proposed that the biological oxidation of iron involves a transfer of electrons from iron to cytochrome c, cytochrome a 1 , and oxygen in that sequence.

A rapid assay for 5-amino-4-imidazelecar☐amide ribotide transformylase

Abstract A rapid spectrophotometric assay for 5-amino-4-imidazolecar☐amide ribotide transformylase has been devised. The assay consists of monitoring the tetrahydrofolate generated during the enzyme-catalyzed reaction which results in an increase in absorbance at 298 nm. The formation of tetrahydrofolate shows an absolute depedence on 5-amino-4-imidazolecar☐amide ribotide, 10-formyltetrahydrofolate, and the enzyme.

Cytochromes of Bacillus megaterium and Bacillus subtilis

Abstract Treatment of Bacillus subtilis and Bacillus megaterium cells by ordinary procedures failed to extract any soluble cytochrome from the cell. Sonoration produced small fragments which contained the cytochrome complement and were sedimentable in a centrifugal field of 144,000 × g . Difference spectra of such particles showed absorption maxima at 599, 557, 552, 530, and 428 mμ. Digestion of the fragments with lipase liberated a rather typical cytochrome c in that it had absorption maxima in the reduced form at 550, 520, and 415 mμ, formed cyanide and pyridine hemochromogens which were spectroscopically similar to those obtained from mammalian cytochrome c, could be purified in the usual manner by ammonium sulfate fractionation, and had an oxidation potential at pH 7.0 of 0.25 v. As isolated, however, the cytochrome c was readily oxidized by oxygen in the air.

The isolation of N5-methyltetrahydrofolate-homocysteine transmethylase from bovine brain☆

Abstract The enzyme ( N 5 -methyltetrahydrofolate-homocysteine transmethylase) that catalyzes the transfer of a methyl group from N 5 -methyltetrahydrofolate to homocysteine to form methionine has been found in a number of bovine tissues. The level of transmethylase activity was higher in brain than in any other tissue examined including liver and kidney. The brain enzyme has been purified approximately 370-fold. An initial fractionation with protamine sulfate and ammonium sulfate was followed by column chromatography on DEAE-Sephadex and Sephadex G-200. As the specific activity of the transmethylase increased the enzyme showed a greater dependency upon S -adenosyl methionine and a reducing system.

The isolation of serine transhydroxymethylase from bovine brain.

Abstract Serine transhydroxymethylase has been purified 150-fold from bovine brain. The initial fractionation with portamine sulfate and ammonium sulfate was followed by chromatography on DEAE-Cellulose and Sephadex G-200. The cofactor requirements for bovine brain serine transhydroxymethylase included pyridoxal phosphate and tetrahydrofolate. The maximum catalytic activity occurred at pH 7.6 and the Michaelis constant for serine was 6.7 × 10 −4 m .

5-methyltetrahydrofolate: synthesis and utilization in normal and SV40-transformed BHK-21 cells.

Abstract BHK-21 cells and SV40-transformed BHK-21 cells (A-8) grew rapidly in a medium containing methionine (I). BHK-21 cells also proliferated in a methionine-deficient medium (II) supplemented with homocysteine and vitamin B 12 ; however, the A-8 cells did not proliferate and ultimately died in this medium. The 5,10-methylenetetrahydrofolate reductase activity in transformed cells was only 10% of that found in BHK-21 cells. While A-8 cells in medium II could not be maintained on folate or 5-formyltetrahydrofolate, they survived and even proliferated slowly when provided 5-methyltetrahdyrofolate. The low level of the reductase in A-8 cells and the inability of these cells to utilize folate or 5-formyltetrahydrofolate suggests that A-8 cells are unable to synthesize 5-methyltetrahydrofolate at a rate that will satisfy the cellular demands for methionine.

The purification of a soluble cytochrome from pig kidney with spectral properties similar to those of microsomal cytochrome b5

Abstract A soluble cytochrome has been partially purified from pig kidney. The absorption spectra of this cytochrome in both the oxidized and reduced state resembles microsomal cytochrome b5. However, this cytochrome is present in crude homogenates in a soluble form and, therefore, the purification scheme does not involve the use of proteases, lipase or detergents.

Studies on the mechanism of cytotoxicity of 3-deazaguanosine in human cancer cells

SummaryThe mechanism of toxicity of 3-deazaguanosine was studied in a number of human tumor cell lines by determination of the effects of various purine compounds on the growth of the cells in the presence of the drug and by studies of the effects of 3-deazaguanosine on the metabolism of radiolabeled precursors in these cells. The drug was found to be toxic to all of the cell lines tested. The toxicity was reversible with removal of the drug. None of the purine bases tested could restore normal growth after 48h exposure to 3-deazaguanosine; the bases were more effective in preventing cytotoxicity when added simultaneously with the drug. Metabolic studies indicated decreased synthesis of DNA, variable inhibition of de novo purine synthesis, and complete inhibition of the enzyme guanosine monophosphate reductase by 3-deazaguanosine.

Novel nucleoside inhibitors of guanosine metabolism as antitumor agents

A detailed study of the inhibition of DR and TR in the SkLu-1 line of human lung adenocarcinoma has shown that TR significantly inhibits this tumor line, probably via inhibition of IMP dehydrogenase by the corresponding TAD analog of NAD. DR exhibited a similar degree of inhibition in this cell line. In a system devised to detect the inhibition of cloning efficiency of the SkLu cells, DR showed a 50% inhibition at 4 X 10(-3) M and TR at 1 X 10(-4) M. When DR and TR were used in combination, the ID50 was decreased to 3 X 10(-5) M. The study of DR in a number of human carcinoma cell lines revealed that de novo purine biosynthesis was significantly inhibited; however, in the SkLu-1 lung carcinoma cells this inhibition was not observed. The synergism observed in this cell line is presently viewed as potentially due to both agents acting on IMP dehydrogenase at different sites.

Serine transhydroxymethylase: a simplified radioactive assay; purification and stabilization of enzyme activity employing Affi-Gel Blue

An improved radioactive assay has been developed for serine transhydroxymethylase. This assay involves the direct measurement of the [14C]HCHO which is generated when [3- 14C]-serine is employed as the substrate. The new assay eliminates the need for a solvent extraction of a [14C]HCHO-dimedon adduct which is the basis of the assay devised by Taylor and Weissbach. The enzyme has been purified employing Affi-Gel Blue. The purified enzyme retains full activity when bound to this affinity chromatography matrix and can be stored in this state at 4 degrees indefinitely.