A fluorometric method for the estimation of tryptophan

The various colorimetric methods now employed for the estimation of tryptophan are not specific for tryptophan but give colored products with many compounds containing the indole nucleus, including indole itself. In order to facilitate studies of the enzymatic synthesis of tryptophan from indole and serine by extracts of Neurospora (1), a search for a rapid, quantitative method for the estimation of tryptophan in the presence of indole was undertaken. Tauber (2) reported that tryptophan gives a green fluorescence when treated with 70 to 72 per cent perchloric acid at room temperature. Modification of this procedure has led to a method for the rapid estimation of tryptophan, without preliminary extraction of indole, in hydrolyzed or unhydrolyzed tryptophan-containing materials

Extraction Methods and an Investigation of Drosophila Lipids

In earlier work (8) we extracted lipids from dried, macerated Drosophila melanogaster with ether, but later, working with larger quantities of undried flies, we found that most of the phospholipids were autolyzed. Kates' studies (2) led him to suggest n-propanol or isopropanol for lipid extraction (isopropanol was his later choice (6, 7)). Attempting to meet the requirements discussed above, we developed a new and relatively simple method of extraction employing n-propanol (9), or chloroformmethanol (2:1). The latter proved to be a more useful solvent. The method will be described in detail below, with results of an examination of Drosophila lipids

The b iosynthesis of histidine: imidazoleglycerol phosphate, imidazoleacteol phosphate, and histidinol phosphate

This is a report on the isolation and characterization of D-erythro-imidazoleglycerol phosphate (IGP), imidazoleacetol phosphate (IAP), and L-histidinol phosphate, which are accumulated in the mycelia of several of these mutants

Purine and pyrimidine bases as growth substances for lactic acid bacteria

In 1936 Richardson (1) showed that uracil was essential for the anaerobic growth of Staphylococcus aureus, but not for aerobic growth of the same organism. Of five strains tested three required uracil, while one required both guanine and uracil for growth. Thymine or cytosine did not replace uracil for this organism. These experiments suggested that hydrolytic products of nucleic acids might become factors limiting growth of various organisms under certain conditions. Bonner and Haagen-Smit (2) in 1939 showed that adenine greatly stimulated growth of leaves under defined conditions, while Möller (3) showed that adenine was required for growth of Streptobacterium plantarum. Pappenheimer and Hottle (4) recently showed that adenine was necessary for the growth of a strain of Group A hemolytic streptococci; it could be replaced by hypoxanthine, guanine, anthine, guanylic acid or adenylic acid. They made the very interesting observation that adenine was unnecessary for growth of this organism if the carbon dioxide tension was maintained at a sufficiently high level

Phospholipides containing amino acids other than serine. I. Detection

In view of the widespread occurrence of the amino acid-containing lipides and the unique course of their metabolism during development of Drosophila, we have carried out extensive investigations concerned with their isolation and chemical nature. The present report is concerned primarily with techniques and procedures developed to insure removal of non-lipide contaminants from preparations of these lipides

The accumulation of orotic acid by a pyrimidineless mutant of Neurospora

The discovery of orotic acid (1,2-carboxyuracil) in cow's milk by Biscaro and Belloni (1), followed by its identification and synthesis (2-4), led to a number of speculations as to its biological origin and significance (,3,5,6). A definite connection of orotic acid with the biosynthesis of nucleic acid pyrimidines is provided by the finding that orotic acid (7) as well as thymine (8,9) can supplement or replace the folic acid required by certain microorganisms. As suggested by Chattaway (7), it would appear that folic acid has a function in the biosynthesis of pyrimidines. Furthermore, this function is probably concerned in some step prior to the appearance of orotic acid in the biosynthetic series. More recently it was shown by Loring and Pierce (10) that orotic acid could be substituted for uracil in satisfying the growth requirements of some pyrimidineless mutants of the mold Neurospora. Investigations of orotic acid in this laboratory have led to a new method of synthesis of the compound (11) and to some suggestions concerning its relation to the biosynthesis of nucleic acids in Neurospora (12). The results of the present work are in accord with the previous suggestions and provide further evidence on the biological origin and function of orotic acid

A microbiological assay method for p-aminobenzoic acid

Since the establishment of p-aminobenzoic acid as a member of the B vitamin group, a considerable interest has been shown in methods of determination in natural materials. Since known chemical methods are not sufficiently sensitive, it became evident that microbiological tests should be the most practicable. The organism Clostridium acetobutylicum has been used (1) but no general assay procedure has been presented. Several bacterial strains which respond to p-aminobenzoic acid have been investigated in this laboratory, but satisfactory assay procedures with these organisms have not yet been devised. For the discovery of the test organism used in the procedure described in this paper, we are indebted to Dr. Beadle and Dr. Tatum who kindly furnished us with a culture of their p-aminobenzoic acid requiring a mutant strain of Neurospora crassa, designated by them as Neurospora crassa p-aminobenzoicless No. 1633 (2). This mold will grow optimally on a medium consisting of inorganic salts, ammonium tartrate, sucrose, biotin, and p-aminobenzoic acid. For purposes of assay, however, it has proved advantageous to supplement this basal medium with natural extracts which are either naturally low in p-aminobenzoic acid or have been treated to remove it. With such a complex medium, the possibility of interference by toxic substances or stimulatory substances other than p-aminobenzoic acid which might be present in samples to be assayed is reduced to a minimum. Since the completion of a considerable part of the experimental work described in this paper, microbiological assay methods for p-aminobenzoic acid have been published by Landy and Dicken (3) utilizing the organism Acetobacter suboxydans and by Lewis (4) using Lactobacillus arabinosus 17-5

Self-degradation of heat shock proteins

The 70-kDa heat shock protein of Drosophila decays in vivo at a much faster rate than other abundantly labeled proteins. Degradation also occurs in vitro, even during electrophoresis. It appears that this degradation is not mediated by a general protease and that the 70-kDa heat shock protein has a slow proteolytic action upon itself. Heat-induced proteins in CHO cells and a mouse cell line also degrade spontaneously in vitro, as do certain non-heat shock proteins from Drosophila tissues as well as the cell lines

The use of isotopic carbon in a study of the metabolism of anthanilic acid in Neurospora

The finding by Tatum, Banner, and Beadle (l), that the tryptophanless Neurospora mutant strain 10575 accumulates anthranilic acid, which in turn can be utilized for growth of strain 40008, has provided evidence that anthranilic acid is a biochemical precursor of tryptophan in this organism. It has been further established that indole is an intermediate in this conversion (2-5). More recent work with a number of mutants of Neurospora (6-8) has established that tryptophan is a biochemical precursor to niacin with kynurenine and hydroxyanthranilic acid as intermediates. The accumulated evidence has indicated the existence in the mold of the following series of reactions: → Anthranilic acid → indole → tryptophan → kynurenine → 3-hydroxyanthranilic acid → nicotinic acid In the light of this evidence the present work was undertaken to trace the carbon in the carboxyl group of anthranilic acid in order to estimate its contribution as a structural unit in the formation of niacin and tryptophan. The organism chosen for this investigation was a biochemical mutant strain of Neurospora designated as strain 40008. This mutant utilizes anthranilic acid, indole, or tryptophan for growth. The mutant was grown in the presence of anthranilic acid containing Cl4 in the carboxyl group. Niacin and tryptophan were isolated from the mold mycelium and tested for radioactivity