Showing papers in "Journal of Biological Chemistry in 1951"

Abstract: Since 1922 when Wu proposed the use of the Folin phenol reagent for the measurement of proteins, a number of modified analytical procedures utilizing this reagent have been reported for the determination of proteins in serum, in antigen-antibody precipitates, and in insulin. Although the reagent would seem to be recommended by its great sensitivity and the simplicity of procedure possible with its use, it has not found great favor for general biochemical purposes. In the belief that this reagent, nevertheless, has considerable merit for certain application, but that its peculiarities and limitations need to be understood for its fullest exploitation, it has been studied with regard to effects of variations in pH, time of reaction, and concentration of reactants, permissible levels of reagents commonly used in handling proteins, and interfering substances. Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.

Abstract: The quantitative separation of amino acids by chromatography on columns of starch has sheen described in previous publications (l-3). In extending these investigations, a systematic study has been made of the separations of amino acids by elution analysis on columns of synthetic ion exchange resins. With a sulfonated polystyrene resin, Dowex-50, it has been possible to develop a chromatographic technique applicable to a wider variety of problems of biochemical interest. Starch columns possess the disadvantage that fluids of high salt content, such as blood plasma or urine, require desalting prior to chromatography (4). Moreover, contamination of the effluent by traces of carbohydrate, coupled with the relat,ively low capacity of these columns, tends to interfere with the isolation of pure compounds (5). Methods in which columns of Dowex-50 are employed have been found to be free from these difficulties. Ion exchange resins of various types have been used in the past to separate amino acids into groups, acidic, basic, and neutral, and also to separate some of the individual basic or acidic amino acids (cf. (6)). The data of Englis and Fiess (7) indicated that certain of the monoamino acids are adsorbed to different degrees by sulfonic acid type resins, but little information was available as to whether columns of ion exchange resins could yield a degree of resolving power comparable with that obtainable with starch or paper. Recently Partridge and coworkers (8-11) have employed displacement development on ion exchange columns for the separation of amino acids on a preparative scale. Those primary fractions containing more than one amino acid were rechromatographed. Their experiments, together with our results by elution analysis (4’), have demonstrated that synthetic resins are capable of separating most of the common amino acids from one another. For analytical experiments, and for isolation work on a small scale, elution analysis would appear to possess higher resolving power and, for this reason, has been employed in the present studies. In Fig. 1 is shown the result obtained upon passage of a complex mixture of amino acids through a 0.9 X 100 cm. column of Dowex-50. This particular resin (Bauman and Eichorn (12)) is one which is available com-

Abstract: The method used by Potter, Albaum, and others (1, 2) for studying the effects of various inhibitors on cytochrome oxidase has been adapted successfully to the assay of the cytochrome oxidase content of rat tissues. The rate of enzymatic oxidation of reduced cytochrome c is measured. A microadaptation enables the measurement of the cytochrome oxidase content of as little as 4 y (wet weight) of an active tissue such as heart.

Abstract: For the preparation of lipide extracts from tissues, the method of Bloor (I), either in its original form or with slight modifications, has been a standard procedure. This method consists in extracting the tissue with a mixture of ethyl alcohol and ether. Since the extract obtained is known to contain non-lipide contaminants, it is usually taken to dryness and the residue extracted with a solvent, such as chloroform or petroleum ether, which exhibits a highly specific solvent power for lipides. However, the secondary extracts obtained have been shown to contain substances other than lipides (2, 3). In the case of nervous tissue, it has been common experience that all of the lipides present in tissue are not extracted by Bloor’s procedure (4, 5). Thus, different workers have found it necessary to introduce a subsequent extraction of the tissue with another solvent of higher solvent power for lipides than Bloor’s mixture. This second solvent has usually been chloroform (4, 5). The methods thus developed are time-consuming, complicated, and, owing to the fact that they involve protracted treatment of the tissue with boiling solvents, they are open to the general objection that the procedure followed results in changing the chemical nature of some of the lipides. Furthermore, the extracts thus obtained are known to contain non-lipide contaminants (4, 5). This paper describes a simple method for the preparation of extracts of total pure lipides from brain tissue. The method consists of homogenizing the tissue with a chloroform-methanol mixture. The clear lipide extract thus obtained is then washed free of non-lipide contaminants by being placed in contact with a large amount of water. The whole procedure can be run at 0” and thus any danger of chemical changes in lipides is reasonably excluded.

Abstract: To 1.0 cc. of a solution containing 1 to 50 y of a ketohexose, either in free form or in the form of its esters or polymers, or 2 y of triose or glycolic aldehyde, is added 0.2 cc. of a 1.50 per cent solution of cysteine hydrochloride. To this 6 cc. of a mixture of 190 cc. of water and 450 cc. of concentrated sulfuric acid, c.p., are added and immediately afterwards 0.2 cc. of a 0.12 per cent alcoholic solution of carbazole. The mixture is shaken and left standing at room temperature.

Abstract: the objective of the present work was to provide a means for separating and indentifying phosphate esters involved in glycolysis in higher plants. Paper chromatography of phosphate esters has been employed by several workers, most notably Benson et al. (1) and Hanes and Isherwood (2). Benson's procedures were not primarily designed for identification of phosphate esters and gave low Rr values for the phosphate compounds of particular interest to us. The unidimensional methods of Hanes and Isherwood do not result in adequate resolution of the complex mixtures such as are obtained from our plant materials. The present procedure is based on two-dimensional chromatography with successive development in an acid and in a basic solvent. The solvents finally selected gave the best over-all resolution of the intermediates involved in plant glycolysis. Undoubtedly the resolution of certain pairs of compounds may be improved by suitable modifications. We have in addition made certain improvements in the procedure for locating the chromatographed materials.

Abstract: Lipmann and Tuttle (1) discovered that certain bacterial extracts catalyze a rapid interchange of inorganic and acetyl-bound phosphate. They suggested that this exchange might be due in part to a reversibility of the phosphoroclastic decomposition of pyruvate; however, the observation that the exchange was surprisingly unaffected by addition of acetyl acceptors like formate led them to suspect that unknown factors may be also involved. More recently, Stadtman and Barker (2) reinvestigated the phosphate exchange reaction in cell-free extracts of Clostridium kluyveri. They found that substitution of arsenate for phosphate in these extracts resulted in a very rapid and complete hydrolysis of acetyl phosphate. The marked similarity between these reactions and the reactions catalyzed by the bacterial transglucosidase system, previously described by Doudoroff et al. (3, 4), led to the proposal that the phosphate exchange and arsenolysis reactions of acetyl phosphate were catalyzed by an acetyl-transferring enzyme (Stadtman and Barker (2) ; Lipmann (5)). Concurrently, acetyl phosphate, until recently refractive with regard to acetyl donor function, was found to serve as a G precursor in the synthesis of butyrate and hexanoate by extracts of C. kluyveri (6), and, in particular, it showed a surprising activity in the coenzyme A (CoA)-linked citric acid synthesis by extracts of Escherichia coli (7). These observations became of even greater interest when it was found that microbial extracts could activate acetyl phosphate to serve as an acetyl donor for various CoA-dependent acetylation reactions catalyzed by animal enzyme preparations (8-10). Attempts to identify this “activator” function of microbial extracts led to the proposition that the phosphate exchange and arsenolysis system may be responsible for the acetyl transfer from acetyl phosphate. This possibility prompted us to embark

Abstract: Extraction-As the oxidation is essentially non-specific, the plasma extract must be as free of non-lipide reducing substances as possible. All solvents should be tested occasionally for reducing residues remaining after evaporation, and redistilled if necessary. Ether should be maintained peroxide-free. The determinations reported here followed a conventional alcohol-ether extraction and subsequent reextraction in petroleum ether. Although Folch and Van Slyke (2) have shown that this procedure carries through significant amounts of urea, it has been found that urea, even in amounts considerably in excess of what might occur, produces no color change in the dichromate reagent. Methods of extraction based on primary elution of water-soluble substances (Folch and Van Slyke (3); Ahrensl) have been used recently and offer definite advantages. Reagent-20 gm. of K2Crp01, c.p., are powdered in a mortar and added slowly with shaking to 1000 ml. of HzS04, c.p. (sp. gr. 1.84) maintained at a temperature not exceeding 100”. There should be no undissolved residue. If the reagent is protected from contamination and from exposure to direct sunlight, it darkens only very slowly with age. This does not affect the calorimetry, however, as the reagent itself is used as the reference blank. The addition of catalysts, including Hg, Pd, and Ag, had no effect on the oxidizing power of the reagent. Silver, recommended by Bloor (I), should not be used with biological extracts, as the latter may contain enough chloride (4) to interfere with the calorimetry. Oxidation-In dealing with extracts of normal human plasma, an aliquot representing from 0.1 to 0.7 ml. of plasma is transferred to a 25 ml. volumetric flask. The solvent is evaporated under nitrogen (water-pumped)

Abstract: Knowledge concerning the nature of the biologic synthesis of purine or pyrimidine mononucleotides is relatively limited. A significant contribution was the demonstration by Ostern and coworkers (3) of the phosphorylation of adenosine to adenosine-5’-phosphate (A-5’-P) and adenosinetriphosphate (ATP) by means of crude yeast extracts. The energy source for this reaction was supplied by the fermentation of hexose diphosphate or phosphoglyceric acid. These extracts did not act on guanosine or ribose and failed to form yeast adenylic acid. Recently, Sable (4) observed the disappearance of ATP in the presence of crude yeast fractions and adenosine. He suggested the direct phosphorylation of adenosine by ATP and the name adenosine phosphokinase for this enzyme. In order to provide some definitive information on the mechanism of conversion of adenosine to adenosine-5’-phosphate, we have purified this activity from yeast autolysates. An enzyme preparation has been obtained which catalyzes the reaction

Abstract: In 1938, Meyerhof, Ohlmeyer, and Mohle (1) reported that diphosphopyridine nucleotide (DPN) reacted with cyanide or bisulfite to form complexes having absorption spectra resembling that obtained by enzymatic reduction of DPN. They proposed that addition of cyanide or bisulfite occurred at the double bond between nitrogen and carbon in the pyridinium ring. In the present paper, it is shown that the ability to form a complex with cyanide is a general property of N-substituted nicotinamide compounds.

Abstract: Chemical studies initiated in the laboratories of Reichstein, Kendall, and Wintersteiner (1) resulted in the isolation of twenty-eight crystalline steroids from adrenal gland extracts. Twelve of the compounds possessed an a-ketol side chain and, of these, six proved to be biologically active. The finding of 11-desoxycorticosterone, one of these active steroids, by Reichstein and von Euw (2) has been corroborated neither in the other two laboratories nor by subsequent workers. The methods used by these workers for the fractionation of the extracts and isolation of the crystalline compounds were such that the possibility of chemical alteration of some of the more labile steroids could not bi excluded. These methods, in addition, gave no reliable data as to the quantitative relationships existing among the different compounds found in the gland. The recent development in our laboratories of paper chromatographic and spectrophotometric techniques for the identification of adrenocortical steroids has been reported (3, 4). A procedure for the fractionation of complex corticoid mixtures and a systematic method for the identification of the separated compounds based upon these techniques are herein described. These methods offer several advantages over those used by previous investigators: (1) Samples are subjected to a minimum of handling; (2) components present in very low concentrations can be isolated and identified; and (3) compounds very closely related in chemical structure can be separated and differentiated. Analysis of a commercial preparation of beef adrenal extract thereby indicated the presence of at least twelve cr-ketolic steroids, of which eight, including 11-desoxycorticosterone, have been identified. Of the remaining four, two may represent new compounds. A preliminary report of these findings has already been made (5).

Abstract: This paper reports the isolation from brain tissue of a substance to which the name of strandin has been given for descriptive purposes. This term has been chosen because, when dried from aqueous solution, strandin has the property of forming long strands that show perfect orientation under polarized light. As isolated from brain by any one of the three procedures described below, strandin is an electrophoretically homogeneous compound. By ultracentrifuge studies, it is shown to have a main component with a minimal molecular weight of 250,000. It is soluble in water and chloroform and is extracted quantitatively from the tissue with chloroform-methanol mixtures (1). Chemically, it can be classified as a lipide, since among its constituents are found fatty acids and sphingosine or a sphingosine-like substance. However, many of its properties are quite different from those of a typical lipide. Strandin is found in gray matter in relatively large concentration; i.e., 6 to 7 mg. per gm. of wet tissue. It is found in white matter at one-tenth its concentration in gray matter and in brain tumors in concentrations larger than in white matter and smaller than in gray matter. In other tissues that have been studied, namely, heart, skeletal muscle, uterus, lung, liver and kidney, strandin is found in very small amounts; i.e.,