DETECTEON AAAA {EA—iAAAAC‘AERLZA’ATADN AA A 0‘1 ”’AFNTM AAA cam AAAAAAAAA. AAAAS 0A REEUC ED AAACAAAAAAAAAAAA: AAAAAAAAA—L AAAAAHCAAAAAADE MFA; BY AAAAA‘AA AEAAA‘ .EAAS Thesis for AAAe Degree of M. S. MACHAGAN STATE UNWERSATY RALPH LEONARD SOMACK 1969 OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: - . '"""""“"""""""" »,;:, ,,,,, , Place in book return to remove ' w“ ”/3"- ~ charge from circulation records ABSTRACT DETECTION AND CHARACTERIZATION OF A COMPONENT(S) IN COMMERCIAL LOTS OF REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE OXIDIZAELE BY INTACT YEAST CELLS By Ralph Leonard Somack Intact yeast cells were found to oxidize some component(s) of reduced nicotinamide adenine dinucleotide (NADH) solutions. This oxidation was also observed with solutions of oxidized nicotinamide adenine dinucleotide (NAD) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) as substrates; but only one commercial lot of each of these compounds was tested. The extent of the oxygen uptake observed varied from lot to lot (0.25 to 1.13 umoles of O2 uptake/mg NADH). The respir- atory quotient of the oxidation with NADH was observed to be in the order of 0.67, and no free amino nitrogen was released during the oxidation. The extent of this oxidation was as high at pH “.0 as at pH 7.0, even though the NADH in the solution was all in the form of the acid modification product at pH “.0. The oxidizable component(s) was separated from the NADH by the use of anion exchange resin; and, thus, proven to be a contaminant(s) of the preparation. This compound(s) was found to be neutral at both acid and basic pHs; as indicated by its failure Ralph Leonard Somack to adsorb to either anion or cation resins at these pHs. The eluted component(s) expressed no character— istic absorption spectrum between 220—UHO nm. 02 uptake was not observed when the eluted component(s) was dried at either acid or basic pHs under a vacuum at Mo C; thus indicating that the component(s) was either volatile or unstable under these conditions. Inhibition of both 0 uptake and CO evolution was 2 2 observed when the component(s) was oxidized by yeast in the presence of azide, malonate or iodoacetate. This indicated that metabolism of the component(s) was dependent on the electron transport system, the tri- carboxylic acid cycle and sulfhydryl containing enzymes. The low levels of the oxidizable component(s) present, the high cost of NADH preparations, and the difficulty in concentrating the component(s) precluded further efforts toward identification at this time. DETECTION AND CHARACTERIZATION OF A COMPONENT(S) IN COMMERCIAL LOTS OF REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE OXIDIZABLE BY INTACT YEAST CELLS By Ralph Leonard Somack A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1969 . M “x‘ V» o 5) ‘\ \ \. \ 2v u\ \ *3 K) K) (3 'r‘\ @‘W -LV- DEDICATION This thesis is dedicated to my wife, Diana, love, her understanding, and her patience. ii for ACKNOWLEDGMENTS I would like to express my sincere appreciation to Dr. R. N. Costilow for his patient guidance throughout this investigation, and for his critical suggestions during the preparation of this thesis. The author is indebted to Dr. H. L. Sadoff and Dr. R. R. Brubaker for their valuable suggestions and concern during the course of this research and my graduate study, and for the use of their laboratories and equipment. Sincere thanks are also extended to Dr. D. Bing for his advice and technical assistance. iii TABLE OF CONTENTS DEDICATION ACKNOWLEDGMENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION REVIEW OF LITERATURE Oxidation of NADH and NADPH Formation and Properties of the Acid Product of NADH and NADPH EXPERIMENTAL METHODS Culture and Cultural Techniques Oxidation Techniques Assay and Cleavage of NADH Purification of the Oxidizable Contaminant RESULTS Studies on the Oxidation of NADH by Yeast Cells . . . . . . . . Nature of the Oxidation Purity Analyses of NADH Preparations Effects of Cleavage of NADH on Oxidation by Yeast Cells Spectrum Before and After Oxidation Studies With the NADH Contaminant Separation on Anion Exchange Resin Free Amino Nitrogen Determination Effects of Substrate Drying on the Oxidation Further Purification with Cation Exchange Resin . Absorption Spectrum of the Contaminant . iv Page ii iii Vi Vii 00 O\ O\U'IU1 U1 LA) 00 11 1A 19 19 21 26 26 Page Studies Using Inhibitors of Cellular Metabolism . . . . . . . . . . . 26 DISCUSSION . . . . . . . . . . . . . . 31 SUMMARY . . . . . . . . . . . . . . . 37 BIBLIOGRAPHY . . . . . . . . . . . . . 39 LIST OF TABLES Table Page 1. Oxidation by yeast of various brands of NADH tested for spectrophotometric and functional purity . . . . . . . . . . . . . l3 2. Separation of an oxidizable contaminant from NADH by anion exchange chromatography. . . 2O 3. Determination of free amino nitrogen before and after oxidation of fraction I by yeast . . 22 A. Effect of various treatments of NADH and frac- tion I on 02 uptake by yeast. . . . . . 23 5. Effect of azide, malonate and iodoacetate on the oxidation of fraction I by yeast cells . 28 vi Figure 1. LIST OF FIGURES Effect of NADH on 02 uptake and CO2 evolu- tion by yeast cells . . . . . Oxidation of various brands of NADH Oxidation of NADPH, NADP, and NAD Phosphodiesterase cleavage of NADH Oxidation of NADH and of phosphodiesterase treated NADH by yeast . . . A. Spectrum of NADH and the NADH acid product. B. Spectrum of the NADH acid prOduct after oxidation by yeast cells . . Oxidation of fraction I by yeast cells at pH 4.0. . . . . . . . . Oxidation of fraction I by yeast cells at pH 7.0. . . . . . . . . Oxidation of fraction I by yeast cells after passage through a cation exchange column Vii Page 12 15 16 18 18 2A 25 27 INTRODUCTION Commercially prepared oxidized and reduced nico- tinamide adenine dinucleotide (NAD and NADH respectively) have been used in a great number of scientific investi- gations. These and other pyridine nucleotides are involved in a large number of oxidation—reduction reactions catalyzed by dehydrogenases. Anderson (1), in his studies on the primary site of inhibition of yeast respiration by sorbic acid, found that sorbate inhibited aerobic oxygen uptake by yeast cells at pH 4.0 when solutions of commercial NADH and reduced nicotinamide adenine dinucleotide phosphate (NADPH) were used as substrates. These oxidations were markedly inhibited by both KCN and azide indicating that the oxidation proceeded via the cytochrome system. Since these coenzymes are known to be converted to products inactive in dehydrogena e reactions under acid conditions O (A (ll, 20), it was unlikely that these coenzymes were metabolized in the manner outlined by And rson. The present study was initiated in an attempt to identify the nature of these oxidations. REVIEW OF LITERATURE Oxidation of NADH and NADPH Anderson (1), showed that solutions of NADH and NADPH supported oxygen uptake at acid pH by yeast cells. The oxidation, which was observed in the presence of 33 umoles of NADH, was shown to be inhibited 100% by KCN 1 (6.7 x 10— M) and 68% by sorbic acid (5.3 x 10-3M). The oxidation observed with NADPH solutions was inhibited 2M), 100% by azide (7.7 x iO’3N), 3 33% by atabrin (5.3 x 10— and 88% by sorbic acid (5.3 x 10- M). Further evidence that the oxidation prOceeded via electron transport (ETS) came from the observation that methylene blue was reduced when cytochrome oxidase was inhibited by KCN with an NADH solution as substrate. The experiment was performed with yeast cells at pH 5.1 — 5.3. Studies with 2-4 dinitrophenol (DNP) indicated that the oxygen uptake by whole cells observed with NADH solutions required active transport since DNP lowered the resulting Qog, while it failed to affect the rates of endogenous respiration or glucose oxidation. As Anderson pointed out, this would be expected since substrate phosphory— lation occurs via glycolysis during glucose metabolism. Studies with crude extraCts of yeast cells showed that these preparations also oxidized NADH and that the oxidation was sensative to azide, antimycin A and KCN. PU Although the oxygen uptake with intact cells was inhibited by CO, there was no significant inhibition of oxygen uptake with the crude yeast extracts (Anderson, 1963, unpublished data). These results were explained on the basis of the peroxidase activity observed in the isolated crude yeast mitochondria preparations but not in the intact cells. Formation and Properties of The Acid Product of NADH and NADPH NADH, NADPH and other nl—substituted dihydropy— ridines have been shown to be unstable in acid (11, 20). The characteristic 3H0 nm absorbtion peaks of the reduced coenzymes are lost with the concurrent formation of a new peak at 290 nm. This latter peak disappears rapidly at pH 1 but can be stabilized by the addition of bisulfite or by neutralization immediately after the formation of the primary acid product. The primary acid product decomposes relatively slowly between pH 3 and 5. Burton and Kaplan (A) have suggested that the primary acid product is formed by the opening of the heterocyclic ring yielding an amino aldehyde. Bisulfite is thought to stabilize the acid product by addition through the oxygenated grouping of the aldehyde at the 6 position of the ring. The results presented by Anderson (1) are complicated by the fact that two different systems seem to have been operative. Since NADH and NADPH are rapidly converted to enzymically inactive coenzymes under acid conditions, these would have been inactive in the ETS oxidations carried out by Anderson with whole yeast cells. However, his experiments with crude yeast extracts were carried out at neutral pH, and these would have involved true coenzyme oxidations in the systems studied. EXPERIMENTAL METHODS Culture and Cultural Techniques The yeast used in these experiments was from two sources. In one experiment, a stock strain of baker's yeast from our laboratories was used to produce fresh cells in the following manner: 250 ml volumes of dextrose broth (Difco) were adjusted to pH “.0 with tartaric acid, innoculated with stock yeast and allowed to grow in shake flasks at 30 C for 2A hours. The cells were harvested by centrifugation, washed two times with 0.1 M KH2P0u and stored at 0 C until use. In all other experiments, baker's yeast purchased locally was air dried and maintained in the freezer. Just prior to use, the dried yeast was washed three times with distilled water, re—suspended in distilled water and incubated on a rotary shaker for three hours to lower the endogenous metabolic rate. Cell weights are reported as dry weights and were determined by placing 1 ml of cell suspension at 110 C for 24 hours. Oxidation Techniques Standard Warburg techniques as described by Umbreit et a1. (17) were employed for measurements of 02 uptake and CO2 reaction mixture were added to the main compartment of evolution. All components of the the Warburg flask except substrates which were tipped in from a side arm after thermal equilibrium was attained. Air was used as the gas phase in all experiments. Carbon dioxide was determined by the direct method; and 0.2 m1 of 20% KOH along with a strip of filter paper was placed in the center well of the cups used to measure oxygen uptake. Reaction mixtures were adjusted to the pH of the experiment with potassium phthalate, glycine— hydrochloride buffer or potassium phosphate buffer. The pH was determined at the termination of the experiment (final pH). In each case where used, the respiratory quotient (RQ) was defined as umoles of CO2 evolved/ umole 02 consumed. Assay and Cleavage of NADH The NADH and related compounds were obtained from various commercial sources as specified in the Results, and were stored at 0 C until use. A modification of the lactic dehydrogenase assay of Kornberg (13) was employed to measure the coenzymatic purity of a number of commercial preparations of NADH. Snake venom phosphodiesterase was purchased from Sigma Chemical Company and was used to split NADH by the method of Razzel and Khorana (15). Purification of the Oxidizable Contaminant Purification of an oxidizable fraction(s) present in NADH was achieved with 200—A00 mesh Dowex 1—X8, OH— form. The resin was washed overnight in A N NaOH followed by five volumes of distilled water. Further purification was achieved with 200—“00 mesh Dowex 50W—XA, H+ form. The resin was activated for use in the same way as the Dowex l-X8 except that 5 N HCl was substituted for the NaOH. Columns of about 0.5 x 3 cm were used with each resin. The columns were washed with at least ten volumes of distilled water prior to use. RESULTS Studies on The Oxidation of NADH by Yeast Cells Nature of the oxidation Varying amounts of 0 uptake and C0 evolution 2 2 by yeast cells were noted when similar concentrations of various commercial brands of NADH were added as substrate. In earlier work, the pH of the reaction mixtures was below 5 suggesting that the acid modification product of NADH was the species being oxidized. Figure 1 shows the oxidation observed with two commercial brands of NADH. The respiratory quotient (RQ), adjusted for endogenous activity, for the oxidation of the lot obtained from Sigma was 0.62 while the RQ for that from Boehringer and Soehne was 0.72. Yeast cells for these oxidations were from two different sources, as indicated. A direct comparison of the extent of O uptake by commercial 2 dried yeast was then made using a number of different lots of NADH from different sources. As is evident in Fig. 2, the yeast oxidized some component(s) of all lots, but there was considerable variation in the extent of oxida— tion among lots. 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