Harold J. Strecker

Glutamate dehydrogenase in nuclear and mitochondrial fractions of rat liver.

Abstract Two glutamate dehydrogenases appear to be present in rat liver, one associated with nuclei, and the other with mitochondria. The two enzymes differ in regard to activation by inorganic phosphate, in the effects of pH on activity, in apparent Km values for some of the substrates and in the shape of curve obtained by a plot of NAD concentration versus activity.

Studies of the effect of ionic compounds on the stability of glutamate dehydrogenase

Abstract 1. 1. Glutamate dehydrogenase ( l -glutamate: NAD(P) oxidoreductase (deaminating), EC 1.4.1.3), in solution in the absence of protective ions, is rapidly inactivated as the pH of the solution is raised to 8 or higher. The addition of some ionic compounds greatly retards this alkaline inactivation. 2. 2. Alkaline phosphate and ammonium salts are among the best protective agents; Tris is one of the least effective. 3. 3. The inactivation of glutamate dehydrogenase in aqueous solutions appears to pass through stages which include conversion of an active form, relatively resistant to inhibition by progesterone, dicumarol, o- phenanthroline , thyroxine and fluorenyl-acetamide, to another active form which is more susceptible to the action of these inhibitors. 4. 4. The rapidity of these changes in the glutamate dehydrogenase molecule is related to the nature of the salt solution used to suspend the crystalline enzyme and to that used to dissolve and dilute this suspension.

ALTERNATE PATHWAYS OF PYRIDINE NUCLEOTIDE OXIDATION IN CEREBRAL TISSUE

THE oxidation of reduced pyridine nucleotides by mammalian tissue has been studied with preparations chiefly derived from heart and skeletal muscle, kidney, and liver (for reviews see CHANCE, 1954; VELICK, 1956; MAHLER, 1956). These studies have provided evidence for two alternate pathways of DPNH and TPNH oxidation. One, principally responsible for the coupling of metabolic oxidations to the esterification of inorganic phosphate, appears to be associated with mitochondria1 particles and is strongly inhibited by antimycin A and amylobarbitone (Amytal). The other pathway, whose function is as yet unknown, appears to be associated with microsomal particles and is not inhibited by either antimycin A or amylobarbitone. Some investigators have suggested that in addition liver mitochondria contain. an external oxidative pathway for DPNH which is insensitive to antimycin A and amylobarbitone and is non-phosphorylating. The possible interrelationships among these systems have received both experimental and theoretical attention (LEHNINGER, 1951, 1954; ERNSTER, JALLING, Low and LINDBERG, 1955; DE DUVE, PRESSMAN, GIANETTO, WA-ITIAUX and APPELMANS, 1955; ERNSTER and LINDBERG, 1958). Prior to the present time, studies of these electron transport mechanisms have not been pursued extensively with tissues of the nervous system. BRODY, WANG and BAIN (1952) have studied the distribution of DPNH-cytochrome c reductase and of cytochrome oxidase in four subcellular fractions separated from rat cerebral cortex by a modification of the SCHNEIDER and HOGEBOOM (1950) method of differential centrifugation developed for liver. The cytological nature of their preparations was not completely determined. It seemed especially interesting to study in cerebral tissue alternative pathways of pyridine nucleotide oxidation, since cerebral tissue function is so strikingly dependent on pyridine nucleotide linked glucose metabolism source of energy (MCILWAIN, 1955). We are reporting here some of the results of our studies in which we have attempted to determine the existence of these alternate pathways in nervous tissue, as a their subcellular distribution, and some of their biochemical characteristics.

Purification and some properties of a brain diaphorase

Abstract During the course of our studies of the oxidation of reduced pyridine nucleotides by brain tissue ( Englard and Strecker, 1956 ; Giuditta and Strecker, 1958 , Giuditta and Strecker, 1959 ) we have extracted two enzymes from ox cerebral cortex which catalyzed the oxidation of both DPNH and TPNH by various electron acceptors (Levine et al, 1960). One of these enzymes has been highly purified and appears to differ in some characteristics from other enzymes of animal origin with diaphorase activity. In view of recent reports by Martius (1959) and Ernster et al (1960) on somewhat similar enzymes found in rat and beef liver, we are reporting at this time some of the properties of the brain enzyme.

PURIFICATION AND SOME PROPERTIES OF A SECOND BRAIN DIAPHORASE

PREVIOUS communications ( ENGLARD and STRECKER, 1956; LEVINE et al., 1960; GIUDITTA and STRECKER, 1960) from this laboratory have reported the presence in brain of soluble, dicumarol sensitive, cnzymes which catalyse the oxidation of reduced pyridine nucleotides. One of theseenzymescan beeasily extracted from brain homogenates with either 0.25 M-Sucrose or water. The partial purification and some properties of this enzyme have been previously described (GIUDITTA and STRECKER, 1961). Exhaustive extraction of protein from brain homogenates with water still leaves intact and apparcntly bound to the particulate matter another enzyme which catalyses the oxidation of reduced pyridine nucleotides. This second enzyme can be brought into solution by treatment of the homogenate with phosphate buffer. For convenience in discussing these two enzymes we are tentatively retaining the name diaphorase introduced by ADLER et a/. (1937). To distinguish one from the other, the enzyme extracted with water will be referred to as brain diaphorase I, and the enzyme extracted with phosphate buffer, brain diaphorase 11. This paper describes the purification and some properties of diaphorase 11. The data indicate that the two enzymes are similar with respect to specificity of electron donors and acceptors, in ease of dissociation of the flavin component, and in susceptibility to inhibitors. They differ in stability to low pH, in kinetic constants, and in sensitivity to inhibitors.

Hydrolysis of proteins during dialysis and ultrafiltration

During the course of studies with crystalline glutamate dehydrogenase (EC 1.4.1.3) it was observed that dialysis of solutions of this enzyme resulted in the loss of significant amounts of protein. At pH 8 to 9 and temperature of 4’C, as much as 20% of protein, as determined with a Folin reagent [ 1 ] , was not recovered after 12 hr of dialysis. The procedure caused almost total enzyme inactivation. Passage of intact molecules through the Cellophane casing (Visking division of Union Carbide Corporation) would appear to be excluded, since at the concentration used the molecular weight has been estimated to be about 2 X lo6 [2-41. The dialysate, which was found to contain ultraviolet-absorbing, ninhydrinpositive [5] material, was concentrated in vucuo after freezing, subjected to electrophoresis in 1.5 M HCOOH at 8000 V for 2 hr on Whatman 3 mM paper strips; these were then treated with 0.25% ninhydrin in acetone [6]. After overnight development at room temperature, a number of ninhydrin-positive areas, some corresponding to known amino acids, were readily apparent. Treatment of the concentrated dialysate, before paper electrophoresis, with 6 N HCI at 110°C for 24 hr resulted in a 3-4 fold increase of total ninhydrin value and additional ninhydrinpositive areas on the paper after electrophoresis, suggesting that peptides were present as well as amino acids. Essentially the same results were obtained when solutions of glutamate dehydrogenase (previously passed through a Sephadex G-25 column) were

A stimulatory factor for mitochondrial NADH oxidase

Abstract This laboratory has reported previously that fragmentation of brain mitochondrial fractions resulted in a 7–25 fold increase of the antimycin A-sensitive NADH oxidase, provided that ionic compounds such as inorganic phosphate were present in the incubation media used for enzymatic determination ( Giuditta and Strecker, 1959 ). The oxidation of NADH and of succinate by the Keilin-Hartree heart muscle preparation has been shown also to be stimulated by ionic compounds and by globin ( Keilin and Hartree, 1949 ; Slater, 1949 ; Bonner, 1954 ). Similar observations have been made now with liver and heart mitochondria. Evidence is presented to indicate that the stimulatory effects obtained with added ionic compounds depends at least partially on interaction with a heat stable non-dialyzable factor, which appears to be present exclusively in the mitochondrial fractions of heart, liver or brain homogenates.

Mechanism of Action of Glutamate Dehydrogenase from Various Sources

Among pyridine nucleotide-dependent dehydrogenases, L-glutamate dehydrogenase has been extensively studied, because of its key role in metabolic pathways inter-converting ammo acids and carbohydrates, as well as the complexity of its molecular structure, probably related to control mechanisms.

STIMULATION OF MITOCHONDRIAL NADH OXIDASE BY BRAIN MITOCHONDRIAL EXTRACTS.

MITOCHONDRIAL preparations of various tissues are known to oxidize added NADH very slowly unless they are altered by exposure to a hypotonic medium, by sonic irradiation, by freezing and thawing, by treatment with detergents, or by other relatively drastic procedures (LEHNINGER, 1951 ; CHANCE and WILLIAMS, 1955; ERNSTER, JALLING, Low and LINDBERG, 1955; MALEY, 1957; VIGNAIS and VIGNAIS, 1957; GIUDITTA and STRECKER, 1959; DEVLIN and BEDELL, 1960; BALTSCHEFFSKY, FUDGE and ARWIDSSON, 1960; BORST, 1962). This unmasking of activity has been assumed to depend on the increased mitochondrial permeability to the nicotinamide nucleotide after damage to the mitochondrial membrane (LEHNINGER, 1951 ; LEHNINGER, SUDDUTH and WISE, 1960). Some experimental data, however, are not readily explained in terms of a permeability barrier to exogenous nicotinamide nucleotides. Thus, NADH generated continuously by dehydrogenases added to incubation media external to the mitochondria is readily oxidized by the antimycin A-sensitive respiratory chain (ERNSTER et ul., 1955 ; VIGNAIS and VIGNAIS, 1957; MALEY, 1957). Further, the addition of either /?hydroxybutyrate or acetoacetate results in a 4to 6-fold stimulation of the oxidation of exogenous NADH by rat liver mitochondria (DEVLIN and BEDELL, 1960). Previous work from this laboratory demonstrated that fragmented brain mitochondria, in which presumably no permeability barriers existed, and in which respiratory control was absent, catalysed the oxidation of exogenous NADH only slightly more rapidly than did intact mitochondria unless ionic compounds were added to the incubation media (GIUDITTA and STRECKER, 1959). Studies with the KeilinHartree heart preparation also have indicated that the rate of oxidation of both succinate and NADH is slow in the absence of added ionic compounds (KEILIN and HARTREE, 1949; SLATER, 1949; BONNER, 1954). To gain some insight into the factors regulating the oxidation of NADH by fragmented mitochondria, a study has been made of this electrolyte effect. The data presented below provide evidence for the presence in the mitochondria1 fractions of brain, liver and heart, of a component or components which modify the rate of oxidation of NADH by the antimycin A-sensitive pathway of the respiratory chain.

Some properties of denatured horse heart cytochrome c

Abstract A cytochrome c derivative has been prepared by treating horse heart cytochrome c at pH 11 at 65 °. This material contains two types of iron. One is associated with the chromophoric center of the protein. The other is nonchromophoric and endows the molecule with an ascorbate oxidase property. Spectral observations indicate protein configurational changes, as well as changes in the accessibility of the chromophoric iron to reducing agents. This derivative no longer can function as part of the electron transport chain, but does competitively inhibit cytochrome c in the succinoxidase and the succinate cytochrome c reductase systems, but not in the NADH oxidase system.