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Multiple deletions of mtDNA have not only been implicated in aging, but also in a wide variety of pathological conditions. The enzyme system used in long-PCR makes it possible to synthesize the entire mitochondrial genome (16.5 kb), exposing the multiple deletions in mtDNAs implicated in and, at least partially, responsible for these pathologies. But it is not the number or type of anomalous mtDNA that is crucial, rather it is their frequency relative to the number of intact copies of the mitochondrial genome. Our work exposes the necessity of quantitating the number of normal mitochondrial DNAs. The accuracy of the technique and the small sample size required permit one to detect multiple deletions, located in a specific organ, and simultaneously measure the fraction of intact molecules. This fraction can then be correlated with mitochondrial dysfunction to serve both as an indicator of tissue aging and a monitor of an impending myopathy.

A magnesium-dependent adenylyl cyclase activity was found in each of the four lobes of the dogfish pituitary. An unusual feature of the enzyme system of the three lobes of the pars distalis was a marked enhancement of fluoride-activation in the presence of calcium ions at concentrations usually found to inhibit adenylyl cyclase systems. With the neurointermediate lobe enzyme the fluoride-activation was inhibited by calcium ions. In the absence of magnesium ions the enzyme activity of the pars distalis was supported by calcium ions, the calcium-supported activity being greater than the magnesium-supported activity.

Dihydrofolate reductase was inactivated when incubated with the 27,000 × g pellet of lymphoma. Inactive enzyme was shown to be bound to the microsomal fraction and could be reactivated by urea. No acid-soluble material was produced nor did any change in the molecular weight of the enzyme molecule occur.

Washed microsomes prepared from animal tissues possess the capacity to reduce 1-nitrosoadamantane to N-hydroxy-1-aminoadamantane in the presence of an NADPH generating system under aerobic conditions. No reduction of N-hydroxy-1-aminoadamantane to 1-aminoadamantane (amantadine) or of 2-adamantanone to 2-adamantanol occurred under these conditions. Reduced pyridine nucleotide cofactor is needed for the metabolic reaction. Maximum activity was obtained using washed microsomes from rabbit liver. The results obtained imply that the microsomal reduction of a C-nitroso compound involves a different enzyme system than that of a ketone reduction.

The enzyme system for the synthesis of the pteridine pigment, sepiapterin, from 2-amino-4-hydroxy-6-(D- erythro -1',2',3'-trihydroxyprophyl) triphosphate (dihydroneopterin triphosphate) has been found in extracts of Drosophila melanogaster . NADP + or NADPH and Mg 2+ are required for this enzymatic transformation. No sepiapterin is produced when dihydroneopterin is supplied as substrate in place of dihydroneopterin triphosphate.

A transplantable rat fibrosarcoma (in both ascites and solid forms) has been shown to contain and synthesize large amounts of hyaluronic acid. A particulate hyaluronic acid synthesizing system has been isolated from the solid fibrosarcoma and some characteristics of the enzyme system are detailed. The enzyme complex transferred GlcUA or GlcNAc from UDP-GlcUA or UDP-GlcNAc at a rate of approximately 60 nmole/hr/mg protein to extend hyaluronic acid chains by approximately 45,000 daltons.

The reaction by which thyroxine (T 4 ) undergoes monodeiodination in the nonphenolic ring to form 3,3′,5′-triiodothyronine (reverse-T 3 ) has been studied in rat liver. In whole homogenate the conversion of T 4 to rT 3 is obscured by rapid degradation of the product, whereas in cytosol accumulation of rT 3 is linear for 60 min of incubation. In the cytosol system, the rate of rT 3 formation is maximal at pH 8.2, is thiol-dependent, is inactivated by heat, but is not inhibited by anaerobiosis or absence of light. Propylthiouracil is a potent inhibitor of the reaction. The higher pH-optimum of the rT 3 -forming system compared to that of T 4 -to-T 3 conversion indicates that the former reaction is mediated by an enzyme which is distinct from that controlling 3,5,3′-triiodothyronine (T 3 ) formation.

Previous communications have reported an enzyme system in homogenate of rat testis ( Eisenberg and Bolden, 1963 ) which catalyzes the cyclization of the glucose chain to myo-inositol ( Eisenberg, Bolden and Loewus, 1964 ). A study of this reaction in yeast ( Chen and Charalampous, 1965 ) has shown that glucose-6-P and myo-inositol-1-P are intermediates in the cyclization. Since myo-inositol-1-P can exist in enantiomeric forms it was of interest in proposing a mechanism of cyclization to determine which optical isomer is involved. Although the L configuration has been observed repeatedly among the inositol phosphates derived from phosphoinositides from many natural sources ( Rapport and Norton, 1962 ), the D isomer has been known only synthetically ( Ballou and Pizer, 1959 ). Evidence that the intermediate in the formation of inositol in the rat is D-myo-inositol-1-P is presented in this paper.

In recent years attention has been given to an adenosine triphosphatase (ATPase), which has been implicated in active cation transport across membranes. This enzyme system has been extensively studied in crab nerve (Skou 1957 1960), red blood cell membranes (Post 1959; Dunham and Glynn 1961) and cerebral tissues (Jarnefelt 1961; Aldridge 1962; Schwartz et al 1962; Skou 1962). The present report is concerned with the preparation and some properties of an active sodium and potassium-dependent ATPase from heart muscle.

An enzyme system present in the stromal fraction of spinach chloroplasts specifically elongated medium chain trienoic fatty acids to α-linolenic acid with either acetyl or malonyl CoA. Medium chain and long chain saturated and monounsaturated fatty acids as well as α-linolenic acid were ineffective substrates for elongation. These results fulfill an essential requirement for the proposed two pathway systems for polyunsaturation in plants.

This report concerns the synthesis of ribonucleic acid, RNA, by an enzyme system from pea embryos. The enzyme, as prepared, requires as substrate the four riboside triphosphates, ATP, CTP, GTP and UTP, and contains not only protein but a substantial fraction of the cellular DNA.

The in vitro incubation of a crude enzyme extract from bone with lathyritic porcine tendon resulted in the formation of all the reducible components previously demonstrated to be involved as the inter molecular crosslinks responsible for the stabilization of the collagen fibre. It is likely that the enzyme involved is a pyridoxal phosphate cupro-protein. Indirect support for the probable involvement of pyridoxal and copper in the enzyme system has been obtained by a series of model experiments demonstrating that the pyridoxal-copper system is capable of producing the crosslink precursors allysine and hydroxyallysine.

Bremer and Greenberg (1961) observed that the in vitro addition of d,l-(N,N-dimethyl) distearoyl-α-cephalin enhanced the incorporation of C14-methyl into choline by the microsome methylating system, while commercial dipalmitoyl-α-cephalin was inactive. Gibson, Wilson and Udenfriend (1961) similarly observed a lack of C14-methyl incorporation with brain phosphatidylethanolamine or phosphatidyl serine. In this report evidence is presented to show that the same enzyme system will methylate monomethyl 1-(α)-distearoyl cephalin, but not 1-(α)-distearoyl cephalin. +

Reaction mechanisms involved in the action of rabbit muscle lactate dehydrogenase were investigated during experiments on the effect of deuterium on the enzyme system. Results indicate that the mechanism involves one or more ternary complexes. (C.H.)

Summary Cytokinin, [8- 14 C]Benzylaminopurine, metabolism in tomato pericarp was followed during a 3 h period utilizing thin layer chromatography and visualization by fluorography. Fluorography indicated the formation of at least 7 metabolites during 3 h. Cytokinin metabolism was reduced by approximately 40% in 3 h by the presence of 250μM metyrapone, an inhibitor of cytochrome P-450 related enzyme systems. In the presence of metyrapone, the number of radioactive metabolites on the thin layer plate was reduced from 7 to 4 and 2 of these were unique to the metyrapone-treated sample. These data suggest the initial step in benzylaminopurine metabolism in tomato pericarp may be mediated by a cytochrome P-450 related enzyme system which is altered in the presence of metyrapone.

Four isomers of 8,15-diHETE as well as 14,15-diHETEs are isolated and characterized after exposure of 15-HPETE to hemoglobin. It is found that 83% of the C-8 oxygen atoms in 8(R), 15(S)-diHETE and 8(S), 15(S)-diHETE, and 41% of the C-8 oxygen atoms in 8(R), 15(S)-11Z-diHETE and 8(S), 15(S)-11Z-diHETE are derived from H2(18)O. These results suggest that hemoglobin catalyzes the transformation of 15-HPETE into these products via a free radical process, possibly involving the intermediacy of 14,15-LTA. Intact human leukocytes contain a distinct enzyme system for catalyzing the conversion of 15-HPETE into 14,15-LTA. This enzyme activity is inhibited by ETYA and is rapidly denatured upon homogenization of the intact leukocytes.

Carcinogenic arylhydroxamic acids (N-hydroxy-2-acetylamino-fluorene, N-hydroxy-4-acetylaminobiphenyl, and trans -N-hydroxy-4-acetyl-aminostilbene) are irreversible inhibitors of rat and hamster hepatic N,O-acyltransferase. The kinetic characteristics of this inhibition are consistent with suicide inactivation of the enzyme. This finding appears to be the first example of carcinogens serving as suicide substrates for an enzyme system which is responsible for their bioactivation.

Agents such as N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and its derivatives, chlorpromazine and amitriptyline that interact with calcium-regulated modulator protein were found to inhibit not only Ca2+ dependent cyclic nucleotide phosphodiesterase but also Ca2+-Mg2+-ATPase of human erythrocyte ghosts. I50 values of modulator interacting agents for testis modulator-activated, brain modulator-activated and erythrocyte modulator-activated-ATPase are indistinguishable. However, I50 of W-7 for troponin C-activated-ATPase is lower than that for modulator-activated ATPase. The specificity of these agents toward modulator-related enzyme reaction is also shown by the negative effect on modulator-unrelated enzyme system such as erythrocyte ghost protein kinase and Mg2+-ATPase. These agents provide a useful tool for elucidating the physiological role of modulator.

Apo- and holo-fatty acid synthetases of pigeon liver were separated by affinity gel chromatography under conditions similar to, but not identical to, those used in separating subunits I and II of [14C]pantetheine-labeled fatty acid synthetase complex [ Lornitzo et al. , J. Biol. Chem. 249 , 1654 (1974) ]. When [14C]pantetheine-labeled fatty acid synthetases were separated, the enzymatically active holo form contained all of the [14C] label. Incubation of the apo-pigeon liver fatty acid synthetase complex with CoA, ATP and a partially purified pigeon liver soluble enzyme system, from which fatty acid synthetase had been removed, resulted in the formation of holo-enzyme. Activation of apo-fatty acid synthetase could also be achieved by replacing the apo-(4′-phosphopantetheine-less) acyl carrier protein with holo-acyl carrier protein. It is evident, therefore, that the inactive apo-fatty acid synthetase lacks a 4′-phosphopantetheine group.

Compound 102804 isolated from Bacillus cereus has been found to be a potent inhibitor of the N5-methyltetrahydrofolate-homocysteine transmethylase isolated from Escherichia coli B. This inhibition was noted when 102804 was added to the enzyme reaction mixture after the reaction started or concurrently with the preparation of the mixture. Chemically inactivated 102804 has no activity as an inhibitor of this enzyme system.

The effects of alloxan-diabetes on the partial reaction of (Na++K+)-ATPase, K+-activated para-nitrophenylphosphatase, and on ouabain binding were studied in isolated adult dog heart myocytes. The Km of K+-activated para-nitrophenylphosphatase for K+ activation was increased from 2.5 to 7.7 mM with no change in Vmax. The Scatchard plots for ouabain binding between control and diabetic animals were indistinguishable. These results indicate that in acute diabetes induced by alloxan, the number of Na+-K+ pumping sites in the heart is not altered but the affinity of the system for K+ is decreased. It is suggested that the decrease in K+ affinity of the (Na++K+)-ATPase enzyme system is at least in part responsible for the altered K+ homeostasis in the diabetic state.

Clostridium M-E ferredoxin has been chemically modified by replacing the only aromatic amino acid residue it contains, Tyr2, with leucine. The resulting Clostridium M-E [Leu2]ferredoxin, which is devoid of aromatic amino acid residues, is as active as the native Clostridium M-E ferredoxin or as Clostridium acidi-urici ferredoxin as an electron carrier in the phosphoroclastic enzyme system.

It has generally been accepted that the production of sulfate from cysteine (or cystine) occurs through the intermediate formation of cysteinesulfinic acid (CSA) as first postulated by Pirie (1934) . The evidence for the central role of CSA has been largely based on the demonstration that various tissue preparations readily oxidize it ( Fromageot et al., 1948 ; Medes and Floyd, 1942 ; and Singer and Kearney, 1956 ). Also, CSA has been demonstrated in normal rat brain (Begeret and Chatnagner, 1954) and S35 CSA has been isolated from tissues of rats injected with S35 cysteine ( Chapeville and Fromageot, 1955 ). However, no enzyme system has been described which is capable of producing CSA from cysteine, and CSA has not been isolated in the in vitro studies of cystine oxidation to sulfate. The present study involves a system in which S35 cysteine was oxidized to S 35 O 4 = by rat liver mitochondria under conditions where added CSA remained essentially unmetabolized. Also, there was no formation of S35 CSA while S 35 O 4 = was being actively produced. The present system seems to be of quantitative importance for the matabolism of cystine in vitro .

An enzyme system isolated from Pseudomonas oleovorans has been previously shown by others to catalyze the ω-hydroxylation of fatty acids and alkanes. The same system is now shown to catalyze the epoxidation of olefins in the presence of NADH and molecular oxygen. The identities of the reaction products were established by gas chromatographic analyses and chemical tests. On the basis of cofactor oxidation rates, 1,7-octadiene was more reactive than either 1-octene or octane.

Enzymatic conversion of 2-tridecanone to undecyl acetate has been achieved with a crude cell-free system prepared from Pseudomonas aeruginosa . The activities of several other enzymes present in unfractionated, unsupplemented supernatants of this bacterium also are reported.

Previous studies showed that in Escherichia coli detachment of adenylyl groups from the adenylylated form of glutamine synthetase is catalyzed by a complex deadenylylation enzyme system composed of at least two protein components, and is activated by α-ketoglutarate, ATP and UTP. It is now found that cleavage of the energy-rich adenylyl-0-tyrosyl bond of adenylylated glutamine synthetase is coupled with the esterification of orthophosphate to form ADP as the deadenylylation product. No deadenylylation occurs in the absence of orthophosphate. When arsenate is substituted for orthophosphate, AMP is the only product of deadenylylation.