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The concentrations of glycolate (hydroxyacetate) and lactate are significantly elevated above control values in urines from streptozotocin-diabetic rats, regardless of whether data are expressed in terms of μg/ml urine or μg/day. The same levels of oxalate and glyoxylate are excreted in 24 h in the urines from normal and diabetic rats. Lactate levels are elevated above control values in serum from streptozotocin-diabetic rats. The elevation of glycolate levels in diabetic rat urine compared to control values occurs regardless of diet and regardless of whether rats were fed or fasted during the 24 h urine collection period. Rat liver glycolate oxidase may be used to assay glycolate concentrations in the presence of up to 500 μg/ml l -lactate when pH 8.6 Tris-Cl is used as buffer. Results obtained with this assay compare qualitatively with the standard colorimetric assay using 2,7-dihydroxynaphthlene for glycolate determination. Beef liver glycolate oxidase is not effective for use in glycolate assays. The identity of urinary glycolate was confirmed by gas-liquid and by paper chromatography.

A spectrophotofluorometric assay was used to measure tryptophan oxygenase activity in several species. The fluorescent assay depends on the conversion of the product of the reaction, N-formyl- l -kynurenine, to anthranilate by means of the coupling enzymes kynurenine formamidase and kynureninase. These enzymes are easily obtained from l -tryptophan-induced N. crassa; and the product, anthranilate, is readily separated by organic extraction from other tryptophan catabolites and easily identified fluorometrically. With this assay, tryptophan oxygenase has been demonstrated in vitro for the first time in N. crassa.

A particulate enzyme preparation obtained from rat liver catalyzes the transfer of glucosamine from UDP- N -acetyl- 14 C-glucosamine to endogenous protein acceptor(s). This transfer activity is found to be much higher in smooth microsomes than in rough microsomes. Unlabeled GDP-mannose stimulates incorporation, while other sugar nucleotides tested do not. The endogenous acceptor appears to be compartmentalized within the microsomes, but can be released by treatment with detergents or ultrasonic vibrations. Pronase digestion converts the products of this reaction from trichloroacetic acid-insoluble proteins to trichloroacetic acid-soluble glycopeptides. The radioactive label is incorporated as glucosamine, as identified by cochromatography on paper with authentic glucosamine, following acid hydrolysis of pronase glycopeptides.

The phospholipides extracted from rabbit liver slices which have been exposed to dl -leucine or dl -tyrosine were found to incorporate these amino acids as a part of their molecular structure. The amino acids are chemically bound to the phospholipides and could not be separated from the lipide complex by electrophoresis. The amino acids could be demonstrated in the aqueous fraction of the acid hydrolysis of the phospholipides by paper chromatography. Phospholipides extracted from liver cells which had been heated at 60 ° for 45 min. prior to incubation with leucine-1-C 14 did not incorporate the radioactive compound and there was no in vitro adsorption of C 14 -labeled leucine by the purified phospholipides.

The lipids of freeze-dried liver mitochondria of nonirradiated and x-ray irradiated rats have been examined by chemical analysis, paper chromatography, infrared spectroscopy, and gas-liquid chromatography. A polyglycerolphosphatide fraction of great similarity with bis(phosphatidic) acid has been isolated and tentatively characterized. Fatty acid analysis suggests that this fraction is not composed of a single lipid but of a group of lipids of different fatty acid composition. Ionizing irradiation of the animals causes a significant increase of the mitochondrial polyglycerolphosphatides. As the other fractions were not similarly affected by the irradiation, polyglycerolphosphatide became one of the major lipid fractions of the liver mitochondria of irradiated rats.

The isolation of geranyl geranyl pyrophosphate synthetases from carrot root and pig liver and the properties of these enzymes are reported. Each of the enzymes catalyzes the formation of geranyl geranyl pyrophosphate from isopentenyl-4-C 14 and farnesyl pyrophosphates. The product of the reaction has been characterized by gas-liquid and paper chromatography and derivative formation. An absolute metal requirement has been demonstrated for the reaction and Mn ++ is the most effective metal in stimulating activity. The pH optimum of the reaction is 6.7–6.8 and 6.9–7.0, respectively, for the carrot root and pig liver enzymes. K m values calculated for farnesyl pyrophosphate and isopentenyl pyrophosphate are 1 × 10 −4 M and 1 × 10 −5 M respectively, for the carrot root enzyme, and 7.6 × 10 −5 M and 6.6 × 10 −6 M , respectively, for the pig liver enzyme.

Coenzyme A-glutathione disulfide has been demonstrated to be a normal constituent of rat liver in an amount of 36 nmoles per gram. The normal levels of two enzymes which act on coenzyme A-glutathione were established. They are glutathione: coenzyme A-glutathione transhydrogenase and coenzyme A-glutathione reductase. The level of coenzyme A-glutathione did not change in diabetic rats nor did the activities of either of these enzymes if they were expressed as units per mg of protein. Methods are described for the preparation of [14C]coenzyme A, [14C]coenzyme A disulfide, and [14C]coenzyme A-glutathione disulfide, each labeled in the adenine ring, as well as [3H]coenzyme A-glutathione disulfide labeled in the glutathione moiety.

The isolation and partial purification of farnesyl pyrophosphate synthetase of pig liver are described. This enzyme does not have isopentenyl pyrophosphate isomerase, geranyl pyrophosphate synthetase, or geranyl geranyl pyrophosphate synthetase activities. Farnesyl pyrophosphate synthetase catalyzes the formation of farnesyl pyrophosphate through the condensation of geranyl and isopentenyl pyrophosphates. The product of this reaction has been characterized by paper and gas-liquid chromatography. A pH of 7.0 is optimum for the reaction, and Km values for geranyl and isopentenyl pyrophosphates are 4.0 × 10−6 and 2.0 × 10−6M, respectively. An absolute metal requirement has been established for this reaction and Mn++ is approximately 40 times more effective than Mg++ at low ion concentrations. At pH 7.0, 2 × 10−3M iodoacetamide does not inhibit the reaction, but 1 × 10−5M p-hydroxy mercuribenzoate completely inactivates the enzyme.

The intraperitoneal injection of large amounts of pyridine nucleotide coenzymes into mice results in an elevated level of liver pyridine nucleotides. These pyridine nucleotides are mainly present in the oxidized form. The highest levels of liver coenzymes are observed after the administration of DPNH. Administration of the coenzymes per os also resulted in an increase in liver DPN + . Studies with radioactive coenzymes indicate that the pyridine nucleotides may enter the liver without cleavage.

Cystathionine has been identified by paper chromatography as a constituent of the liver following the feeding of both l -methionine and l -serine to a fasting rat. l -Methionine alone was ineffective.

A sensitive assay has been devised for glutathione- S -arene oxidase transferase using as substrates naphthalene-1,2-oxide or styrene oxide along with [ 35 S]glutathione. Activity of the order of 2–3 nmoles of conjugate formed during a 5-min incubation can be detected. This yields about 2000 cpm above a blank of about 1500 cpm. Transferase activity was found mainly in liver and kidney but was also present in most other tissues of rats. Glutathione- S -arene oxide transferase has been purified 70- to 80-fold from sheep liver 100,000 g supernatants using the conventional procedures. After electrofocusing, enzyme activity separated into two major peaks and two or three minor peaks, ranging in isoelectric point from pH 6.5 to 7.5. Activities assayed with naphthalene-1,2-oxide or styrene oxide as substrates were found to almost parallel each other in all the peaks. The sheep liver transferase required neither metal ions nor cofactors such as FAD, pyridoxal-phosphate and thiamine pyrophosphate. The molecular weight of the transferase has been estimated to be about 40,000. K m values for glutathione, naphthalene-1,2-oxide, and styrene oxide are 1.6, 0.11, and 0.13 m m , respectively. K m values for glutathione decreased with increasing pH, whereas the K m values for naphthalene-1,2-oxide were independent of pH in the range of 6.5–8.

β-N-Acetylhexosaminidase A and B were partially purified from normal human liver using DEAE-cellulose column chromatography. Hexosaminidase B was also purified from the livers of patients who had died of Tay-Sachs disease. The hexosaminidase fractions were tested for their ability to hydrolyze the amino sugar moiety of synthetic substrates and of three amino sugar-containing glycolipids, GA2, globoside, and GM2. Hexosaminidase A and B had similar ability to catalyze the hydrolysis of synthetic N-acetylglycosaminides. The hexosaminidase B from the liver of a Tay-Sachs disease patient had a slightly higher Michaelis constant toward the same substrates as the control hexosaminidase B. Radioactively labeled GA2, N-acetyl-[1-14C] galactosaminyl (β 1 → 4)-galactosyl (β 1 → 4)-glucosyl (β 1 → 1)-ceramide was prepared biosynthetically. Hexosaminidase A and B from normal liver had identical Michaelis constants for the hydrolysis of GA2; whereas, the hexosaminidase B from a Tay-Sachs disease patient possessed a higher Michaelis constant. Globoside was hydrolyzed by both hexosaminidase A and B, although hexosaminidase B has a significantly lower Michaelis constant than the hexosaminidase A. Under the conditions of incubation neither hexosaminidase A nor B catalyzed the hydrolysis of the N-acetylgalactosaminyl residue from ganglioside GM2. However, in the presence of a neuraminidase preparation from Clostridium perfringens plus hexosaminidase A or B at pH 3.8 there was significant enzymatic hydrolysis of GM2 to ceramide lactose. Both GM2 and globoside were found to be excellent competitive inhibitors of GA2 hydrolysis by hexosaminidase B. These results are discussed with respect to the accumulation of these glycolipids in two GM2 ganglioside-storage diseases.

Fructose diphosphate aldolases have been isolated as crystalline proteins from rat liver, muscle and the Novikoff hepatoma. All three enzymes exhibit identical properties in sedimentation velocity and sedimentation equilibrium ultracentrifugation, corresponding to a molecular weight of 158,000. Dissociation in guanidinium chloride yields subunits of molecular weight approximately 40,000 in each case. Tumor and muscle aldolases exhibit identical electrophoretic properties on cellulose acetate or disc gel which are markedly different from those of liver aldolase. The tumor and muscle enzymes are 50 times more active with fructose-1,6-P2 than with fructose-1-P and treatment of these enzymes with carboxypeptidase A to remove the COOH-terminal tyrosine residues decreases the rate of cleavage of fructose-1,6-p2 so that it becomes equal to the rate of cleavage of fructose-1-P. Native rat liver aldolase, on the other hand, cleaves both substrates at equal rates and digestion with carboxypeptidase, while also liberating tyrosine from the COOH-terminus, does not affect the substrate specificity. The amino acid compositions of muscle and heppatoma aldolases were found to be identical and to differ significantly from that of the rat liver aldolase. Tryptic fingerprints of muscle and tumor aldolases are identical and differ significantly from that of liver aldolase. However, a number of peptides, including the active site peptide, are similar or identical in all three aldolases. The results provide support for the conclusion that the liver aldolase is replaced by muscle aldolase during the development of the Novikoff hepatoma.

The hydroxylation of a variety of specifically deuterated aromatic substrates by liver microsomes has been investigated. Labeled substrates which cannot readily ionize by loss of a proton from the ring substituent display deuterium retentions varying from 40 to 64% upon hydroxylation at the ring position bearing the isotopic label. These substrates include compounds with either electron-donating or electron attracting substituents (anisole-4- 2 H, diphenyl ether-4- 2 H, biphenyl-4- 2 H, N -methyl- N -(phenyl-4- 2 H)-benzenesulfonamide, toluene-4- 2 H, fluorobenzene-4- 2 H, chlorobromobenzene-4- 2 H, benzonitrile-4- 2 H, benzamide-4- 2 H, and nitrobenzene-4- 2 H). Substrates that can, however, ionize by loss of a proton from the ring substituent exhibit lower retentions ranging from 0 to 30% at pH 8. This class of compounds includes phenols such as salicylic-5- 2 H acid, aniline-4- 2 H, N -(phenyl-4- 2 H)-benzenesulfonamide, and various N -acylanilines-4- 2 H. As the acidity of the amide hydrogen increases in the acylanilines, migration and retention of deuterium decreases. A mechanism is presented which accommodates the retention values observed for both classes of substrates. The degree of retention for acetanilide-4- 2 H is strongly dependent upon the pH of the incubating medium ranging from 21 (pH 10) to 44% (pH 6). N -(Phenyl-4- 2 H)-benzamide shows a similar pH dependence whereas nonionizable substrates (biphenyl-4- 2 H or anisole-4- 2 H) do not display this dependence. The relative reactivity of the substrates studied toward the microsomal hydroxylating system correlates well with their reactivity toward chemical reagents which cause electrophilic substitution: i.e., reactive or electron-rich rings act as the best substrates. In chemical terms, the microsomal hydroxylating system acts as though it is a weak, selective electrophile.

The metabolism of d - and l -lysine has been studied in the chicken using both 14C and 15N labels. In contrast to the rat, the chicken actively metabolized d -lysine and l -pipecolate. The data indicate that d -lysine is degraded via conversion to pipecolate, α-aminoadipate, α-ketoadipate, and eventually to CO2. The α-nitrogen atom of d -lysine is the first one removed from the carbon skeleton by this route. l -Lysine is metabolized by two alternate pathways which converge at α-aminoadipate. The major pathway, which results in the retention of the α-nitrogen atom of l -lysine in α-aminoadipate, includes saccharopine as a precursor of α-aminoadipate and is thus identical to l -lysine catabolism in rats. The pathway which is indicated to be the minor one includes l -pipecolate as a precursor of α-aminoadipate and results in the retention of the ϵ-nitrogen atom of l -lysine in α-aminoadipate. Even though saccharopine is indicated to be an intermediate in l -lysine breakdown, a large percentage of an intramuscularly injected dose is excreted unchanged in the urine.

The oxidation of both N1-methylnicotinamide and pyridoxal appears to be catalyzed by a single liver enzyme, aldehyde oxidase (aldehyde:oxygen oxidoreductase, EC 1.2.3.1). This notion is based on the following evidence: (1) Inbred strains of mice having characteristic and different levels of N1-methylnicotinamide oxidase activity also differ in their abilities to oxidize pyridoxal; the levels of the two activities have been found to vary among the strains in a parallel fashion. (2) The Km values of the N1-methylnicotinamide oxidizing activity for N1-methylnicotinamide and pyridoxal oxidizing activity for pyridoxal also vary among the inbred strains of mice in a parallel fashion. (3) The genetic determinants of pyridoxal and N1-methylnicotinamide oxidizing activity in the mouse do not segregate in the F2 generation or in backcrosses to the parental strains. (4) The levels of N1-methylnicotinamide and pyridoxal oxidizing activities in male mice increase in a parallel fashion upon the attainment of sexual maturity. (5) Competition of N1-methylnicotinamide and pyridoxal for oxidation in vivo has been demonstrated in the rat. (6) The inactivation of N1-methylnicotinamide oxidase activity which is connected with catalytic performance results in an equal loss of pyridoxal oxidase activity. A parallel loss in activity for both substrates also results from the catalysis of pyridoxal oxidation. In the course of these studies, a genetic analysis of aldehyde oxidase in the C58/J and C57B1/6J strains of mice was conducted. The earlier conclusion that aldehyde oxidase is controlled by a single autosomal locus with alleles acting in a codominant manner has been confirmed.

In man and rat two enzymes have been found to catalyze the formation of the pyridones of N 1 -methylnicotinamide in vivo . Aldehyde oxidase, the sole apparent functionary in most mammalian species, shares its role with xanthine oxidase in these two species. Although purified xanthine oxidase had previously been shown to be capable of catalyzing the formation of N 1 -methyl-2-pyridone-5-carboxamide (2-pyridone) at pH values above 8.0, this is the first documentation of such catalysis in vivo . The aldehyde oxidases of both man and the rat are like all other mammalian aldehyde oxidases in that they catalyze the formation of both 2-pyridone and N 1 -methyl-4-pyridone-3-carboxamide (4-pyridone). The human and the rat liver enzymes differ from other mammalian aldehyde oxidases in that the proportions of the products vary as a function of the inactivation of these enzymes which occurs during catalysis. All of these results are compatible with the idea that, in mammals, a single enzyme, aldehyde oxidase, is capable of catalyzing the oxidation of N 1 -methylnicotinamide to two alternate products and that in the rat and man xanthine oxidase can contribute to the formation of some additional 2-pyridone.