Tyrosine aminotransferase activity of frog (Rana esculenta) liver—III. A circannual study

• 1.1. A circannual study of tyrosine aminotransferase and other metabolic enzymes in frog liver is reported. The subcellular distribution of all enzymatic activities under investigation was also studied.
• 2.2. Results show significant oscillations of all enzymatic activities throughout the year; in particular tyrosine aminotransferase has a marked summer maximum.
• 3.3. The subcellular distribution of tyrosine aminotransferase shows significant variations: the soluble activity of the enzyme presents a bimodal circannual distribution, which has its counterpart in an increased activity of heavier fractions.

     

NAD(P)H dehydrogenase from rabbit and rat liver: Purification and some properties

• 1.1. NAD(P)H dehydrogenase from rabbit liver was purified to electrophoretic homogeneity using a procedure also found applicable for the rat liver enzyme.
• 2.2. Rabbit and rat liver enzymes showed different behaviour in isoelectric focusing and different K_m values and turnover numbers.
• 3.3. Both enzymes were inhibited to similar extents by warfarin.
• 4.4. The rabbit enzyme is composed of two subunits of mol. wt 27,000 and contained 1 FAD group per subunit.
• 5.5. Some absorption and circular dichroism properties of the rat enzyme are shown.

     

Effects of high dietary methionine on activities of selected enzymes in the liver of kittens (felis domesticus)

• 1.1. Growing male kittens were fed an 18% casein diet supplemented with 2, 3, or 4% l-methionine (MET) for 6 weeks.
• 2.2. Free MET concentration in liver increased 30-fold and cystathionine two- to three-fold; the activity of adenosyl-MET transferase and cystathionase also increased but remained lower than previously found in rats.
• 3.3. Taurine concentration in liver decreased in cats fed excess MET and appeared to depend on taurine intake.
• 4.4. Alanine aminotransferase activity was high in all groups while serine dehydratase activity was very low.
• 5.5. Pyruvate kinase and malic enzyme activities which are normally low in cat liver increased after excess MET. Also, glucose 6-phosphate and 6-phosphogluconate dehydrogenases increased.
• 6.6. Cat liver metabolism showed limited adaptation to an excess dietary intake of methionine compared to that found in rats.

     

A quantitative study of the subcellular distribution of aspartate aminotransferase isozymes in rat liver

• 1.1. Between 15 and 19% of the aspartate aminotransferase activity of rat liver is of cytosolic origin; the remainder is localised entirely in the mitochondria.
• 2.2. Mitochondria do not contain detectable levels of the cytosolic isozyme or vice versa.
• 3.3. In solutions of low ionic strength, damaged mitochondria release only small amounts of aspartate aminotransferase. but the enzyme is released quantitatively by exposure to high concentrations of salt.

     

Subunit homologies of lactate dehydrogenase (LDH) isoenzymes between amphibians (Xenopus laevis) and mammals (Wistar rat)

• 1.1. The subunit distribution and subunit homologies of LDH isoenzymes were studied in the amphibian Xenopus laevis and in Wistar rats.
• 2.2. Several of the 11–15 isoenzymes of the pattern in Xenopus, separable by vertical starch gel electrophoresis, were purified, hybridized, and the cross-reaction of antibodies against the most positively charged isoenzyme with the isoenzymes present in tissue extracts of both species was tested.
• 3.3. The isoenzyme with the highest positive charge in Xenopus is a M-homotetramer homologous to mammalian LDH₅.
• 4.4. The multibanded pattern of Xenopus LDH isoenzymes is very probably due to heterozygoty of the gene locus controlling the synthesis of M-subunits rather than to an epigenetic subbanding phenomenon.

     

A study of glycerolphosphate acyltransferase in rat liver mitochondria-submitochondrial localization and some properties of the solubilized enzyme

• 1.1. Glycerolphosphate acyltransferase (GPAT) was solubilized from the rat liver mitochondrial membranes using sodium cholate. Dithiothreitol was necessary to stabilize the solubilized enzyme on storage.
• 2.2. Unlike the enzyme in situ in mitochondrial membranes, the solubilized mitochondrial GPAT was susceptible to inhibition by N-ethylmaleimide; a property more characteristic of the distinct microsomal form of GPAT.
• 3.3. Solubilized mitochondrial GPAT retained its very high preference for saturated acyl-CoA substrate (palmitoyl-CoA) and had no activity whatever with any tested concentration of the unsaturated substrate oleoyl-CoA.
• 4.4. Solubilization increased the affinity of mitochondrial GPAT for palmitoyl-CoA whilst decreasing the K_m for glycerol phosphate.
• 5.5. After separation of liver mitochondrial outer and inner membranes and estimation of cross-contamination by appropriate markers it was concluded that the mitochondrial inner membrane contains significant GPAT activity. This was established with preparations from fed, 48 hr-starved and streptozotocin-diabetic rats.

     

Cooperative atp inhibition and fructose-1,6-biphosphate activation of rat liver pyruvate kinase

• 1.1. For the determination of relationship between FDP and ATP in the rat liver pyruvate kinase regulation, kinelic studies have been carried out at several ATP and FDP concentrations.
• 2.2. The results obtained on FDP activation show a great cooperativity for FDP saturation with a Hill coefficient of h = 2.79.
• 3.3. Kinetic studies on ATP inhibition also show a great cooperativity for ATP saturation (h = 2.84) at high FDP concentrations.
• 4.4. These results may contribute to explain the regulation of rat liver pyruvate kinase accounting for the activity of this enzyme at high FDP concentrations modulated by small changes in ATP concentrations.

     

Purification and partial characterization of the alkaline phosphatase from Allolobophora caliginosa

• 1.1. A purification of the enzyme from the starting material was achieved by means of butanol and acetone fractionations and, successively, by DEAE cellulose and Sephadex G-200 chromatographies.
• 2.2. Two enzymatic forms were separated; they showed various similar characteristics but differed greatly in specific activity.
• 3.3. It is probable that in A. caliginosa a sole alkaline phosphatase form exists and the less active fraction is partly denatured enzyme.
• 4.4. It is not completely possible to exclude the existence of two isoenzymes.

     

Uptake by rat liver of circulating amyloglucosidase in vivo and in perfusion

• 1.1. Following administration of purified amyloglucosidase from Aspergillus niger to rats, the enzyme accumulated in a liver lysosome-containing fraction. However, there was not extensive breakdown of liver glycogen. Most of the enzyme disappeared from blood over several hours.
• 2.2. In the isolated perfused liver, uptake of amyloglucosidase added to the perfusion medium was less extensive than that in the intact animal. There was no associated alteration of liver glycogen content or structure.
• 3.3. These results suggest that amyloglucosidase which can accumulate in liver is not accessible to the bulk of liver glycogen.

     

Reductive metabolism of D-glyceraldehyde,L-glyceraldehyde and dihydroxyacetone in rat liver: Identification of alcohol dehydrogenase as the main responsible enzyme

• 1.1. The paper describes NADH- and NADPH-dependent enzyme activities in rat liver which catalyse the reduction of the following substrates: d-glyceraldehyde, l-glyceraldehyde and dihydroxyacetone. Test conditions for the optimal rates of the oxidoreductase reactions are described.
• 2.2. As a test of metabolic relevance of these activities the hormonal status of the rats was changed by pretreatment with alloxan.
• 3.3. This lowers all described activities if the concentration of blood glucose is increased. But there is also a range of elevated activities which are not associated with changes in the glucose concentration.
• 4.4. It is shown that rat liver alcohol dehydrogenase (EC 1.1.1.1) 2 is the enzyme which catalyses the reduction of the substrates named above and also of acetaldehyde with NADH and NADPH. The preparation and characterization of the enzyme are described.

     

DNase-I-like enzyme from the carp liver—Inhibition by muscle and endogenous actin

• 1.1. DNase-I-like activity occurs in the carp (Cyprinus carpio) liver cytosol (supernatant 105,000g).
• 2.2. The enzyme resembles DNase I from bovine pancreas in respect to the molecular mass (~31 kDa), pH (7.4) and ion requirements (Mg²⁺, Ca²⁺) and the ability to degrade native as well as denatured DNA.
• 3.3. As judged by comparison of DNase zymograms obtained after native- and SDS-PAGE, the enzyme occurs in the three molecular forms of similar molecular weight and different charges.
• 4.4. All these forms are inhibited by rabbit skeletal muscle actin as well as by endogenous actin isolated from the carp liver cytosol.
• 5.5. DNase from the carp liver cytosol does not interact with the antibodies directed against DNase I from bovine pancreas and against DNase I from the rat and bovine parotid glands.

     

Detergent solubilisation of rat liver particulate cyclic AMP phosphodiesterase

• 1.1. Detergent solubilisation of particulate rat liver low K_m cyclic AMP phosphodiesterase in the presence of protease inhibitors yields a form of the enzyme with a larger molecular weight than the form solubilised by protease treatment.
• 2.2. The detergent solubilised enzyme could be partially purified by anion exchange chromatography.
• 3.3. It displayed a marked tendency to precipitate from solution when detergent was removed.

     

Studies on tryptophan pyrrolase of Schistocerca gregaria försk. (Orthoptera)

• 1.The trytophan pyrrolase activity of central fat bodies of S. gregoria hoppera was studied.
• 2.The enzyme system appears to be similar to that of mammalian liver.
• 3.The enzyme was localized only in central fat bodies.
• 4.Extracts of other body parts can mimic an enzyme activity because of a degradation of ommochromes in the enzyme test.

     

Comparative study of mammalian uridine kinases in the soluble and Zn-precipitated form

• 1.1. Uridine kinase partially purified from various tissues of higher organisms can be precipitated by means of Zn²⁺-ions without a substantial loss of enzyme activity.
• 2.2. Using an extract from Escherichia coli a similar procedure resulted in the inactivation of uridine kinase.
• 3.3. Uridine kinases from various tissues of mice and rats differ in their thermal stabilities during incubation as cell-free extracts, partially purified enzymes as well as Zn-insolubilized and freeze-dried enzyme preparations.
• 4.4. The highest thermal stability displays Zn-complexed uridine kinase prepared from the kidney and the lowest stability enzyme precipitated from the liver.

     

Host-parasite interaction of tyrosine metabolism in rats infected with trypanosoma brucei

• 1.1. p-Hydroxyphenylpyruvate, the by-product of tyrosine metabolism by T. brusei, was found to be non-toxic to the rat.
• 2.2. A model is proposed for host-parasite interaction in the metabolism of tyrosine.
• 3.3. Oxygen uptake data suggest a conversion of parasite respiration to the Kreb's cycle during glucose deprivation conditions in the host, fueled by products of tyrosine metabolism.

     

Separation of β-N-acetylglucosaminidase isoenzymes from liver and plasma of six mammalian species by DEAE-cellulose chromatography

• 1.1. The β-N-acetylglucosaminidase (EC 3.2.1.30) activities of human, pig, calf, lamb, rat and rabbit liver and plasma have been investigated.
• 2.2. All preparations had maximum activity between pH 4.0 and 4.5 and K_m values with the substrate 4-methylumbelliferyl-2-acetamido-2-deoxy-β-d-glucopyranoside ranged from 0.54 to 2.54 mM.
• 3.3. The isoenzyme profiles of liver and plasma β-N-acetylglucosaminidase activity were compared using DEAE-cellulose chromatography. In all species the major anionic component of liver (β-N-acetylglucosaminidase A) was eluted at a higher salt concentration than the most anionic plasma isoenzyme.
• 4.4. The plasma β-N-acetylglucosaminidase A isoenzyme of all species contained sialic acid residues whereas only the rabbit, pig and calf liver isoenzymes were sialylated.

     

Human GMP synthetase

• 1.1. The specific activity of GMP synthetase was measured in several human tissues and found to be highest in cultured skin fibroblasts, followed by bone marrow, leukocytes, erythrocytes. placenta, and liver.
• 2.2. The enzyme from fibroblasts was purified approximately 50-fold by ammonium sulfate fractionation and gel filtration.
• 3.3. The K_m values were determined to be 4.9μM for XMP, 270μM for ATP. and 340 μM for glutamine.
• 4.4. Ammonium sulfate could replace glutamine as the amino donor but was much less efficient.
• 5.5. The enzyme was specific for ATP as the energy source.
• 6.6. Unlike the calf thymus enzyme, the human enzyme has no requirement for a reduced sulfhydryl compound.
• 7.7. Human GMP synthetase is inhibited by ATP, dATP, azaserine, and hydroxylamine.

     

Kinetic study of the inhibition of rat liver ornithine decarboxylase by diamines; considerations on the mechanism of interaction between enzyme and inhibitor

• 1.1. Partially purified rat liver ornithine decarboxylase is inhibited by several diamines including putrescine, 1,3-diaminopropane, cadaverine and p-phenylenediamine.
• 2.2. The inhibition is dependent on pH, being strong at pH above 8 and negligible below pH 6.5.
• 3.3. The kinetic study of the inhibition showed that while the aromatic diamine behaved as a simple competitive inhibitor, the aliphatic diamines presented a more complex pattern of inhibition in which two molecules of inhibitor might bind to the enzyme active site.
• 4.4. The K_I values for the different inhibitors were calculated and the degree of affinity for the enzyme was p-phenylenediamine > putrescine > cadaverine > 1,3-diaminopropane.
• 5.5. A molecular mechanism explaining how one or two molecules of inhibitor can bind to the enzyme is proposed.

     

Mixed function oxidase activity in the harbour seal (Phoca vitulina) from sable is., N.S.

• 1.1. One adult male, eight pups (including two full term foetuses) and nine adult female harbour seals (Phoca vitulina) were analysed for indices of mixed function oxidase (MFO) activity.
• 2.2. MFO activity was present in liver samples, but was at or below detection limits in samples of kidney, lung and pancreas.
• 3.3. Hepatic ethoxyresorufin O-de-ethylase and benzo[a]pyrene hydroxylase activities were similar to those reported in other seals and in other mammals.
• 4.4. Cytochromes P-450 and b₅ concentrations were slightly lower than those observed in other mammals.
• 5.5. MFO activities in newborn pups and foetuses were significantly lower than those in adult females.
• 6.6. No qualitative differences in cytochrome P-450 isozyme distribution between foetal and adult samples could be discerned by electrophoresis.