The activity and distribution of gamma-glutamyl transpeptidase (y-GT) in human foetal organs

Summary The activity and distribution of y-GT was investigated in a number of organs from human foetuses aged from 14 to 24 weeks post menstruationem. Over this period, enzyme activity increased in the kidney, pancreas and thymus, but decreased in the small intestine. No trend could be established for the liver, although activity was high. In the lung, spleen, brain and adrenals, y-GT was either detectable at very low levels or could not be demonstrated. The possible relationship between y-GT activity in some human tumours and the enzymes level in the corresponding foetal organs is discussed.This work was supported by a grant from the ASFC, SwitzerlandHolder of a Royal Society European Science Exchange Programme Fellowship     

The activity and distribution of gamma-glutamyl transpeptidase (y-GT) in human lung cancers serially transplanted in nude mice.

Gamma-glutamyl transpeptidase (y-GT) activity and distribution were investigated in different types of human lung cancers (three epidermoid carcinomas, one large cell carcinoma) which were maintained by serial transplantation in nude mice. All transplanted tumour fragments were positive for the enzyme. In the epidermoid carcinomas, y-GT levels were related to the degree of tumour differentiation. Enzyme activity in tumour fragments was always higher than that found in normal adult human lung tissue, and was, in general, maintained throughout the transplantation series.     

The activity and distribution of gamma-glutamyl transpeptidase (y-GT) in human lung cancers serially transplanted in nude mice

Summary Gamma-glutamyl transpeptidase (y-GT) activity and distribution were investigated in different types of human lung cancers (three epidermoid carcinomas, one large cell carcinoma) which were maintained by serial transplantation in nude mice. All transplanted tumour fragments were positive for the enzyme. In the epidermoid carcinomas, y-GT levels were related to the degree of tumour differentiation. Enzyme activity in tumour fragments was always higher than that found in normal adult human lung tissue, and was, in general, maintained throughout the transplantation series.This work was supported by a grant from A.S.F.C., Switzerland, and the Jubiläumsspende 1970 der Zürcher KantonalbankN.F. was supported during part of the work by a Royal Society European Science Exchange Programme Fellowship     

Metabolism of retinaldehyde and other aldehydes in soluble extracts of human liver and kidney

Purification and characterization of enzymes metabolizing retinaldehyde, propionaldehyde, and octanaldehyde from four human livers and three kidneys were done to identify enzymes metabolizing retinaldehyde and their relationship to enzymes metabolizing other aldehydes. The tissue fractionation patterns from human liver and kidney were the same, indicating presence of the same enzymes in human liver and kidney. Moreover, in both organs the major NAD(+)-dependent retinaldehyde activity copurified with the propionaldehyde and octanaldehyde activities; in both organs the major NAD(+)-dependent retinaldehyde activity was associated with the E1 isozyme (coded for by aldh1 gene) of human aldehyde dehydrogenase. A small amount of NAD(+)-dependent retinaldehyde activity was associated with the E2 isozyme (product of aldh2 gene) of aldehyde dehydrogenase. Some NAD(+)-independent retinaldehyde activity in both organs was associated with aldehyde oxidase, which could be easily separated from dehydrogenases. Employing cellular retinoid-binding protein (CRBP), purified from human liver, demonstrated that E1 isozyme (but not E2 isozyme) could utilize CRBP-bound retinaldehyde as substrate, a feature thought to be specific to retinaldehyde dehydrogenases. This is the first report of CRBP-bound retinaldehyde functioning as substrate for aldehyde dehydrogenase of broad substrate specificity. Thus, it is concluded that in the human organism, retinaldehyde dehydrogenase (coded for by raldH1 gene) and broad substrate specificity E1 (a member of EC 1. 2.1.3 aldehyde dehydrogenase family) are the same enzyme. These results suggest that the E1 isozyme may be more important to alcoholism than the acetaldehyde-metabolizing enzyme, E2, because competition between acetaldehyde and retinaldehyde could result in abnormalities associated with vitamin A metabolism and alcoholism.     

Magnesium-dependent sphingomyelinase.

An enzyme which requires divalent metals and hydrolyses sphingomyelin to ceramide and phosphorylcholine is present in rat and human brain and practically absent from other organs. The greatest activity is associated with the microsomal fraction. It had an optimal pH at about 7.4, required magnesium or manganese ions and was completely inhibited by EDTA. Triton X-100 was required for optimal activity and this detergent could also be used to partly solubilize the enzyme from rat brain microsomes. Lecithin was hydrolyzed at only 2% of the corresponding rate of hydrolysis of sphingomyelin.     

Measurement of 1-aspartamido-beta-N-acetylglucosamine amidohydrolase activity in human tissues.

The activity of 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglucosylaminase, EC 3.5.1.26) was measured in normal and diseased human liver, brain and kidney. Organs from patients with aspartylglucosaminuria show very little activity. Crude homogenates of human organs show a reaction catalysed by a complex enzyme system. With homogenate, the formation of product was linear with time up to about 6 h. Reaction times longer than 6-7h resulted in a decrease in the total concentration of product. This phenomenon was not found with the partially purified enzyme fraction. Linearity of the enzyme activity with different protein concentrations was found, independent of the incubation time. Longer incubation of the crude homogenate resulted in the utilization of the product, N-acetylglucosamine. This phenomenon was not observed with the partially purified enzyme fraction. This amidase from human organs differs from that obtained from other sources and apparently represents a rather complex enzyme system.     

Indole-3-Pyruvic Acid as an Intermediate in the Conversion of Tryptophan to Indole-3-Acetic Acid. II. Distribution of Tryptophan Transaminase Activity in Plants

Extracts from different organs of 30 plant species belonging to 16 families have been analysed for tryptophan transaminase activity. Only the brown alga Fucus spiralis was found to be devoid of the enzymes. Among the other plants tested, a difference in activity of two orders of magnitude was recorded. None of the genera or families investigated could be considered as particularly rich or poor sources of the enzyme. Extracts from leaves and stem tips contained generally more transaminase activity than extracts from stems and roots. The results are discussed in relation to other reports on the occurrence of the enzyme in plants.     

OCCURRENCE AND LOCALIZATION OF BRAIN PHENOLSULPHOTRANSFERASE

—Rat brain contains the enzyme which forms sulphate conjugates of phenols, phenolsulphotransferase (EC 2.8.2.1), but the physiological role of the enzyme is unclear. The enzyme is unevenly distributed in rat brain, with the activity 13 times higher in the hypothalamus than in the cerebellum. Phenolsulphotransferase does not seem to be primarily located in glial cells. Cultured cells (type C6 astrocytoma) derived from rat glia had less than 1 per cent of the phenolsulphotransferase activity of whole rat brain. Sulphate conjugation of neutral compounds may be important in their removal from brain. The pineal and pituitary glands, areas outside the blood-brain barrier had very low phenolsulphotransferase activity. The activity of the enzyme in brain varied widely among different species: rabbit and rat had much higher levels of activity than mouse or frog; the activity in human brain was intermediate. Phenolsulphotransferase also occurred in other organs, including liver, heart, testes, lung, spleen, salivary glands, and intact or decentralized superior cervical ganglion. There was no correlation of enzyme activity with adrenergic or cholinergic innervation, or with the known roles of various tissues in drug metabolism or detoxification. The enzyme activity does not seem to be under neuronal control since ganglionectomy did not affect the phenolsulphotransferase activity of salivary glands. The precise localization of phenolsulphotransferase remains to be established, as well as the physiological importance of sulphate conjugation of phenols in brain and other organs.     

Subregional and Intracellular Distribution of NADP-Linked Malic Enzyme in Human Brain

High total activity (expressed as μmol/min/g of wet tissue or per milligram of DNA) and differential subregional distribution of NADP-linked malic enzyme was found in autopsy specimens of human brain. Striatum showed the highest activity of malic enzyme, which was two to five-fold higher than that in other human organs tested. High activity was also found in frontal cortex, while the lowest activity of the enzyme in the central nervous system was found in cerebellum, substantia alba, and corpus callosum. In striatum, frontal cortex, pens, and cerebellum more than 80% of total malic enzyme activity was localized in the mitochondrial fraction, while in substantia alba and corpus callosum approximately 60% of the enzyme activity was present in the mitochondrial fraction. Relatively high specific activity of malic enzyme was found in a crude mitochondrial fraction isolated from various regions of human brain. The highest specific activity was found in the mitochondria isolated from striatum (more than 100 nmol/min/mg of mitochondrial protein); the lowest, but still high (approximately 32 nmol/min/mg of mitochondrial protein) was present in corpus callosum. These data and the different ratios of citrate synthase to mitochondrial malic enzyme activities found in different regions of brain suggest that human brain mitochondria, like the mitochondria isolated from other mammalian brains, are extremely heterogenous. A possible role of mitochondrial malic enzyme in human brain metabolism is discussed.     

Cholinesterase activity in tissues of some crab species from the Japan Sea

The comparative study of the cholinesterase activity in some crab species was carried out for the first time with use of a set of thiocholine substrates. The substrate specificity was studied in stellar nerve, heart, and hemolymph of three crab species. The crab hemolymph was shown to be characterized by the highest enzyme activity. The enzyme from various crab organs has different structure o substrate specificity. Properties of crab enzymes were compared with acetylcholinesterase (AChE) of human blood erythrocytes, butyrylcholinesterase (BuChE) of horse blood serum, enzyme of squids and bivalve molluscs. The obtained data allow the conclusion to be made on differences in properties of enzymes both at the interspecies and at the tissue levels.     

Electroimmunochemical quantification of UDP-glucuronosyltransferase in rat liver microsomes

Microsomal UDPglucuronosyltransferase(1-naphthol), an enzyme form previously shown to be selectively inducible in rat liver by 3-methylcholanthrene-type inducers, was purified to apparent homogeneity. Rabbit antibodies against this enzyme form precipitated UDPglucuronosyltransferase activities towards 1-naphthol and 4-methylumbelliferone faster and to greater extents than enzyme activities towards bilirubin, oestrone and 4-hydroxybiphenyl. Ouchterlony double-diffusion analysis showed immunochemical similarity of the rat liver enzyme with the enzymes from other organs of the rat (kidney, testes) and the mouse liver but not with the enzyme from cat and human liver. Electroimmunochemical quantification of the enzyme indicated that its level was enhanced 1.3-fold and 2.5-fold in liver microsomes from phenobarbital-treated and 3-methylcholanthrene-treated rats, respectively. The results indicate that 3-methylcholanthrene treatment increases the enzyme level of rat liver microsomal UDPglucuronosyltransferase(1-naphthol). Despite phospholipid-dependence of its catalytic activity microsomal enzyme activity appears to be a good index of the enzyme level.     

Organ Specificity of Isoforms of Starch Branching Enzyme (Q-Enzyme) in Rice

The activity and isoenzymes of starch branching enzyme or Q-enzymein the developing endosperm were compared with those in theleaf blade, leaf sheath, culm and root of rice plants. Q-enzymefrom each of these organs could be resolved into two fractions,QE I and QE II, by column chromatography on DEAE cellulose.However, the ratio of the activity of QE I to that of QE IIvaried considerably among the organs. The Q-enzyme from theendosperm was specific for that organ in that the enzyme activity,on the basis of either fresh weight or soluble protein content,was about 100- to 1,000-fold higher than those from the otherorgans. Moreover, in the endosperm, the activity of QE I wasmarkably higher than that of QE II as compared with the relativelevels in other organs. Native polyacrylamide gel electrophoresisfollowed by activity staining revealed that the QE II fractionwas composed of multiple isoforms. The endosperm contained twoisoforms, QE IIa and QE IIb. After electrophoresis on a nativepolyacrylamide gel, QE IIa was detected only in the extractof endosperm, whereas QE IIb was present in extract of all organsexamined. The antiserum raised against QE IIa from the endospermcross-reacted to a considerable extent with QE IIb from thesame organ. However, the antiserum failed to recognize any isoformsof QE II from the other organs. ¹ Present address: National Institute of Sericultural and EntomologicalScience, Tsukuba, Ibaraki, 305 Japan.     

Immunohistochemical detection of betaine-homocysteine S-methyltransferase in human, pig, and rat liver and kidney

Betaine-homocysteine S-methyltransferase (BHMT) has been shown to be expressed at high levels in the livers of all vertebrate species tested. It has also been shown to be abundant in primate and pig kidney but notably very low in rat kidney and essentially absent from the other major organs of monogastric animals. We recently showed by enzyme activity and Western analysis that pig kidney BHMT was only expressed in the cortex and was absent from the medulla. Using immunohistochemical detection, we report here that in human, pig, and rat kidney, BHMT is expressed in the proximal tubules of the cortex. Immunohistochemical staining for BHMT in human, pig, and rat liver indicate high expression in hepatocytes. The staining patterns are consistent with cytosolic expression in both organs.     

The distribution of sorbitol dehydrogenase in embryonic and adult chicken tissues.

Sorbitol dehydrogenase activity (SDH) has been determined in various organs of embryonic and adult chickens. SDH is present in 24-hour embryos, and its activity continues to rise during the next 48 hours. During embryonic development and after hatching, regional differences in SDH activity are demonstrable in the organs of the animal. These differences concern both level of enzyme activity and temporal changes with development. No correlation could be established between enzymatic activity and the fructose concentration of the organs studied.     

Monoacylglycerol hydrolase in human platelets.

In the present paper we show for the first time monoacylglycerol hydrolase in human platelets. No monoacylglycerol hydrolase activity could be demonstrated in the other blood cells. The monoacylglycerol hydrolase of platelets could not be released from the cells by heparin, thus the enzyme is distinct from the postheparin plasma lipases. The enzyme could be solubilized by a non-ionic detergent, Triton X-100. The solubilized monoacylglycerol hydrolase from platelets was optimally active at pH between 7 and 8 and at ionic strength corresponding to [NaCl] between 0.1 and 0.3 M. The optimal assay temperature was 37 degrees C. The enzyme activity was sensitive to HgCl2 but not to NaF. Accordingly, it was stabilized by 2-mercaptoethanol.     

Anandamide amidohydrolase (fatty acid amide hydrolase)

Anandamide (N-arachidonoylethanolamine) loses its cannabimimetic activity when it is hydrolyzed to arachidonic acid and ethanolamine by the catalysis of an enzyme referred to as anandamide amidohydrolase or fatty acid amide hydrolase. Cravatt's group and our group cloned cDNA of the enzyme from rat, human, mouse and pig, and the primary structures revealed that the enzymes belong to an amidase family characterized by the amidase signature sequence. The recombinant enzyme acted not only as an amidase for anandamide and oleamide, but also as an esterase for 2-arachidonoylglycerol. The reversibility of the enzymatic anandamide hydrolysis and synthesis was also confirmed with a purified recombinant enzyme. Several fatty acid derivatives like methyl arachidonyl fluorophosphonate potently inhibited the enzyme. The enzyme was distributed widely in mammalian organs such as liver, small intestine and brain. However, the anandamide hydrolyzing enzyme found in human megakaryoblastic cells was catalytically distinct from the previously known enzyme.     

Immunoaffinity purification and immunoassay determination of human erythrocyte acylphosphatase

Specific anti-human erythrocyte acylphosphatase antibodies were raised in rabbits, purified by affinity chromatography, and used to develop an enzyme purification procedure based on an immunoaffinity chromatography step. This procedure permitted the rapid purification of the enzyme, with a high final yield and with a specific activity very similar to that found for the enzyme purified by the standard procedure. The noncompetitive enzyme-linked immunoadsorbent assay developed with the affinity-purified antibodies was very specific and sensitive in that a positive reaction could be detected in the presence of antigen amounts of as little as 0.01 ng/ml. By this assay the enzyme content was determined in normal cells, tissues, and organs as well as in blood samples from hemopathy-affected patients. This test could possibly have clinical applications.     

Characterization of Quinolinic Acid Phosphoribosyltransferase in Human Blood and Observations in Huntington''s Disease

Quinolinic acid (QUIN), an excitotoxic compound present in the mammalian CNS and periphery, has been hypothetically linked to human neurodegenerative disorders such as Huntington's disease and epilepsy. Quinolinic acid phosphoribosyltransferase (QPRT), the catabolic enzyme of QUIN, is found in the CNS and peripheral organs where it may be a major influence on the tissue levels of QUIN. We have measured QPRT activity in human blood as a means of assessing one aspect of QUIN metabolism in humans. The enzyme was present in blood cells, platelets having a sixfold greater activity than erythrocytes, but was essentially absent from the plasma. In a blood cell fraction, enzyme activity was potently inhibited by phthalic acid (IC50 = 6.1 microM). Kinetic analyses conducted over a range of QUIN concentrations yielded Km values of 1.89-3.75 microM and Vmax values of 33.4-72.5 fmol nicotinic acid mononucleotide/h/mg protein. Enzyme activity varied 2.2-fold between normal individuals, was reasonably constant over a series of sampling intervals, and showed some diminution when blood was stored for 1 month at -20 degrees C. No differences of enzyme activity in erythrocytes or platelets were apparent between three Huntington's disease patients and their unaffected spouses. These data indicate that measurements of QPRT activities in blood are a convenient means to monitor QUIN metabolism in human subjects and that a deficiency of the enzyme is not apparent in Huntington's disease.