Annemarie Herzfeld

Enzymes of orithine metabolism in adult developing rat intestine

Abstract The levels of 11 enzymes, most of them involved in the metabolism of orithine, were measured in whole upper intestine, or in duodenum, small intestine and colon of adult rats. The developmental formations in small intestine of arginase, orithine aminotransferase, and orithine transcarbamylase were compared with those in liver. Changes with age (late gestation to adult) of the intestinal activities of pyrroline-5-carboxylate reductase, proline oxidase and glutamyl transpeptidase are also described. The results suggests that the proximal part of the intestine is well endowed with enzymes involved in the conversion of ornithine to proline as well as to citrulline. Fetal intestine is rich in proline oxidase and pyrroline-5-carboxylate reductase. The peak levels of ornithine aminotraferase found in intestine in the first 3 postnatal weeks were higher than seen in any other rat tissue. Some of the properties of arginase, ornithine aminotransferase and pyrroline-5-carboxylate reductase in small intestine were compared with those in liver. Isozymes of arginase in small intestine differed from those in liver; the kinetic properties of ornithine aminotransferase were similar in the two tissues. In intestine of 14-day-old rats, the orithine aminotransferase reaction was reversible, forming ornithine from pyrroline-5-carboxylate. The intestinal pyrroline-5-carboxylate reductase was cold-labile as was the hepatic enzyme in rat.

Aspartate aminotransferase in rat tissues: Changes with growth and hormones

Abstract Levels of the two isozymal forms of aspartate aminotransferase (EC 2.6.1.1) were determined in normal and neoplastic rat tissues. Of the adult tissues with high total aspartate aminostransferase activity heart and muscle contain twice as much soluble than particulate isozyme while in liver and kidney (and tumors whose total activity is low) the particulate isozyme predominates. Mammary gland begins to exhibit aspartate aminotransferase activity after parturition and the soluble isozyme continues to rise throughout lactation. Fetal tissues have generally low aspartate aminotransferase activity; the different rates of its accumulation during gestation and postnatal life determined in liver, kidney and heart. The administration of thyroxine 2 days before birth, or that hydrocortisone 2 days before birth, promotes the accumulation of liver aspartate aminotransferase. Estradiol inhibits the developmental formation of the enzyme in liver and prevents the hydrocortisone induction of soluble aspartate aminostransferase in liver of adult males. The results indicate that the amounts of the two forms of aspartate aminotransferase are subject to different physiological control in different tissues. The response of the liver isozymes to hormones depends on the stage of development and after maturity of the sex of the animal.

Defective ornithine metabolism in cultured skin fibroblasts from patients with the syndrome of hyperornithinemia, hyperammonemia and homocitrullinuria☆

The syndrome of hyperornithinemia, hyperammonemia, and homocitrullinuria (HHH) is a metabolic disorder resulting in protein intolerance and mental retardation. The primary metabolic defect has yet to be determined. We studied some aspects of ornithine metabolism in cultured skin fibroblasts from two patients from two patients with the HHH syndrome. The fibroblasts failed to incorporate 14C-label from ornithine into protein, a defect also observed in fibroblasts from patients with gyrate atrophy of the choroid and retina and a deficiency of ornithine aminotransferase activity. The defect can be corrected in heterokaryons formed between these two types of fibroblasts. These findings indicate that fibroblasts are suitable for further studying the underlying metabolic defect in HHH syndrome. The combination of the ornithine incorporation assay and genetic complementation analysis provide a confirmatory test for the diagnosis of this syndrome.

The Dedifferentiated Pattern of Enzymes in Livers of Tumor-bearing Rats

Summary Seven days after the s.c. implantation of a neoplasm, there are significant decreases of ornithine aminotransferase and glucokinase, as well as an increase of hexokinase in livers of adult rats. The concentration of 10 additional enzymes changes in the course of the following week, while the levels of nine other enzymes and the liver size remain normal. Growth of tumors in suckling rats inhibits the appearance of liver enzymes that normally emerge at this stage of differentiation. An implanted tumor inhibits the developmental formation in liver of ornithine aminotransferase, glucokinase, glutamine synthetase, and malate-NADP dehydrogenase, but it does not prevent glucocorticoids from inducing a premature rise of ornithine aminotransferase. The enzymes that increase in host liver upon tumor transplantation are among those that are relatively high in fast-growing hepatomas and in fetal liver. Those that decrease are those that are low or absent in hepatomas and fetal liver. Thus, by gain or partial loss of these enzyme activities, the quantitative pattern of enzymes in host liver diverges from normal adult liver toward that of immature liver and the well-differentiated hepatomas.

Human colon tumors. Enzymic and histological characteristics

In samples of colonic adenocarcinomas, the mean activities of thymidine kinase, glucose‐6‐phosphate dehydrogenase, phosphoserine phosphatase and pyrroline‐5‐carboxylate reductase were several fold higher than those of non‐neoplastic colon. The presence of considerable, told labile pyrroline‐5‐carboxylate reductase activity provided an additional criterion for distinguishing tumors from the control tissue. Deviations from the pattern of enzymes in normal colon were much more pronounced in the five moderately well‐differentiated than in the single well‐differentiated adenocarcinoma.

Enzyme activities in human fetal and neoplastic tissues

The concentrations of ten or 12 enzymes involved in the metabolism of DNA, collagen, amino acids, or glucose have been determined in variants of human intestinal and pulmonary tissues. In comparison to nonneoplastic adult colon, normal fetal colon had elevated concentrations of thymidine kinase, peptidyl proline hydroxylase, phosphoserine phosphatase, ornithine transcarbamylase, γ‐glutamyl transpeptidase, and ornithine aminotransferase. Raised activities of the first five of these enzymes, and of hexokinase, glucose‐6‐phosphate dehydrogenase, and pyrroline‐5‐carboxylate reductase distinguishes neoplastic from nonneoplastic sections of adult colon. Study of a wide range of pulmonary specimens permitted comparisons of different types of tumors, and revealed some subtle differences between lungs of noncancer patients and nonneoplastic portions of host lungs. The concentrations of eight previously identified enzymic indicators were less in moderately or well differentiated than in poorly differentiated pulmonary adenocarcinomas. The latter differed from epidermoid carcinomas (also poorly differentiated) by containing lower concentrations of thymidine kinase (both soluble and particulate) and hexokinase.

Phosphoserine phosphatase distribution in normal and neoplastic rat tissues

Abstract Optimal conditions for assay of phosphoserine phosphatase by phosphate release were determined in dialyzed rat tissue extracts. Hydrolysis that was specifically inhibited by 10 m m l -serine was used to measure the enzyme activity. Nonspecific reactions were minimal. Phospho- d -serine was the preferred substrate since it avoided inhibition by the product of the reaction. Significant activities were present in normal adult kidney, liver, spleen, and brain, and extremely low activities in heart and breast. Relatively high activities in fetal liver, lactating mammary gland, and tumors suggested a parallel between enzyme level and tissue growth or protein synthesis. Marked activity changes occurred physiologically with development of liver and kidney.

Glutamate Dehydrogenase, Alanine Aminotransferase, Thymidine Kinase, and Arginase in Fetal and Adult Human and Rat Liver

Extract: In fetal livers of both man and rat thymidine kinase activity was 12 times higher than in the adult, glutamate dehydrogenase and arginase were present at 20–50% of their adult values, whereas alanine aminotransferase activity was only an insignificant fraction of that in the adult. Although the developmental changes for the four enzymes were quantitatively similar in both species, qualitatively there were some significant differences.In adult human liver, glutamate dehydrogenase activity was distributed almost equally between the cytosol and particles; the concentration of only the soluble enzyme increased after birth. In rat liver, glutamate dehydrogenase remained exclusively a particulate enzyme. The soluble hepatic alanine aminotransferase activity rose in both species after birth (from less than 2 U/g to 41–57 U/g, respectively). Thymidine kinase was wholly soluble in the fetal livers; only in adult human liver was additional activity (at least 50% of the total) found in the particles. Arginase isozymes, identical and apparently the same single isozyme in fetal and adult rat liver, show an ontogenetic change in man. A shift from a single form, common to human fetal liver and fetal kidney, to at least two variants in adult human liver, indicates another complexity of the fully differentiated liver in man.Speculation: It is unlikely that the occurrence of particulate thymidine kinase and soluble glutamate dehydrogenase in man is restricted to liver. Their relatively late appearance in life suggests that their further study would be of interest to both ontogeny and phylogeny.

Defective ornithine metabolism in the syndrome of hyperornithinaemia, hyperammonaemia and homocitrullinuria

The syndrome of hyperornithinaemia, hyperammonaemia and homocitrullinuria (HHH) is characterized by protein intolerance, mental retardation, seizures and episodic attacks of ataxia and lethargy (Shih et aL, 1969). The basic metabolic defect in this disorder is still unknown. This is in contrast to another hyperornithinaemic syndrome associated with gyrate atrophy of the choroid and retina (GA) in which a deficiency of ornithine aminotransferase (OAT) (EC 2.6~1.13) is well established. In addition, there are two other hyperornithinaemic syndromes. Bickel et al. (1978) described two siblings with liver disease and a partial hepatic OAT deficiency. Another patient studied by Rennert et al. (I976) had clinical features similar to that of HHH patients and was also found to have OAT deficiency. Ornithine can be metabolized through three different routes (Figure 1). It is involved in urea synthesis via ornithine carbamyltransferase (OCT) (EC 2.1.3.3), and is also a precursor of polyamines (via ornithine decarboxylase; EC 4.1.I.17). Ornithine is catabolized primarily by its conversion to pyrrotine-5-carboxylate (PC) via OAT, a mitochondrial enzyme, and subsequent formation of proline (via PC reductase; EC 1.5.1.1) or glutamate (via PC dehydrogenase; EC 1.5.1.12). The incorporation of the ornithine carbon skeleton into protein is dependent upon the proper functioning of the OAT pathway. A block at any of several enzymes involved in ornithine metabolism (Figure 1) could be the primary defect in HHH. The following enzyme defects have been considered, investigated and ruled out either by dissimilar metabolite patterns or by measurement of enzyme activity: ornithine aminotransferase (Shih and Schulman, 1970); carbamylphosphate synthase and

Amino acid metabolizing enzymes in rat submaxillary gland, normal or neoplastic, and in pancreas.

The activities of 12 enzymes, many related to ornithine metabolism, were measured in rat submaxillary gland, submaxillary gland tumors and pancreas. In submaxillary gland, the activities of arginase, ornithine aminotransferase, pyrroline-5-carboxylate reductase and glutamine synthetase were high, but no ornithine transcarbamylase or proline oxidase could be detected. In the fetal submaxillary gland, arginase was at almost adult levels while ornithine aminotransferase reached 50% of its adult value postnatally. Submaxillary tumors deviated from their cognate tissue by lower levels of amino acid metabolizing enzymes and by high concentrations of thymidine kinase. In pancreas, none of the pyrroline-5-carboxylate metabolizing enzymes were as high as in either liver or submaxillary gland. The outstanding activities were those of gamma-glutamyl transpeptidase and glutamate dehydrogenase. Although arginase activities in submaxillary gland and pancreas were quantitatively similar, they differed qualitatively: submaxillary gland contained the same variant as liver while the pancreatic isozymes resembled those of other nonhepatic tissues.