Purine salvage enzyme activities in normal and neoplastic human tissues

The enzymatic pattern of five enzymes involved in the purine salvage pathway, namely purine nucleoside phosphorylase (EC 2.4.2.1), adenosine deaminase (EC 3.5.4.4), 5'-nucleotidase (EC 3.1.3.5), alkaline phosphatase (EC 3.1.3.1), and hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) has been evaluated both in human intestinal and breast carcinomas and compared to that of normal tissues. A higher level of hypoxanthine-guanine phosphoribosyltransferase was associated with tumor tissues. This metabolic alteration should lead to an elevated synthesis of nucleotides in cancer cells, might confer selective growth advantages to neoplastic tissues, and account, at least in part, for the difficulties encountered in the chemotherapy of human tumors, by using compounds affecting only the purine de novo biosynthesis.     

Endothelin-converting enzymes.

The putative endothelin-converting enzyme (ECE) has been the focus of intense research, both within academia and the pharmaceutical industry. Interest in ECE stems mainly from the hypothesis that development of inhibitors of ECE will provide an effective means of preventing production of endothelin in circumstances where it may play a pathogenic role. Both an aspartic and a metalloprotease have been identified that have characteristics of this putative enzyme. Evidence suggests that the metalloprotease, which is inhibited by phosphoramidon, may be the physiologically relevant converting enzyme. However, it remains to be demonstrated conclusively that any inhibitor of an ECE activity directly alters endogenous endothelin production and/or the pathogenesis of a disease condition in which endothelin is thought to play a primary role.     

Immunohistochemical study of angiotensin-converting enzyme in human tissues using monoclonal antibodies

Summary The localization of angiotensin-converting enzyme (ACE) in human tissues has been studied by the PAP-method with the use of monoclonal antibody 9B9 against human lung ACE. The enzyme was detected on the surface of endothelial cells in lung, myocardium, liver, intestine and testis as well as in the epithelial cells of the kidney proximal tubules and intestine. The monoclonal antibody 9B9 did not react with ACE in the epithelial cells of the testis seminiferous tubules. These data suggest that the antibody 9B9 recognizes epitope which is shared by the ACE molecule of endothelial cells and renal and intestinal epithelial cells but is not present in testicular ACE, or is not accessible there to the antibody.     

Immunohistochemical study of angiotensin-converting enzyme in human tissues using monoclonal antibodies

The localization of angiotensin-converting enzyme (ACE) in human tissues has been studied by the PAP-method with the use of monoclonal antibody 9 B9 against human lung ACE. The enzyme was detected on the surface of endothelial cells in lung, myocardium, liver, intestine and testis as well as in the epithelial cells of the kidney proximal tubules and intestine. The monoclonal antibody 9 B9 did not react with ACE in the epithelial cells of the testis seminiferous tubules. These data suggest that the antibody 9 B9 recognizes epitope which is shared by the ACE molecule of endothelial cells and renal and intestinal epithelial cells but is not present in testicular ACE, or is not accessible there to the antibody.     

Distribution of post-proline cleaving enzyme in human brain and the peripheral tissues

Summary We have studied the distribution of post-propline cleaving enzyme activity in the various tissues in humans using 7-(succinyl-Gly-Pro)-4-methylcoumarinamide as substrate. The post-propline cleaving enzyme activity was high in muscle, testes, kidney and submandibular gland, but was low in the heart, mesenterium and aorta. In the brain, relatively high post-propline cleaving enzyme activity was observed in the cerebral cortex, but other brain regions showed a very low enzyme activity.On Sephadex G-100 column chromatography, enzyme activity in human kidney showed a major peak and a minor peak. The major peak coincided with the enzyme in human cerebral cortex, but was different from human serum enzyme. Diisopropylfluorophosphate, a serine protease inhibitor, strongly inhibited the enzyme activity of each active fraction. The enzyme in the cerebral cortex and kidney was inhibited by heavy metals and thiol blocking agents. However, inhibition of enzyme activity in the serum was not observed with such inhibitors. Therefore, we suppose that post-proline cleaving enzyme activity in the brain is similar, if not identical, to that in the kidney.     

Eicosanoid forming enzyme mRNA in human tissues. Analysis by quantitative polymerase chain reaction

The key enzymes in the formation of eicosanoids, including leukocyte 5-lipoxygenase (5LX), platelet 12-lipoxygenase (12LX), reticulocyte 15-lipoxygenase (15LX), prostaglandin G/H synthase cyclooxygenase, and leukotriene A4 (LTA) hydrolase have been studied extensively in recent years. Little is known, however, about the regulation of these enzymes at the gene level. We have developed a quantitative polymerase chain reaction (PCR) assay to quantify the mRNAs for these five enzymes, as well as for cytoplasmic beta-actin (bACT) mRNA. Human erythroleukemia (HEL) cells, which display megakaryocytic/erythroid characteristics, were selected as a source of RNA to characterize the assay. These cells expressed mRNA for bACT, LTA, cyclooxygenase, and 12LX (in decreasing order). mRNA for 5LX and 15LX was undetectable. Bronchoalveolar lavage fluid cells obtained from asthmatic patients, primarily alveolar macrophages, contained mRNA for bACT, LTA, 5LX, cyclooxygenase, and 15LX (in decreasing order). Treatment of HEL cells with phorbol 12-myristate 13-acetate or steroid administration to asthmatic patients apparently selectively regulated certain of these target genes. The utility of this assay in quantifying mRNA for the various target genes in blood cells, including platelets from patients with chronic myelogenous leukemia, has also been demonstrated. Studies on the regulation of genes for enzymes involved in the leukotriene and prostaglandin biosynthetic pathways, especially when only small tissue samples are available, will be facilitated with this approach.     

Angiotensin-converting enzyme from human tissues. Physicochemical, catalytic, and immunological properties

Angiotensin-converting enzyme was purified from human lung, kidney, testis, blood plasma, and seminal plasma using a facile two-step protocol which included affinity chromatography on Sepharose-bound lisinopril followed by either gel filtration or hydroxylapatite chromatography. Molecular mass for converting enzyme from all sources except testis was 140 kDa. That from testis consisted of both a 90- and a 140-kDa form in a 4:1 ratio. Detergent-extracted membrane-bound converting enzyme aggregated on gel filtration chromatography, while trypsin-extracted and soluble converting enzyme did not. Comparison of detergent-extracted and trypsin-extracted membrane-bound converting enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing indicated that the membrane binding sequence contributed minimally to the size and charge of the enzyme. Catalytic and kinetic properties assessed by interaction with substrates, inhibitors, and anti-converting enzyme immunoglobulin were similar for all forms and sources of converting enzyme. Enzyme-linked immunosorbent assay revealed only partial homology between the 90- and 140-kDa forms of the enzyme.     

A novel enzyme immunoassay specific for ABCA1 protein quantification in human tissues and cells

ATP-binding cassette transporter A1 (ABCA1) mediates the transport of cholesterol and phospholipids from cells to lipid-poor HDL and maintains cellular lipid homeostasis. Impaired ABCA1 function plays a role in lipid disorders, cardiovascular disease, atherosclerosis, and metabolic disorders. Despite the clinical importance of ABCA1, no method is available for quantifying ABCA1 protein. We developed a sensitive indirect competitive ELISA for measuring ABCA1 protein in human tissues using a commercial ABCA1 peptide and a polyclonal anti-ABCA1 antibody. The ELISA has a detection limit of 8 ng/well (0.08 mg/l) with a working range of 9-1000 ng/well (0.09-10 mg/l). Intra- and interassay coefficient of variations (CVs) were 6.4% and 9.6%, respectively. Good linearity (r = 0.97-0.99) was recorded in serial dilutions of human arterial and placental crude membrane preparations, and fibroblast lysates. The ELISA measurements for ABCA1 quantification in reference arterial tissues corresponded well with immunoblot analysis. The assay performance and clinical utility was evaluated with arterial tissues obtained from 15 controls and 44 patients with atherosclerotic plaques. ABCA1 protein concentrations in tissue lysates were significantly lower in patients (n = 24) as compared with controls (n = 5; 9.37 +/- 0.82 vs. 17.03 +/- 4.25 microg/g tissue; P < 0.01). The novel ELISA enables the quantification of ABCA1 protein in human tissues and confirms previous semiquantitative immunoblot results.     

The effect of storage on enzyme activities in tissues

The stability to storage at -20 degrees C of 29 enzymes of rat tissues was measured for periods of up to 100 days. The enzymes could be divided into four groups: (1) those that increased in activity with storage; (2) those that showed transient rises subsequently declining to the original activity or below; (3) those that showed no significant change; (4) those that declined in activity. Details of methods used and of results obtained have been deposited as Supplementary Publication SUP 50038 (19 pages) at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem.J. (1973), 131, 5.     

Selenoproteins in human thyroid tissues

The aim of the present work was to clarify whether the activities of selenoenzymes can serve as markers for different tumors or goiters, as classified by histological criteria. The following parameters were determined: 1) selenium content of plasma (Se), 2) activities of the selenoenzymes: plasma glutathione peroxidase (plGSHPx), cytosolic glutathione peroxidase (cGSHPx), type I and type II iodothyronine deiodinases (ID-I, ID-II), thioredoxin reductase (THRR) in human thyroid tissues. The material came from follicular neoplasm, papillary carcinoma, struma nodosa, struma lymphomatosis Hashimoto, other thyroid surgery specimens, and normal tissues. There was no difference in Se nor in plGSHPx between patients and healthy volunteers. No significant differences were found for any parameter in thyroid carcinoma versus normal or goitrous thyroid tissue. In the whole group of thyroid surgery specimens the statistically significant correlations were found between ID-I and ID-II and between THRR and selenoperoxidases. Principal components analysis confirmed the above correlation and moreover revealed correlation between Se and plGSHPx, but did not detect any clear distinction between patients with the different diagnoses.