Comparison between avian and human prolyl 4-hydroxylases: studies on the holomeric enzymes and their constituent subunits.

Prolyl 4-hydroxylase, a key enzyme in collagen biosynthesis, catalyzes the conversion of selected prolyl residues to trans-hydroxyproline in nascent or completed pro-alpha chains of procollagen. The enzyme is a tetramer composed of two nonidentical subunits, designated alpha and beta. To compare the enzyme and its subunits from different sources, the chick embryo and human placental prolyl 4-hydroxylases were purified to homogeneity and their physicochemical and immunological properties were determined. Both enzymes were glycoproteins with estimated apparent molecular weights ranging between 400 and 600 kDa. Amino acid and carbohydrate analyses showed slight differences between the two holomeric enzymes, consistent with their deduced amino acid sequences from their respective cDNAs. Human placental prolyl 4-hydroxylase contained more tightly bound iron than the chick embryo enzyme. Immunodiffusion of the human placental enzyme with antibodies raised against the purified chick embryo prolyl 4-hydroxylase demonstrated partial identity, indicating different antigenic determinants in their tertiary structures. The enzymes could be separated by high-resolution capillary electrophoresis, indicating differential charge densities for the native chick embryo and human placental proteins. Electrophoretic studies revealed that the human prolyl 4-hydroxylase is a tetrameric enzyme containing two nonidentical subunits of about 64 and 62 kDa, in a ratio of approximately 1 to 2, designated alpha and beta, respectively. In contrast, the chick embryo alpha and beta subunit ratio was 1 to 1. Notably, the human alpha subunit was partially degraded when subjected to electrophoresis under denaturing conditions. Analogously, when the chick embryo enzyme was subjected to limited proteolysis, selective degradation of the alpha subunit was observed. Finally, only the alpha subunit was bound to Concanavalin A demonstrating that the alpha subunits of prolyl 4-hydroxylase in both species were glycosylated. Using biochemical techniques, these results demonstrated that the 4-trans-hydroxy-L-proline residues in human placental collagens are synthesized by an enzyme whose primary structure and immunological properties differ from those of the previously well-characterized chick embryo enzyme, consistent with their recently deduced primary structures from cDNA sequences.     

The plasma membrane of epididymal epithelial cells has a specific receptor which binds to androgen-binding protein and sex steroid-binding protein.

The binding of [3H] delta 6-testosterone photoaffinity-labelled rat androgen-binding protein (rABP) has been studied in an enriched fraction of plasma membranes of epithelial epididymal cells in immature (15 days) and adult rats (40 days). The binding was maximal in less than 30 min and more rapid at 4 degrees C than at 34 degrees C. It was calcium and pH dependent. Scatchard plots of the binding data gave curvilinear plots with two types of binding sites corresponding to a K(ass1) of 18.2 nM-1 and K(ass2) of 1.6 nM-1 (2.2 x 10(11) sites/mg protein and 5.4 x 10(11) sites/mg protein, respectively). In adult rats, only one type of binding site was found, with a K(ass) of 3.7 nM-1 (4.5 x 10(11) sites/mg protein). The number of receptors was 5-fold lower in the cauda than in the caput of the epididymis. The pretreatment of the isolated intact cells with streptozotocin induced a 45% reduction of the binding. Only unlabelled rABP and hSBP (human sex steroid-binding protein) but not other proteins (lactotransferrin, serotransferrin, asialofetuine, fetuine and bovine serum albumin) competed with the labelled ligand to bind plasma membranes. The membrane fraction was solubilized by triton X-100. Its incubation with labelled rABP and hSBP provoked the elution of the tracer as an aggregate into the void volume fraction of superose 6B mini-gel filtration columns. Structural homology between hSBP and rABP could be responsible for the common behaviour of the steroid-carrier molecules for the ABP receptor of rat epididymal epithelial cells.     

Malignant ascites of serous papillary ovarian adenocarcinoma. An immunocytochemical study of the tumor cells

In 17 malignant peritoneal effusions due to papillary serous adenocarcinoma of the ovary, the reaction patterns of the tumor cells to monoclonal antibodies (MAbs) against surface antigens were studied and compared with the reaction patterns of mesothelial cells in the same effusions. The following surface markers were used with the adhesive slide method: epithelial membrane antigen (EMA), human epithelium-specific cell surface antigen (HEA-125), human endothelial antigen (BMA-120), carcinoembryonic antigen (CEA 3-13), an antibody against natural killer cells and cytotoxic cells (BMA-070), granulocyte antigen (Leu M1) and leukocyte antigen of class I (HLA-1). In all cases, from 30% to 95% of the tumor cells reacted with EMA and HEA-125. Tumor cells showed a positive staining with CEA 3-13 in only five cases. In all cases, from 75% to 95% of the tumor cells reacted positively with BMA-120. The reactivity of a few mesothelial cells with EMA and of all mesothelial cells with BMA-120 did not interfere with the identification of positive tumor cells since the reaction patterns were different. Interestingly, our study demonstrated that BMA-070, an MAb identifying natural killer cells and cytotoxic cells, is also a most useful tumor marker. The same was found to be true for Leu M1, an MAb originally thought to react only with granulocytes. The tumor cells showed a partial or total loss of the expression of HLA-1 reactivity. Since all cases were immunocytochemically positive for tumor cells while conventional cytology was positive in only 13 of the cases, the immunocytochemical analysis of malignant peritoneal effusions due to papillary serous adenocarcinoma of the ovary seems able to improve the cytologic diagnosis of the fluids.     

The value of the immunoperoxidase slide assay in the diagnosis of malignant pleural effusions in breast cancer

Whether immunocytochemical studies of malignant pleural effusions due to breast cancer would increase the diagnostic yield as compared with conventional effusion cytology was examined in 30 cases with biopsy-proven metastatic spread to the pleura. Conventional cytology was performed on air-dried smears as well as on cytocentrifuge preparations stained with the May-Grünwald-Giemsa stain. Immunocytochemistry was performed with monoclonal antibodies against carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA) and human leukocyte antigen (HLA) and the peroxidase-antiperoxidase technique on glass slides after Ficoll-Hypaque centrifugation. By conventional cytology, 13 cases (43%) were positive for malignant cells, 6 cases (20%) were suspicious, and 11 cases (37%) were negative. In marked contrast, all 30 cases were immunocytologically positive for malignancy. Tumor cells in all cases demonstrated a positive reaction for EMA. Some mesothelial cells were also positive for EMA, but their reaction pattern was clearly distinguishable from that of the tumor cells. Twenty-one cases (70%) also showed CEA-positive tumor cells; mesothelial cells never reacted with CEA. Some tumor cells showed a loss of HLA expression. In conclusion, this immunocytologic method can be recommended as a routine procedure for greatly increasing the diagnostic yield of cytology in pleural effusions due to breast cancer.     

Correlation between serum enzymes and serum unsaturated vitamin B12 binding proteins in primary liver carcinoma

The serum unsaturated vitamin B12-binding capacity (UBBC), unsaturated transcobalamin (UTC) I, UTC II, UTC III levels, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase activities and bilirubin concentration were estimated in 61 patients with liver diseases (31 with hepatoma, 30 with viral hepatitis). The levels of serum cobalamin, UTC I, UTC III, UBBC, alanine and aspartate aminotransferases, and bilirubin were raised in both hepatoma and viral hepatitis patients. Serum UTC II was reduced in both conditions. Alkaline phosphatase activity was significantly increased in hepatoma. Four significant correlations were observed among these parameters in the hepatoma patients while only one significant correlation was observed in viral hepatitis.     

Effects of ethanol feeding on hepatic lipid synthesis

Rats were fed a high-fat, liquid diet containing either 36% of total calories as ethanol or an isocaloric amount of sucrose, for a period up to 35 days. At different time intervals we measured the effects of ethanol administration on the activities of a number of key enzymes involved in hepatic lipid synthesis. At the start of the experimental period the activities of acetyl-CoA carboxylase and fatty acid synthase, measured in liver homogenates, increased in the control as well as in the ethanol-fed group. After 35 days these enzyme activities were still elevated but there were no significant differences between the two groups. In hepatocytes isolated from controls as well as from ethanol-fed rats, short-term incubations with ethanol induced an increase in the rate of fatty acid synthesis and in the activities of acetyl-CoA carboxylase and fatty acid synthase. However, no alterations in the regulation of these enzymes by short-term modulators of lipogenesis were apparent in hepatocytes isolated from alcohol-treated animals. The results do not indicate a major role for the enzymes of de novo fatty acid synthesis in the development of the alcoholic fatty liver. The amount of liver triacylglycerols increased in ethanol-fed rats during the entire treatment period, whereas the hepatic levels of phosphatidylcholine and phosphatidylethanolamine were not affected by ethanol ingestion. Ethanol administration for less than 2 weeks increased the activities of phosphatidate phosphohydrolase, diacylglycerol acyltransferase, and microsomal phosphocholine cytidylyltransferase, whereas the cytosolic activity of phosphocholine cytidylyltransferase was slightly decreased. Upon prolonged ethanol administration the activities of these enzymes were slowly restored to control values after 35 days, suggesting development of some kind of adaptation. It is interesting that, although the activities of phosphatidate phosphohydrolase and diacylglycerol acyltransferase were restored to the levels found in the control rats, this effect was not accompanied by a stabilization or decrease of the concentration of hepatic triacylglycerols.     

Aromatase activity in human skin fibroblasts: characterization by an enzymatic method

Human skin fibroblasts were incubated for 24 h with 10(-6) M androstenedione and the estrone + estradiol released in the culture medium were measured by an enzymatic assay. Aromatase activity was expressed as pmol (estrone + estradiol) formed in the medium per mg cell protein per day. Using this method we were able to investigate the kinetic properties of aromatase in different cell strains and its stimulation by dexamethasone. Values of 92 nM and 9.1 pmol/mg protein/day were obtained respectively for Km and Vmax in cultured fibroblasts derived from genital skin of normal prepubertal subjects. In patients with complete androgen insensitivity syndrome CAIS, the Km was 156 nM and the Vmax 42 pmol/mg protein/day. Aromatase activity varied from 7.9 +/- 1.2 pmol/mg protein/day (mean +/- SD; n = 19) in normal prepubertal boys to 24.5 +/- 4.7 pmol/mg protein/day (mean +/- SD; n = 11) in those from normal postpubertal boys. The values were even higher in fibroblasts cultured from genital skin of prepubertal patients with CAIS. Cell concentrations did not modify the pattern of estrogen formation and aromatase activity did not vary with serial subcultures. The stimulatory effect of dexamethasone on aromatase activity in cultured fibroblasts was measured after preincubation of the cells for 48 h with dexamethasone, by determining estrogen formation after 24 h incubation of the cells with androstenedione 10(-6) M using this enzymatic method. This data suggest that aromatase activity measured in cultured fibroblasts could be a useful tool for studying extraglandular estrogen formation in physiological and pathological conditions.     

Autolysis of cell walls in pea epicotyls during growth. Enzymatic activities involved

Pisum sativum L. (cv. Lincoln) epicotyl cell walls show autohydrolysis and release into the incubation medium up to 120 μg of sugar per mg of cell wall dry weight in 30 h. Cell walls from younger epicotyls with high growth capacity showed higher auto-lytic capacity than older epicotyls. This suggests that both processes, growth and au-tolysis, are related. The proteins responsible for autolysis were extracted from the wall fraction with high saline solution (3 M LiCl) and enzymatic activities associated with the proteins were studied. The highest activity corresponded to α-galactosidase; lower activities were found for β-galactosidase, a-arabinosidase and exoglucanase. Changes in enzymatic activities and changes in the proportion of sugars released in autolysis by cell walls during the growth of epicotyls support the notion that α-galac-tosidase is one of the enzymes involved in the process of autolysis, and that the liberation of arabinose and galactose in this process occurs as arabinogalactan.