Galactosamine-induced alpha 1-antitrypsin deficiency in rats. Alterations in plasma glycoproteins and alpha 1-antitrypsin carbohydrate composition.

Administration of D-galactosamine (GalNH2) is known to produce alterations in plasma glycoprotein levels, including alpha 1-antitrypsin. The authors have studied the effects of GalNH2 on circulating protein bound carbohydrates and on the plasma concentrations of two alpha 1-antiproteases, transferrin, IgG, and albumin in rats. The alpha 1-antiproteases from GalNH2-treated rats were isolated and their molecular weight, isoelectric point, and carbohydrate composition compared with those of control rat alpha 1-antiproteases. Total plasma protein, albumin, and transferrin levels in the GalNH2-treated rats do not differ significantly from those of control rats. Plasma protein-bound carbohydrate is decreased significantly in the experimental animals, compared with controls: sialic acid decreased 60%, neutral sugars decreased 43%, and amino sugars decreased 38%. The concentrations of alpha 1-antitrypsin (AAT) and a higher molecular weight alpha 1-antiprotease designated AP2 are decreased by 79% and 38%, respectively. AAT isolated from the plasma of GalNH2-treated rats contains 2-3 fewer moles of sialic acid, 3 fewer moles of neutral sugar, and 2 fewer moles of amino sugar per mole of antiprotease than AAT isolated from controls. AP2 from GalNH2-treated rats contains 1 fewer mole each of sialic acid, neutral sugar, and amino sugar per mole of antiprotease than AP2 from controls. These alterations are similar to those seen in humans with genetically determined alpha 1-antiprotease deficiency.

Altered glycosidase activity in the liver of rats with galactosamine-induced alpha 1-antiprotease deficiency.

Previous studies have shown that chronic administration of D-galactosamine (GalNH2) in rats produces alpha 1-antiprotease (AAP) deficiency and causes accumulation of aberrantly glycosylated AAP in hepatic granules. In order to examine the disordered mechanism which produces this altered glycosylation, the activities of 6 glycosidases in liver homogenates of control and AAP-deficient rats were determined. GalNH2 treatment increases acid pH glycosidase activity, while it decreases intermediate pH alpha-mannosidase and alpha-glucosidase activities. beta-D-Glucosidase, beta-D-mannosidase and beta-D-N-acetylglucosaminidase activities, measured at acid pH, increase more than 2-fold in the GalNH2-treated rats compared to controls. alpha-D-Glucosidase activity measured at intermediate pH decreases 2.5-fold in the experimental rats. alpha-L-Fucosidase and acid phosphatase activities are not significantly changed by GalNH2 treatment. alpha-D-Mannosidase activity can be separated into 2 fractions by ion exchange chromatography. Acid pH alpha-D-mannosidase is increased nearly 2-fold in the GalNH2-treated rats. Intermediate pH alpha-D-mannosidase optimum is decreased alpha-D-mannosidase activities have been observed in humans with AAP deficiency. alpha-Glucosidases and alpha-mannosidases play a crucial role in glycoprotein synthesis. The altered synthesis and structure of AAP in GalNH2-induced AAP deficiency may be a reflection of altered enzyme activities.

Vitamin A deficiency and serum or plasma fibronectin in the rat and in human subjects.

Fibronectin, a high-molecular-weight glycoprotein occurring in plasma and on the surface of many cells, is involved in cell adhesion and other cell-surface phenomena. Vitamin A deficiency in rats resulted in a threefold increase in the concentration of fibronectin in serum, as measured immunochemically. In vitamin A-depleted human subjects, on the other hand, no correlation could be found between plasma retinol and fibronectin levels.

Radioimmunoassay of a glycoprotein associated with malignancy.

A glycoprotein associated with malignancy was purified from the 0.6M perchloric acid-soluble fraction of serum obtained from cancer patients. The purified glycoprotein contained sialic acid, which was responsible for binding to wheat-germ agglutinin-Sepharose. Gel electrophoresis showed one band with an apparent Mr of 50 000-55 000, and the isoelectric point was 4.4 +/- 0.1. The glycoprotein could be distinguished from carcinoembryonic antigen and alpha-fetoprotein. Iodination of this material with chloramine-T permitted development of a radioimmunoassay.

Retinoids and phorbol esters alter release of fibronectin from enucleated cells.

Addition of 12-tetradecanoylphorbol 13-acetate (TPA) to cultures of intact Swiss mouse 3T3 fibroblasts induced a dose-dependent increase in ornithine decarboxylase (OrnDCase) activity. Over the same concentration range, 10(-9) to 10(-6) M, TPA induced the release of radioactively labeled fibronectin (FN) from the cells into the culture medium. Retinoic acid, a derivative of vitamin A, inhibited in a dose-dependent manner both the increase in OrnDCase activity and the release of FN induced by TPA. To examine the effects of TPA and retinoic acid in enucleated cells, the cells were treated with 7.5 micrograms of cytochalasin B per ml of medium during centrifugation at 10,000 X g for 35 min at 37 degrees C. In a series of five experiments, the treated cells were 94.7 +/- 4.8% (+/- SEM) enucleated as measured by [3H]thymidine incorporation and verified by Giesma staining for nuclei. In the enucleated cells, TPA did not induce increased OrnDCase activity but did induce FN release in a dose-dependent fashion over the same concentration range effective for FN release from intact cells. Moreover, addition of retinoic acid to the enucleated cells inhibited the phorbol ester-induced release of FN in a dose-dependent manner. A series of phorbol ester derivatives showed the same order of activity in causing FN release from the enucleated cells as reported for inducing OrnDcase activity in intact cells or in promoting mouse skin tumors in vivo. Similarly, several retinoids were tested for their ability to inhibit the phorbol ester-induced release of FN from enucleated cells. The efficacy of the retinoids in preventing FN release paralleled their activity in inhibiting phorbol ester-induced OrnDCase activity and skin tumor promotion, as reported in the literature. We conclude that at least one aspect of tumor promotion induced by phorbol esters--the loss of FN--does not require the cell nucleus, and further, that retinoids can inhibit this aspect of tumor promotion without nuclear involvement.