Erwin Regoeczi

Elimination of asialofetuin and asialoorosomucoid by the intact rat quantitative aspects of the hepatic clearance mechanism

The capacity of the liver to eliminate asialofetuin and asialoorosomucoid was investigated in intact rats. From plasma radioactivity curve measurements and assays on tissue homogenates the liver is shown to be able to dispose of an average of 19.8 microgram of asialofetuin/min per 100 g body weight. No other major route is identified for the disappearance of asialofetuin from the plasma, although trace amounts of the protein were detectable in the urine. From analyses of the plasma radioactivity curves the elimination process for asialoorosomucoid appears to be comparatively complex because of the existence of extrahepatic disposal routes. Quantification of labelled asialoorosomucoid in liver homogenates indicates, however, that the hepatic clearance rate for asialoorosomucoid is similar to that for asialofetuin. Urinary excretion significantly contributes to the disappearance of asialoorosomucoid from the plasma but the hepatic and renal routes do not account for all the protein lost from this compartment. At plasma concentrations above the maximal eliminative capacity of the liver, the hepatic clearance of asialofetuin obeys zero-order kinetics and is remarkably constant. Elimination of a quantity of asialoglycoprotein which exceeds the calculated total number of binding sites in the liver does not reduce the efficiency of the pathway, and studies of [3H]leucine incorporation indicate that the lectin, unlike the bound asialoglycoprotein, is not destroyed in the elimination process. Cytochalasin B (80 microgram/100 g body wt.) had no measureable effect on the hepatic clearance of asialofetuin. Administration of colchicine (10 mg/100 g body wt.) resulted in transitory accumulations of asialoorosomucoid in the liver, presumably due to interference with the intracellular transport of the endocytised protein.

Synthesis of antithrombin III and alpha-1-antitrypsin by the perfused rat liver

Livers isolated from control or turpentine-injected rats were perfused for 3 h with human red cells suspended in Krebs-Henseleit solution containing bovine serum albumin, dextran, glucose, heparin, cortisol, insulin, a mixture of 20 amino acids and [3H]leucine. Changes in the concentrations of antithrombin III and alpha-1-antitrypsin were evaluated by rocket immunoelectrophoresis using specific antisera, and incorporation of the 3H radioactivity into the total protein, albumin, antitrhombin III and alpha-1-antitrypsin in the perfusate was measured. The results indicate that both antithrombin III and alpha-1-antitrypsin are synthesized in the liver. Local inflammation induced in the liver donors moderately stimulated the synthesis of alpha-1-antitrypsin but it affected only marginally that of antithrombin III.

The proteolytic nature of commercial samples of galactose oxidase. Purification of the enzyme by a simple affinity method.

Several commercially available samples of galactose oxidase (D-galactose: oxygen 6-oxidoreductase, EC 1.1.3.9) were found to contain high proteolytic activity on proteins such as fibrinogen, transferrin, albumin and casein. A simple, efficient method was devised for the purification of galactose oxidase which relies on the affinity of the enzyme for agarose (Sepharose 6B). The purified galactose oxidase was recovered in high yield free from proteolytic activity. The enzymic affinity for Sepharose and Sephadex was investigated to clarify the absorption mechanism.

Tritiation of Commercial Heparins by Reaction with NaB3H4: Chemical Analysis and Biological Properties of the Product

Abstract A simple method to tritiate commercial samples of heparin is described. Heparin is reacted with NaB3H4 at pH 8.0 for 3 h at room temperature, after which the 3H-labeled mucopolysaccharide is isolated by gel filtration. Using NaB3H4 of low specific radioactivity (227–555 mCi/mmol), [3H]heparins containing 3.7–8.0 × 105 dpm/mg are made whereas with NaB3H4 of higher specific activity (5–10 Ci/mmol), preparations of 1.2–1.8 × 107 dpm/mg are obtained. From analysis of [3H]heparin using several techniques (periodate oxidation; mild acid hydrolysis accompanied by ion-exchange chromatography and high-voltage electrophoresis), the labeling procedure largely relies on a small proportion (approximately 4–6%) of heparin molecules possessing a terminal monosaccharide which, on reaction with NaB3H4, is reduced to yield an alditol. Consequently, 3H is incorporated on C1 of this modified terminus. The product, [3H]heparin as the Na salt, is very stable under normal conditions of treatment and storage. The behavior of [3H]heparin when chromatographed on Sepharose-antithrombin III and on Sepharose-thrombin compares closely with that of unlabeled heparin. However, gel filtration on Sephadex G-200 reveals that [3H]heparin ( V e V 0 = 2.19 ) chromatographs more slowly than native heparin ( V e V 0 = 2.08 ), a feature which may reflect the true nature of the constituent molecules present in the heparin sample.

Galactose-specific elimination of human asialotransferrin by the bone marrow in the rabbit

Abstract Rabbit bone marrow accretes and degrades human asialotransferrin in vivo through a mechanism that recognizes the exposed galactose groups in this glycoprotein. After the liver, rabbit bone marrow is the second mammalian tissue now being identified as possessing a galactose-specific pathway for the elimination of a plasma protein. However, comparative studies with desialylated glycoproteins containing bi-, tri-, and tetraantennary glycans (asialofetuin, asialoorosomucoid, chicken α 1 -acid glycoprotein, and rabbit transferrin) indicate that the bone marrow recognizes fewer glycan structures than does the liver. Optimal uptake and degradation of an asialoglycoprotein by the bone marrow requires the presence of biantennary glycans.

On the physicochemical and chemical properties of α1-acid glycoproteins from mammalian and avian plasmas

Abstract We have isolated from the plasma of man, rabbit, pig, rat and chicken α1-acid glycoproteins that show a single band on polyacrylamide gel electrophoresis and a single are in immunodiffusion. Amino acid and carbohydrate compositions are presented. The human protein is very similar to preparations described by earlier workers and, although significant differences in amino acid composition exist among them, the proteins from the other species are assumed to be analogous to it. Ultracentrifugal studies, despite showing single, fairly symmetrical peaks at high speed, produced evidence for the physical heterogeneity of each protein in agreement with some earlier reports. Although many models consisting of mixtures of components were tested, none was found that fitted adequately all observations on any one of the glycoproteins.

Adhesion of Fibroblasts to Polymerizing Fibrin and Retraction of Fibrin Induced by Fibroblasts

Summary Fibroblasts readily adhere to polymerizing fibrin, but not to fibrinogen or fully polymerized fibrin. Conditions for optimal interaction are similar to those known for the platelet-fibrin interaction. Fibroblasts retract fibrin formed by thrombin but not by reptilase. Fibrin retraction induced by fibroblasts is inhibited by agents inhibiting clot retraction induced by platelets. We thank Dr. L. Prevec for generous supplies of fibroblasts and Messrs. A. F. Senyi, K. L. Wong and T. Bistricki for skilled technical assistance.

A simple method for the purification of commercial neuraminidase preparations free from proteases

Abstract A simple method is described for the further purification of commercially prepared neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) (ex Clostridium perfringens filtrate). A single-step ion-exchange chromatography process renders the enzyme free from proteolytic enzymes and much contaminating protein. Consequently when 125I-labelled proteins, such as fibrinogen, casein, caeruloplasmin, are used as substrates, no proteolytic activity is observed. In contrast, the commercial sample is shown to cleave trichloroacetic acid-soluble 125I-labelled peptides from these three proteins. In addition the purified neuraminidase preparation is subject to both polyacrylamide gel electrophoresis and Sephadex gel filtration under various conditions. Heat stability studies show the enzyme to retain approx. 28% activity after 3 h at 58 °C.

Some observations on the affinity chromatography of rabbit plasminogen

Abstract The feasibility of isolating plasminogen allomers from plasma by affinity chromatography in a single stage and the specificity of Sepharose-lysine for plasminogen are examined. It is shown that the method of Castellino can readily be adapted by relatively minor modifications as a one-stage procedure without endangering the quality of the product. The optimal degree of lysine conjugation with Sepharose is defined for this process. Experiments with labelled allomers are described showing the competitive nature of the capture of plasminogen by Sepharose-lysine. The affinity behaviour of plasmin and of complexes of plasmin with serum protease inhibitors are compared to plasminogen. Several other plasma proteins which also exhibit affinity to lysyl groups are identified. Plasminogen obtained in the present study compares well with previously reported analyses for molecular weight, N-terminal amino acid and sialic acid content, but not for neutral carbohydrates.

The relevance of the structure of lysine bound to Sepharose for the affinity of rabbit plasminogen

The features of the structure of lysine, linked to Sepharose by the alpha-amino group, which are important for affinity chromatography of rabbit plasminogem were studied. Nine lysine and lysine-like conjugates, including epsilon-aminohexanoic acid DL-norleucine, DL-alpha-aminoadipic acid, DL-alpha-epsilon-diaminopimelic acid, cadaverine L-ornithine, L-arginine and D-lysine, were prepared; Using labelled rabbit plasminogen added to plasma, the ability of each conjugate to absorb plasminogen and separate the allomeric forms, type I and type II, during epsilon-aminohexanoic acid gradient elution was compared to Sepharose-L-lysine. Plasminogen had no affinity for Sepharose-epsilon-aminohexanoic acid, and was only weakly attracted by Sepharose-norleucine, Sepharose-cadaverine and others. Sepharose-ornithine held a greater attraction to the protein but the strongest binding was obtained with Sepharose-arginine. The affinity of plasminogen type I was always less than type II for the Sepharose-lysine analogues and the recovery of type II greater than type I from Sepharose-ornithine and Sepharose-arginine. Plasminogen affinity was in the order of Sepharose-arginine greater than Sepharose-lysine greater than Sepharose-ornithine. However, because of the present difficulty in recovering plasminogen from Sepharose-arginine the use of Sepharose-lysine in the affinity chromatography of rabbit plasminogen remains unchallenged. It is concluded that binding of rabbit plasminogen to conjugates of lysine and its analogues is determined by the presence of both a free carboxyl and a free amino group and that the distance between these groups is critical.