L. Helgeland

Sex difference in the ferritin content of rat liver

1. 1. The concentration of ferritin in liver from male and female rats at different ages has been determined by a single radial immunodiffusion technique. Low levels of ferritin iron and ferritin protein were found in both sexes from a week after weaning to about 7 weeks of age. From then on the levels increased, and the increase was about twice as rapid in livers from female rats compared to those from male rats. Ferritin from female rats contained slightly more iron than ferritin from males, and the degree of iron saturation increased as the rats of both sexes developed. The identity of apoferritins from male and female liver has been verified by double immunodiffusion and by polyacrylamide gel disc electrophoresis. 2. 2. A new method for small scale purification of ferritin, based on polyacrylamide gel disc electrophoresis, is described. The purity of the isolated ferritin has been verified by disc electrophoresis at different pH and gel concentrations and by immuno-electrophoresis. 3. 3. Incorporation of [14C]leucine into ferritin of isolated perfused male rat liver seemed to be stimulated by preadministration of estradiol-17-β in vivo.

The submicrosomal site for the conversion of prothrombin precursor to biologically active prothrombin in rat liver.

Abstract The submicrosomal site for the conversion of prothrombin precursor to prothrombin in rat lever has been investigated by subcellular fractionation techniques. Prothrombin precursor activity could be detected in the luminal as well as the membrane fraction of the rough microsomes. The corresponding fractions from smooth microsomes did not exhibit any activity. After warfarin treatment of the rats, the concentration of prothrombin precursor in rough microsomes increased rapidly from approx. 2 to 6–8 h, when a plateau was reached. In smooth microsomes, prothrombin precursor activity appeared 1 h after injection of warfarin, and increased to a plateau reached after about 4 h. The total activity of prothrombin precursor at the plateau obtained after warfarin treatment was 4–5 times higher in the rough luminal fraction than in the corresponding smooth fraction. The vitamin K-dependent carboxylase activity was localized to the rough microsomes. The enzyme system was associated with the membrane, mainly at the luminal side, whereas the substrate appeared to be localized in both the luminal and membrane fraction. The results indicate that the conversion of prothrombin precursor to biologically active prothrombin occurs at a late stage in the rough endoplasmic reticulum or at a transition between rough and smooth endoplasmic reticulum.

The distribution of protein-bound carbohydrates in submicrosomal fractions from rat liver

Abstract 1. 1. Subfractions of washed rough and smooth microsomes were prepared by sonication and deoxycholate treatment. The amount of protein-bound mannose, galactose, glucosamine and sialic acid (NANA) in the subfractions has been determined. 2. 2. The vesicle protein obtained from rough and smooth microsomes contained the same amounts of mannose, whereas the galactose and glucosamine content was significantly larger in the vesicle protein from smooth microsomes. The Galsmooth vesicles/Galrough vesicles ratio was 3.3, and GlcNsmooth vesicles/GlcNrough vesicles ratio was 1.9. Sialic acid was found only in the smooth vesicle protein. The results support the “multi-site” hypothesis for attachment of carbohydrate to the polypeptide chain. 3. 3. Mannose and glucosamine were found in similar amounts in total protein from rough and smooth membranes. The Galsmooth membranes/Galrough membranes ratio was 3.3, and the NANAsmooth membranes/NANArough membranes ratio was 2.7. 4. 4. The amino acid composition and the disc electrophoretic pattern of the vesicle proteins from rough and smooth microsomes were similar. A high degree of similarity in amino acid composition and the disc electrophoretic pattern was also found for the membrane proteins from rough and smooth microsomes, although different from that obtained for the vesicle protein.

Incorporation of radioactive glucosamine into submicrosomal fractions isolated from rat liver

Abstract The incorporation of [1- 14 C[glucosamine into various submicrosomal fractions and cell sap from rat liver, as well as into plasmaproteins, has been examined after intraperitoneal injection. It has been shown that after 1 h there is a significant incorporation of protein-bound radioactivity into the submicrosomal fractions. 69% of the total radioactivity was found in the ultrasonic extract of the microsomal pellet, 28% in the subsequent deoxycholate extract and 3% associated with the ribosomes. Of the total radioactivity in the ultrasonic extract and the deoxycholate extract, more than 80% was found in the perchloric acid precipitate and only about 5–10% in the phosphotungstic acid precipitate. 15% of the protein-bound radioactivity in the perchloric acid precipitate of the ultrasonic extract was associated with a sialic acid fraction and about 1% found in the hexose fraction. Most of the remaining radioactivity was associated with glucosamine. The intracellular site for the biosynthesis of glycoproteins is discussed.

Sex and age differences in the hemosiderin content of rat liver

1. 1.|The concentration of hemosiderin iron and hemosiderin protein in liver from male and female rats at different ages has been determined. From about 6–16 weeks of age an increase in the hemosiderin iron as well as in the hemosiderin protein concentration was found in both sexes. The increase was about 2 fold larger in female rats. From about 16–26 weeks of age the concentration levelled off, but the sex difference was maintained. 2. 2.|A new method for purification of hemosiderin has been developed. The method includes sonication of the 10 000 × g pellet, removal of connective tissue, and successive treatments with 4 M LiCl, 8 M urea, 5 M guanidine hydrochloride and 1% sodium dodecylsulphate. A purification of about 3000 fold was obtained. 3. 3.|The purified hemosiderin which contained about 9% iron was characterized by spectral analyses and amino acid composition. The preparation did not contain protoporphyrin, heme, cholesterol, phospholipids, carbohydrates, DNA or RNA.

Biological and immunological activity of prothrombin in rough and smooth microsomes isolated from rat liver

1. The presence of biological and immunological activity of prothrombin in sonicates from rough and smooth microsomes has been investigated. 2. Biological activity of prothrombin was detected in both the rough and smooth microsomal fraction. The specific and the total activity of prothrombin in the sonicates from smooth microsomes were 3-4-fold higher than in the corresponding fraction from rough microsomes. 3. Prothrombin could be identified in both microsomal fractions by double immunodiffusion. 4. The presence of a macromolecular inhibitor of blood coagulation in the microsomes is reported.

On the significance of the carbohydrate moieties of bovine prothrombin for clotting activity

Purified bovine prothrombin has been treated with different mixtures of glycosidases. Upon incubation of the prothrombin for 30 h with a combination of neuraminidase, alpha- and beta-galactosidase and beta-N-acetylglucosaminidase in 4 mM diisopropylfluorophosphate at pH 5.3 and 30 degrees C, about 70% of the carbohydrates were removed without affecting the coagulation activity. All the sialic acid and about half of the mannose, galactose and glucosamine residues were removed by this treatment.

A study of intracellular transport of secretory glycoproteins in rat liver

To study the transport of secretory glycoproteins in the endoplasmic reticulum of rat liver, the distribution of nascent glycoproteins in the membrane and luminal fraction of rough and smooth microsomes has been examined after a short-time incorporation of radioactive glucosamine in vivo. 50--60% of the radioactivity was associated with the membranes of rough and smooth microsomes, whereas about 10% of the serum albumin was found in the same fractions. The relative amount of radioactivity in the membranes was the same whether the luminal content of the microsomal vesicles was released by sonication, French press, Triton X-100, Brij 35 or sodium deoxycholate. The distribution of labeled glycoproteins between the membrane and luminal fraction of rough and smooth microsomes did not change during the time interval of 15--120 min after administration of the isotope. The similarity of the labeling patterns obtained after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis indicated that the same set of glycoproteins were located in the lumen and the membrane of rough and smooth microsomes. A specific precipitation of nascent glycoproteins from both the membrane and luminal fractions of rough and smooth microsomes were obtained with rabbit antiserum against rat serum. The nascent glycoproteins associated with the membranes were not released by high ionic strength or treatment with mercaptoethanol. A slow exchange between [14C]glucosamine-labeled glycoproteins in the lumen and membrane fraction was, however, found.

A Novel Enzyme Immunoassay for Quantitation of Rat Prothrombin in Microsomal Subfractions

An enzyme linked immunoassay (ELISA) for quantitation of rat prothrombin, based on a biotin-streptavidin alkaline phosphatase system is described. The assay utilizes rabbit antiserum raised against purified rat prothrombin. The assay was twenty fold more sensitive than a rat prothrombin assay based on amidolytic activity following activation by Echis carinatus venom. Results obtained with the two assays show good correlation. The ELISA is a valuable tool for quantitation of minute amounts of prothrombin in subcellular fractions and large series of plasma samples.

On the quantification of prothrombin from different species using echis carinatus as activator

The generation of thrombin-like activity from rat, human, bovine and mouse prothrombin by Echis carinatus venom (ECV) treatment was compared using a partially purified system (i.e. whole ECV and isolated prothrombin). A rapid increase in coagulant activity was obtained within 0.5 to 2 min., being constant upon further incubation for 60 min. A large variation in coagulant activity of the ECV generated thrombin from the four species was found, whereas no differences were found for the amidolytic activities. The coagulant activities of the ECV generated thrombin was also low compared with the corresponding thrombin activities obtained by physiological activation. Coagulant activity of the ECV generated thrombin levelled off at increasing concentration of prothrombin in the sample as measured by the one-stage coagulation assay. By measuring amidolytic activity a linear relationship to the concentration of prothrombin was found, however. These findings indicate that ECV converts prothrombin from the four different species to a thrombin-like protein with properties distinct from alpha-thrombin. The lack of linearity in the ECV generated clot activity with increasing concentration of prothrombin could be explained by assuming a dimerization of the thrombin-like protein molecules making them less accessible to fibrinogen. The significance of these observations for the quantification of prothrombin from different species is discussed.