Studies on the site of biosynthesis of acidic glycoproteins of guinea-pig serum

Abstract

1. Studies were carried out to determine the cellular and subcellular site of biosynthesis of components of fraction I, an alpha-globulin fraction containing acidic glycoproteins isolated from guinea-pig serum. l-[U-(14)C]Leucine or -valine and d-[1-(14)C]glucosamine were used as precursors. 2. A lag of about 10min. occurred before appreciable label appeared in fraction I of serum after injection of leucine or glucosamine. Label in fraction I after 60min. labelling with glucosamine was present almost entirely in hexosamine and sialic acid. 3. Site of synthesis was investigated by studies in vivo up to 17min. after injection of precursor. Particulate subcellular fractions isolated from liver, spleen and kidney or homogenates of the latter two tissues were extracted with Lubrol. Extracts were allowed to react by double diffusion with antisera to fraction I or to subfractions isolated from it, and gels were subsequently subjected to radioautography. With either amino acid or glucosamine as precursor, only extracts of the microsome fraction of liver formed precipitin lines that were appreciably radioactive. 4. The role of the microsome fraction of liver in the synthesis of these glycoproteins was confirmed by immunological studies after incubation of liver slices with leucine or glucosamine. Incorporation of leucine was also investigated in a cell-free microsome system. 5. Material was also precipitated from certain Lubrol extracts of liver microsomes by direct addition of antiserum and its radioactivity measured. Degradation of material thus precipitated and use of heterologous immune systems showed that labelling of precipitin lines represented biosynthesis. 6. A study of extraction procedures suggested that the substances present in the microsome fraction of liver that react with specific antisera are associated with membranous structures. 7. Most or all precipitin lines formed by Lubrol extracts of liver microsomes interacted with precipitin lines given by guinea-pig serum or fraction I, immunological identity being apparent with some lines. The microsome-bound substances thus represent serum glycoproteins or precursors of them. 8. The distribution of label in various tissues and in the protein of subcellular fractions of liver after administration of [(14)C]glucosamine to the guinea pig was also studied. Some variation in results obtained with liver was found depending on the fractionation medium used.

Biogenesis of microsomal membrane glycoproteins in rat liver. I. Presence of glycoproteins in microsomes and cytosol.

“…These tests gave a 10-20% precipitation of the total protein-bound [3H]glucosamine. These precipitated proteins can be attributed to the presence of some bound serum proteins whose oligosaccharide chain is incomplete but which are still precipitable by antibiodies to rat serum proteins (39). On the other hand, the fact that no radioactive sialic acid was associated with the immunoprecipitate clearly demonstrated that the membranous sialoproteins represent constitutive components of the microsomal membrane, in addition, after the washing procedure we were unable to obtain any precipitation of dissolved Golgi membranes with rabbit antiserum against rat serum proteins, a finding in agreement with those of Bizzi and Marsh (40).…”

Studies on Acute Phase Proteins of Rat Serum. III. Site of Synthesis of Albumin and α1-Acid Glycoprotein and the Contents of These Proteins in Liver Microsome Fractions from Rats Suffering from Induced Inflammation

“…Double diffusion analysis was based on the technique of Quchterlony (15) as described by Simkin and Jamieson ( 1 1 ) . For preparation of radioautographs following immunodiffusion, gels were washed at room temperature for 2 days with 0.15 M NaCI, 2 days with tap water, and 2 days with distilled water, the wash liquids being changed continuously (11). The gels were dried at 25 "C, numbered with marking ink containing P'7PmCl ( 11 ) , and placed on Ilford Ilfex X-ray film for 3-8 weeks.…”

“…50 angle head was used for all other subcellular fractionation procedures. For preparation of homogenates, tissues were minced and freed of connective tissue using a stainlesssteel tissue mincer (11,14). A motor-driven PotterElvehjem type homogenizer with a polytetrafluoroethylene pestle was used for a11 homogenization procedures.…”

Characterization, Localization, and Biosynthesis of an Interstitial Retinol‐Binding Glycoprotein in the Human Eye

“…Rabbit antiserum against purified bovine IRBP was obtained as described for cytosol retinol-binding protein by Liou et al (1981). lmmunodiffusion according to the methods of Ouchterlony (19.53) and Simkin and Jamieson (1967) was carried out for 72 h at 4°C in 0.9% agarose plates (Miles Laboratories). Twenty-five microliters of antigen (200 pg/ml), rabbit antibovine IRBP serum, or preimmune serum were added to each well.…”