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

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Abstract
Studies were carried out to determine the cellular and subcellular site of biosynthesis of components of fraction I, an a-globulin fraction containing acidic glycoproteins isolated from guinea-pig serum. L-[U-14C] Leucine or -valine and D-[1-14C]gluco-samine were used as precursors. A lag of about 10 min. occurred before appreciable label appeared in fraction I of serum after injection of leucine or glucosamine. Label in fraction I after 60 min. labelling with glucosamine was present almost entirely in hexosamine and sialic acid. Site of synthesis was investigated by studies in vivo up to 17 min. 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 subtractions 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. 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. Material was also precipitated from certain Lubrol extract 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. 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. 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. The distribution of label in various tissues and in the protein of subcellular fractions of liver after administration of [14C]glucosamine to the guinea pig was also studied. Some variation in results obtained with liver was found depending on the fractionation medium used.