Radioactive fucose as a tool for studying glycoprotein secretion

The efficiency and reliability of radioactive fucose as a specific label for newly synthesized glycoproteins were investigated. Young adult male rabbits were injected intravitreally with [3H]-fucose, [3H]-galactose, [3H]-mannose, N-acetyl-[3H]-glucosamine or N-acetyl-[3H]-mannosamine, and killed 40 h after injection. In another series of experiments rabbits were injected with either [3H]-fucose or several tritiated amino acids and the specific activity of the vitreous proteins was determined. Vitreous samples were also processed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and histological sections of retina, ciliary body and lens (the eye components around the vitreous body) were processed for radioautography. The specific activity (counts per minute per microgram of protein) of the glycoproteins labeled with [3H]-fucose was always much higher than that of the proteins labeled with any of the other monosaccharides or any of the amino acids. There was a good correlation between the specific activity of the proteins labeled by any of the above precursors and the density of the vitreous protein bands detected by fluorography. This was also true for the silver grain density on the radioautographs of the histological sections of retina, ciliary body and lens. The contribution of radioautography (after [3H]-fucose administration) to the elucidation of the biogenesis of lysosomal and membrane glycoproteins and to the determination of the intracellular process of protein secretion was reviewed. Radioactive fucose is the precursor of choice for studying glycoprotein secretion because it is specific, efficient and practical for this purpose

Evidence for the presence of a kininogen-like species in a case of total deficiency of low and high molecular weight kininogens

Low and high molecular weight kininogens (LK and HK), containing 409 and 626 amino acids with masses of ~65 and 120 kDa after glycosylation, respectively, are coded by a single gene mapped to the human chromosome 3 by alternative splicing of the transcribed mRNA. The NH2-termini Glu1-Thr383 region, identical in LK and HK, contains bradykinin (BK) moieties Arg363-Arg371. LK, HK and their kinin products Lys-BK and BK are involved in several biologic processes. They are evolutionarily conserved and only 7 patients, all apparently normal, have been reported to lack them. In one of these patients (Williams' trait), a codon mutation (Arg178 ® stop) has been blamed for the absence of LK and HK. However, using Western blots with 2 monoclonal anti-HK antibodies, one that recognizes the region common to LK and HK and the other that recognizes only HK, I detected ~110-kDa bands in the plasma of this LK/HK-deficient patient vs ~120-kDa bands in normal human and ape plasmas. With polyclonal anti-Lys-BK antibody, which strongly detects BK cleaved at its COOH-terminus in purified HK, I detected ~110-kDa bands in the normal and the deficient plasmas. Western blots with a monoclonal anti-prekallikrein (PK) antibody showed that surface activation of PK and distribution of PK activation products, both dependent on HK, were similar in these plasmas. These findings suggest that a mutant gene yielded a kininogen-like species possibly involving aberrant mRNA splicing - structurally different from normal HK, but apparently with the capacity to carry out seemingly vital HK functions.