Characterization of glycoprotein antifreeze biosynthesis in isolated hepatocytes from Pagothenia borchgrevinki.
Incorporation of 14C-leucine and 3H-alanine into TCA-precipitable protein. TCA-soluble protein, and antifreeze glycoproteins (AFGP) was measured in isolated hepatocytes from Pagothenia borchgrevinki Boulenger following acclimation to -1.5 degrees C and +4 degrees C. the rate of 3H-alanine incorporation into AFGP followed Michaelis-Menten kinetics with a Vmax of 4.8 nM X mg protein-1 X h-1 at -1.5 degrees C and 7.5 nM X mg protein-1 X h-1 at +4 degrees C. Km values were 27.9 microM and 41.7 microM at -1.5 degrees C and +4 degrees C, respectively. Incorporation of 14C-leucine into TCA-precipitable protein also showed Michaelis-Menten kinetics with a Vmax of 20 nM X mg protein-1 X hr-1 at 1.5 degrees C and 32.3 nM X mg protein-1 X hr-1 at +4 degrees C. Km values were 83.3 microM at -1.5 degrees C and 125 microM at +4 degrees C. AFGP synthesis was monitored over a 120-h period by radioimmunoassay in cultures of hepatocytes from cold acclimated fish (-1.5 degrees C) incubated at both -1.5 degrees C and +4 degrees C. The estimated Q10 for AFGP from these data is 3.23. Polyacrylamide gel electrophoresis of antifreeze glycoproteins produced by isolated hepatocytes showed that all four antifreeze fractions normally present in the serum of P. borchgrevinki are also synthesized by isolated hepatocytes. The two major conclusions from these experiments were that 1) P. brochgrevinki, unlike many northern fishes, does not show thermal acclimation, and 2) environmental factors responsible for modification of peptide antifreeze synthesis in northern fishes do not elicit changes in AFGP synthesis in P. borchgrevinki.
D000064:Acclimatization; D000409:Alanine / Q000378:metabolism; D000818:Animals; D021301:Antifreeze Proteins; D002250:Carbon Radioisotopes; D005399:Fishes; D005615:Freezing; D006023:Glycoproteins / Q000096:biosynthesis*; D066298:In Vitro Techniques; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D014316:Tritium
D021301:Antifreeze Proteins; D002250:Carbon Radioisotopes; D006023:Glycoproteins; D014316:Tritium; D007930:Leucine; D000409:Alanine