Biosynthesis of N‐Glycolylneuraminic Acid in Porcine Submandibular Glands

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Abstract
1. The morphology of porcine submandibular gland tissue is demonstrated by light and electron microscopy.2. Subcellular particles from porcine submandibular glands were prepared by a procedure adapted to the presence of viscous mucus, and were fractionated on continuous and discontinuous sucrose gradients.3. Six particulate fractions were obtained by discontinuous gradient centrifugation. These were identified by electron microscopy, marker enzymes and nucleic acid content and were designated as follows: (1) plasma membranes, (2) smooth membranes and light Golgi membranes, (3) heavy Golgi membranes, (4) membrane mixture and mitochondria, (5) mitochondria and (6) membrane mixture and mucin residues. The bulk of rough membranes and cell nuclei was found in the 3000 ×g sediment before fractionation.4. Sialyltransferase and N‐acetylneuraminate monooxygenase showed highest activities in the Golgi membrane fractions representing 45% and 36%, respectively, of total tissue activity. The specific activities of these membrane‐bound enzymes were respectively 13 and 10 times higher when compared with the homogenate.5. The acylneuraminic acid/protein ratio was lowest in the Golgi membranes when compared with the other particulate fractions and the cytosol glycoprotein. The molar N‐acetylneuraminic acid/N‐glycolylneuraminic acid ratio was highest in the two Golgi membrane fractions (average 45:55) and in the pool of free acylneuraminic acids (41:59), and lowest in the cytosol glycoprotein (10:90).6. Approximately 2.6% of the total acylneuraminic acids occurring in porcine submandibular glands are freely diffusible, 80% of them representing free N‐acetylneuraminic acid and N‐glycolyl‐neuraminic acid in a molar ratio of about 4:6 and 20% representing cytidylyl‐glycosides of N‐acetylneuraminic acid and N‐glycolylneuraminic acid in a ratio of 6:4.7. In radioactive labelling experiments with surviving slices of porcine submandibular glands the highest specific radioactivities were found in the pool of free acylneuraminic acids, followed by N‐acetylneuraminic acid and N‐glycolylneuraminic acid bound to the Golgi membranes. N‐Acetylneuraminic acid and N‐glycolylneuraminic acid of the cytosol glycoproteins have lowest turnover rates.8. Based on these experiments a model is presented describing two pathways of N‐acetylneuraminic acid modification in the course of glycoprotein biosynthesis. At least 40% of N‐acetylneuraminic acid is hydroxylated before its transfer onto Golgi membranes and linkage to growing glycoprotein molecules and about 50% thereafter. This results in a molar N‐acetylneuraminic acid/N‐glycolyl‐neuraminic acid relationship of 1:9 in the secreted glycoprotein. It is assumed that free N‐acetylneuraminic acid is modified by the soluble cytosol monooxygenase whereas the glycoprotein‐bound N‐acetylneuraminic acid is hydroxylated by the corresponding membrane‐bound enzyme.

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