Abstract
Tris caused the distention of the Golgi cisternae in primary cultured rat hepatocytes and perturbed the functions occurring there. Proteolytic cleavage of precursors of both albumin and complement C3 was inhibited, whereas that of prohaptoglobin was not affected by Tris. These effects on the proteolytic cleavages resemble those of acidotropic amines (Oda, K., and Ikehara, Y. (1985) Eur. J. Biochem. 152, 605-609; Oda, K., Koriyama, Y., Yamada, E., and Ikehara, Y. (1986) Biochem. J. 240, 739-745). However, the effects of Tris significantly differed from acidotropic amines on the basis of its effects on the processing of N-linked oligosaccharides of glycoproteins. Both alpha 1-protease inhibitor and haptoglobin secreted from the Tris-treated cells were found to contain almost equal amounts of endo-beta-N-acetylglucosaminidase H-sensitive and -resistant oligosaccharides, whereas the glycoproteins from both the control and methylamine-treated cells were resistant to the enzyme. The endo-beta-N-acetylglucosaminidase-sensitive oligosaccharides were analyzed to be Man8-5GlcNAc by high resolution gel permeation chromatography, suggesting that trimming of alpha-mannose residues from the precursor Man9GlcNAc2 is incomplete in the Tris-treated cells. On the other hand, Tris did not significantly inhibit incorporation of radioactive monosaccharides (N-acetylglucosamine, galactose, and fucose) into the glycoproteins. However, two-dimensional gel electrophoresis in combination with neuraminidase digestion demonstrated that sialylation was markedly inhibited by Tris. Taken together, our results reveal that Tris inhibits not only the sialic acid addition which takes place in the trans Golgi region, but also the trimming step of high mannose-type oligosaccharides, which is thought to occur before glycoproteins reach the trans Golgi region.
Highlights
Tris caused the distention of the Golgi cisternae in Alternatively, Yamashiro et al (1984) have indicated that the primary cultured rat hepatocytes and perturbed the para Golgi compartment is mildly acidic byfollowing the functions occurring there
As the chase time elapsed, proalbumin was converted to serum albumin in the cell (Fig. 2, lanes 2-4), followedby its release into medium.No proalbumin was secreted from the control cells (Fig.2A, lanes 5 and 6).In the Tris-treated cells, howevera, nother form in addition to serum albumin was found in the medium (Fig. 2B, lanes 5 and 6)
Proteolytic cleavages of proalbumin and pro-C3 are known to occur in the Golgi complex (Edwards et al, 1976;Ikehara et al, 1976;Judah and Quinn, 1976;Misumi et al, 1984;Oda et al, 1986b;Redman et al, 1978),whereas prohaptoglobin undergoes limited proteolysis in the ERbefore reaching the Golgi complex(Hanely et al, 1983;Misumi et al, 1983;Chow et al, 1984;Fries and Lindstrom, 1986)
Summary
Tris Inhibits Secretion of al-Protease Inhibitor-To examine the effect of Tris or methylamine on the secretion of alPI, hepatocytes were pulse-labeled with [3H]leucine in the presence or absence of the amines. As the chase time elapsed, proalbumin was converted to serum albumin in the cell (Fig. 2, lanes 2-4), followedby its release into medium.No proalbumin was secreted from the control cells (Fig.2A, lanes 5 and 6).In the Tris-treated cells, howevera, nother form in addition to serum albumin was found in the medium (Fig. 2B, lanes 5 and 6). Our results further demonstrated that mature C3 with the a and @ subunits markedly accumulated in the cells in response to Tris(Fig. 3, lanes 911), strongly indicating that Tris inhibits the proteolytic cleavage of the precursor, and blocks the release of mature C3 into themedium. N terminus of serum albumin (Russell and Geller, 1975).Fig. pro-C3 and the a and subunits of C3, respectively
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