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Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Molecular Biology Program, Department of Biology


Jacques U. Baenziger, Nancy L. Baenziger, Thomas F. Deuel, Rosalind H. Kornfeld, Linda Pike, Arnold W. Strauss


English (en)

Date of Award


Degree Type

Restricted Access Dissertation

Degree Name

Doctor of Philosophy (PhD)


In this thesis research I have identified and characterized a unique tyrosine sulfotransferase and its substrate in A431 cells. I also established an in vitro assay method for tyrosine sulfotransferase activity, which opened a door towards enzyme purification Sulfation of proteins on tyrosine residues has recently been shown as a widespread occurrence. Most of the known tyrosine sulfated proteins are secretory proteins. The tyrosine sulfotransferase activity was suggested to be localized to the Golgi apparatus. In an effort to understand more about this modification I examined A431 cells and found a protein of Mr 61,000 which could be tyrosine sulfated by cells (in vivo) and by a cell-free sulfation system (in vitro). The sulfated P61 was not detected in the cell medium. Treatment of membranes with high salt, high EDTA, high pH buffer prior to or after cell-free sulfation could not release P61 or its sulfotransferase, suggesting that both substrate and enzyme are integral membrane proteins. Further, I developed a subcellular fractionation method for A431 cells and employed enzyme marker assay and electron microscopy examination to characterize the subcellular fractions. The sulfated P61 and its sulfotransferase were localized to the rough endoplasmic reticulum. A431 cells also have the capacity to sulfate proteoglycans and pNp-GalNAc. The sulfation of carbohydrates occurs in Golgi which is distinct from the site where sulfation of P61 take place. Therefore the tyrosine sulfation activity not only is confined to Golgi but also can occur in RER. And, instead of a secretory protein this RER sulfotransferase is responsible for sulfation of an integral membrane protein. In order to characterize this RER enzyme and for the future to purify enzymes I established an in vitro assay method for tyrosine sulfotransferase activity by using a synthetic peptide with sequence derived from the fourth component of human complement as an accepter. Experimental results showed that the C4 peptide shares the same RER enzyme with P61. The catalytic domain of the enzyme was facing the lumen of the ER. The apparent Km for the peptide was 0.12 mM, and for the sulfate donor, PAPS, was 0.45 uM. In addition, I also examined the tyrosine sulfotransferase activities from rat liver RER and Golgi fractions with the C4 peptide.


Permanent URL: Print version available in library catalog at Call #: LD5791.8 PhD87 L57.

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