Abstract
The properties and developmental regulation of vertebrate polysialyltransferase (PST), an enzyme activity responsible for extension of alpha 2,8-linked sialic acid homopolymers (PSA) associated with the fifth Ig domain of the neural cell adhesion molecule (NCAM). have been studied. The assay for PST used exogenous NCAM as a substrate, with a PSA-specific endoneuraminidase as a control for specificity. Optimal conditions for PST activity at 37 degrees C were found to be pH 6.0 in the presence of divalent cations (Mn2+, 20mM). The enzyme Km was found to increase with increasing polymer length, ranging from 0.7 to 0.07 microns. The developmental regulation both of PST activity and of the addition of PSA to NCAM were studied in chick whole brain, tectum, and cerebellum and found to be precisely coordinated. In each tissue PSA and PST were highest during early stages of morphogenesis, followed by a decrease as development reached completion. The insertion of the VASE exon in the fourth Ig domain of NCAM was also found to parallel closely the developmental down-regulation of PSA, and on this basis could be considered a potential determinant in the specific polysialylation of NCAM. However in direct tests of this hypothesis in transfected cells the presence of VASE did not markedly alter the level of NCAM polysialylation or alter the affinity of PST for the NCAM substrate.
Highlights
The properties and developmental regulation of vertebrate polysialyltransferase (PST), an enzyme activity responsible for extension of a2,8-linked sialic acid homopolymers (PSA) associated with the fifth Ig domain of the neural cell adhesion molecule (NCAM), have been studied
Polysialyltransferase Activity Assay-To evaluate quantitatively the role of PST in the developmental regulation ofNCAM polysialylation, an assay was developed to measure the incorporation of [14C]NeuAc into a2,8-linked Polysialic acid (PSA) chains on exogenous NCAM purified from embryonic brain
In order to clarify further how thi s regu lation occurs, thi s study has sought to determine if th e regulation of PSA expression in vivo is tightly linked to changes in the levels of specific polysialyltransferase activity, to define more clearly the properties of that enzymatic activity, and to explore whether alternative splicin g of th e NCAM polypeptid e might affect its ability to serve as a n acceptor for polysialylation
Summary
Properties and Developmental Regulation of Polysialyltransferase Activity in the Chicken Embryo Brain*. The properties and developmental regulation of vertebrate polysialyltransferase (PST), an enzyme activity responsible for extension of a2,8-linked sialic acid homopolymers (PSA) associated with the fifth Ig domain of the neural cell adhesion molecule (NCAM), have been studied. The enzyme K m was found to increase with increasing polymer length, ranging from 0.7 to 0.07 /LM The developmental regulation both of PST activity and of the addition of PSA to NCAM were studied in chick whole brain, tectum, and cerebellum and found to be precisely coordinated. We have used cell transfection studies to test directly whether the developmentally regulated insertion of the VASE exon [12] into the Ig domain adjacent to the site ofNCAM polysialylation might be a determinant in control of PSA biosynthesis
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