This invention relates to a method for measuring a process for forming sugar chains which participates in metabolism or maintenance of the living body, particularly to a method for measuring glycosyltransferase.
Glycosyltransferase is a basic enzyme widely distributed in organisms from higher animals including human beings to yeasts or bacteria, and it has been said to be an enzyme which participates in biosynthesis of sugar chains in glycoproteins, glycolipids and is responsible for an important role in metabolic maintenance of the living body. On the other hand, it has been reported to exist widely in cytoplasms of higher animals including human beings, primarily in Golgi apparatus, cell membranes and blood.
Also, in recent years, physiological activity of the sugar chain itself, particularly in cell-cell recognition is of foremost interest. Above all, aberrant glycosylation in transformation is furnishing a great topic from the standpoint of tumor markers. The sugar chain sequence and its bonding mode in such sugar chains or oligosugars are formed specifically by glycosyltransferase depending on individual sugars, while on the contrary specificity of glycosyltransferase has been considered to participate greatly in characterizing the sugar chain.
In view of such background, measurement of glycosyltransferase may be said to have an important position not only in basic research of life science but also in applications for medicine or clinical chemistry.
On the other hand, methods for measuring glycosyltransferase up to date have generally been the method in which the sugar moiety of the sugar nucleotide which is the donor is labelled with .sup.3 H, .sup.14 C, and the radioactivity of the acceptor to which its sugar is transferred by glycosyltransferase is measured.
In the present specification, the donor means a substance having been bound to the sugar to be transferred by glycosyltransferase. The acceptor means a substance of which sugar is to be transferred by glycosyltransferase, and the product means a substance in which sugar is transferred to the acceptor by glycosyltransferase.
The above method has two drawbacks:
(1) the labelled sugar nucleotide is limited to .sup.3 H and .sup.14 C, and the donor labelled by .sup.3 H or .sup.14 C is expensive and unstable as well as low in sensitivity. Consequently liquid scintillation measurement is required; and
(2) during separation of the acceptor containing the transferred labelled sugar from the labelled sugar nucleotide, operation such as salting-out, washing, electrophoresis, etc. are required.
The problems in operability and cost have which inhibited wide generalization of this method in clinical tests, etc.
In order to solve these problems, several attempts have been done. In Analy. Biochem. 102, 441 (1980), there is disclosed the method in which, instead of labelling sugar, the nucleotide formed by transfer of sugar is via plural enzymatic reactions finally measured by colorimetry or fluorescent intensity.
However, this method is deficient in sensitivity as compared with the prior art methods.
Also, in Analy. Biohem. 155, 395 (1986), in order to solve above drawback (2), choleratoxin capable of being specifically bound to the acceptor to which the sugar is transferred is used, but this method is devoid of general applicability for glycosyltransferases in general, and also the above drawback (1) has not been solved.