Patent ID: 6040149
Filing Date: 2000-03-21
Classification: C12N,C12Q,G01N

Abstract:
A method of determining whether an agent is capable of specifically inhibiting the ability of lipopolysaccharide to stimulate phosphorylation of the threonine residue in a Thr-Pro- or a Thr-Leu-Pro-containing polypeptide by a ceramide-activated protein kinase having a molecular weight of about 100-110 kD, which comprises:(a) incubating the protein kinase with a reaction mixture containing a predetermined amount of a polypeptide capable of being specifically phosphorylated by the protein kinase, a predetermined amount of lipopolysaccharide, and the agent, under conditions (I) which would permit activity of the protein kinase to be linear with respect to time, lipopolysaccharide concentration and protein kinase concentration in the absence of the agent, and (ii) which would permit the specific phosphorylation by the protein kinase of a predetermined number of the threonine residues in such predetermined amount of the polypeptide in the absence of the agent;(b) quantitatively determining the percentage of such predetermined number of threonine residues which are specifically phosphorylated in the presence of the agent, thereby determining whether the agent is capable of inhibiting the ability of lipopolysaccharide to stimulate the phosphorylation activity of the ceramide-activated protein kinase; and(c) determining whether the agent inhibits the ability of a non-lipopolysaccharide agent to stimulate the phosphorylation activity of the ceramide-activated protein kinase, the non-lipopolysaccharide agent being known to stimulate the activity in the absence of the agent, so as to determine whether the agent is capable of specifically inhibiting the ability of lipopolysaccharide to stimulate the phosphorylation activity of the ceramide-activated protein kinase.