Enzyme Kinetic Assay to Measure the Activity of Tumor M2 Pyruvate Kinase in Breast Cancer Patients.

Abstract

BACKGROUND:

M2 type Pyruvate kinase (PKM2) is the key rate-limiting enzyme of glycolysis, and it mainly exists as a dimer in tumor cells. This study aims to establish an enzyme kinetic assay for serum tumor M2 pyruvate kinase (TuM2-PK), and to evaluate its diagnostic value in breast cancer.

METHODS:

The catalytic kinetics of Pyruvate Kinase (PK) was examined. Its allosteric regulation property and Michaelis constant were then measured. Next, the levels of TuM2-PK in serum were detected and compared with results from an enzyme-linked immunosorbent assay (ELISA). Finally, the levels of TuM2-PK among breast cancer patients, post-mastectomy patients, patients with benign breast diseases, and healthy controls were compared.

RESULTS:

A PK kinetic assay was established in this study. The assay reaction time is 108 seconds, and the optimum pH level is 8.0. In the presence of the allosteric activator fructose 1, 6-bisphosphate (FBP), the Km of PK for phosphoenolpyruvate (PEP) is 0.15 mmol/L and the Vmax is 330 μmol/min. The levels of TuM2-PK in serum obtained by enzyme kinetics are comparable to the ELISA results. Both assays showed that TuM2-PK in breast cancer patients was increased from stage I to IV. Importantly, TuM2-PK levels were significantly different between early and late-stage breast cancer patients (stage I and stage II vs. stage III and IV), as well as between late-stage and non-malignant patients (p<0.05). No statistical difference was found between benign breast disease patients and healthy controls.

CONCLUSIONS:

An enzyme kinetic assay of serum TuM2-PK was successfully established and may be useful for breast cancer diagnosis.

• Authors: Mingxiu Guan, Yingna Tong, Xiaobin Liu, Dong Dong, Yunli Zhou
• Journal: Annals of clinical and laboratory science (Ann Clin Lab Sci) Vol. 47 Issue 6 Pg. 676-686 (Nov 2017) ISSN: 1550-8080 [Electronic] United States
• PMID: 29263041 (Publication Type: Journal Article)
• Copyright: © 2017 by the Association of Clinical Scientists, Inc.

Chemical References

• Fructosediphosphates
• Pyruvate Kinase
• fructose-1,6-diphosphate

Topics

• Adult
• Breast Neoplasms (blood, enzymology, pathology)
• Cell Line, Tumor
• Enzyme Assays (methods)
• Enzyme-Linked Immunosorbent Assay
• Female
• Fructosediphosphates (metabolism)
• Humans
• Kinetics
• Middle Aged
• Pyruvate Kinase (blood, metabolism)
• ROC Curve
• Reproducibility of Results