Isolation and characterization of phosphoglycerate kinase and creatine kinase from bighead carp (Aristichthys nobilis): Potential sources for antitumor agents

Abstract

Bighead carp (Aristichthys nobilis) has garnered significant attention due to its potential health benefits, yet its bioactive protein components remain largely unexplored. In this study, two proteins S3 and Z1 were isolated from Aristichthys nobilis using ammonium sulfate precipitation and serial column chromatography guided by their in vitro antitumor activity. Both proteins were found to be homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a purity exceeding 95% as confirmed by reverse-phase high performance liquid chromatography (RP-HPLC). Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis revealed their precise molecular weights to be 44.335 kDa for S3 and 43.028 kDa for Z1. Their amino acid sequences were elucidated through tandem mass spectroscopy and transcriptome unigene analysis, identifying S3 as phosphoglycerate kinase and Z1 as creatine kinase. Furthermore, secondary structure was measured by circular dichroism and three-dimensional structure was predicted by modeling software. The antitumor potential of S3 was evaluated by an in vitro assay, yielding an IC₅₀ value of 26.3 ± 2.9 μM against the HT-29 cell line. Z1 demonstrated antiproliferative activity in vitro with IC₅₀ values of 21.8 ± 1.4, 22.3 ± 2.1, and 22.3 ± 2.5 μM against HT-29, HeLa, and HepG2 cell lines, respectively. Notably, Z1 was found to enhance glucose metabolism and significantly increase the production of lactic acid and CO₂ in tumor cells. These findings suggest that bighead carp (A. nobilis) could serve as a promising source for both antitumor agents and functional food ingredients.