Multienzyme activity profiling for evaluation of cell‐to‐cell variability of metabolic state

Abstract

Much of what is being done to understand organ functions focuses on characterization of the "types" of cells they include. A recent development has been to try to decipher how single cells of the same type can diverge in their "state." [1][2][3] We already know that variability of state is physiologically meaningful. For example, the metabolic state of hepatocytes varies according to their location along the axis of liver lobules that extends between the central and portal veins, and this diversity of state is a hallmark of normal liver function. 4 This paper focuses on the potential utility of single-cell analysis of enzyme kinetics for probing the diversity of metabolic state of post-mitotic cells of the same type.The nature of state differences between individual cells is now being explored using a plethora of high throughput

Biochemical evidence that the whole compartment activity behavior of GAPDH differs between the cytoplasm and nucleus

“…These findings validated the fractionation method and set the stage for analysis of GAPDH.s and GAPDH.l expression as revealed within a sample by the raw value of the Σ# PSMs for each isoform ( Fig 1G ). Because 96% of total oocyte GAPDH resides in the cytoplasm [ 43 ], GAPDH.s and GAPDH.l are expected to be of similar abundance in whole cytoplasm and the whole oocyte. This is the case.…”

“…One possibility is that the global abundance of some key intermediates of central metabolism is determined predominantly by the biochemical programming of nuclear rather than cytoplasmic GAPDH. We consider this unlikely because in the full-grown oocyte only 4% of all GAPDH is found in the nucleus [ 43 ]. It follows that nucleus-specific tuning of GAPDH activity would not directly dictate the running of modules of metabolism located only in the cytoplasm (glycogen and NMP synthesis, Fig 2 ).…”

“…The volume of cytoplasm contributed to oil isolated nuclei by the nucleoplasmic reticulum and cytoplasmic rim is therefore approximately 1. 5 Gill and Schultz (2022), who also reported on the expression of full-length MDH1 in these compartments). In the present study enzyme activity was analyzed as described for whole oocyte homogenates by Gill and Schultz (2022) Enolase activity in homogenate of whole cytoplasms and the low-speed supernatant of nuclear homogenate.…”

“…5 Gill and Schultz (2022), who also reported on the expression of full-length MDH1 in these compartments). In the present study enzyme activity was analyzed as described for whole oocyte homogenates by Gill and Schultz (2022) Enolase activity in homogenate of whole cytoplasms and the low-speed supernatant of nuclear homogenate. Activity in samples from oil-dissected oocytes was assessed using a colorimetric assay kit (Sigma # MAK178).…”

“…Qualitative analysis was performed by two methods which yielded similar results. The conventional method used SDS-12% polyacrylamide gels and a submarine transfer system for blotting to nitrocellulose [ 43 ]. The updated method used the Bio-Rad Trans-Blot Turbo Transfer System for blotting (Bio-Rad #s 1704150, 1704271).…”