NMR detection of creatine kinase expressed in liver of transgenic mice: determination of free ADP levels.

Abstract

To use the equilibrium established by creatine kinase (CK) to determine hepatic free ADP levels, the transcriptional control elements of the transthyretin gene were used to direct expression of the CK B isozyme to the livers of transgenic mice. Activities of CK ranging from 80-250 pmol per min per g (wet weight) were detected in liver extracts from five founder mice. The CK activity was stably transmitted to subsequent generations. Isozyme gels and immunoblots confirmed that the activity detected in extracts was due to the B isozyme of CK. Immunohistology indicated that the protein was expressed uniformly throughout the liver and was localized primarily to the cytoplasm. 31P NMR spectroscopy was used to detect the metabolic product of the CK reaction, phosphocreatine, demonstrating that the enzyme was active in vivo. The phosphocreatine level fell rapidly during anoxia (tQ12 = 1 min), indicating that the CK reaction was integrated into hepatic energy metabolism. The equilibrium established by CK was used to calculate a hepatic free ADP level of 0.059 ± 0.004 ,umol/g (wet weight). In vivo NMR studies of these mice will be valuable for studying the role of free ADP in regulating liver metabolism.Adenosine diphosphate (ADP) is an important regulator of many metabolic pathways in vitro, including oxidative phosphorylation (1), glycolysis (2), gluconeogenesis (2), and ion transport (3). Due to difficulties in measuring free ADP levels in intact liver it has been impossible to assess the role of this key metabolite in regulating hepatic metabolism in vivo (4). To study changes that may occur during perturbations of liver metabolism, ADP is usually assayed from whole cell extracts or from liver cells after subcellular fractionation into cytoplasmic and mitochondrial compartments (4-6). However, it is generally acknowledged that for metabolites such as ADP, the concentrations of which are low and comparable to the number of intracellular binding sites, measurements from extracts lead to an overestimation of free metabolite levels (4).Hans Krebs introduced the idea of using cellular reactions near equilibrium to determine the levels of free metabolites at low concentrations (7). This technique relies on measuring compounds present in relatively high concentrations that are in chemical equilibrium with the metabolite of interest. Ideally, a single equilibrium using metabolites that can be monitored in vivo should be used. The reaction catalyzed by creatine kinase (CK), phosphocreatine (PCr) + MgADP + H+ = MgATP + creatine, is the method of choice for the determination of ADP (4). The value of the equilibrium constant is known for a variety of conditions, and free ADP levels can be calculated from in vivo nuclear magnetic resonance (NMR) measurements of PCr, ATP, creatine, Mg2+, and pH. This approach has been successfully used to study the role of ADP in metabolic regulation in heart (8), muscle (9), and brain metabolism (10). However, it has not been useful for liver due to the lack of significant levels of CK.In...

Noninvasive evaluation of liver repopulation by transplanted hepatocytes using 31P MRS imaging in mice

“…Donor hepatocytes were isolated from transgenic mice livers that express the brain isoform of the CK enzyme, CK-BB, under the control of liver-specific transthyretin promoter (CK-Tg), 14 using an in situ two-step collagenase perfusion procedure. 20,21 BDF mice were subjected to laparotomy, and 50 Gy of hepatic irradiation was delivered to anterior liver lobes by shielding the caudate lobe and other abdominal organs.…”

“…13 Creatine kinase (CK) is an adenosine triphosphate (ATP)-buffering enzyme not normally expressed in the liver. 14 Koretsky et al 14 showed that CK expressed in hepatocytes of transgenic mice catalyzes the phosphorylation of creatine (Cr), generating phosphocreatine (PCr) that is absent in normal liver and generates a specific 31 P NMR spectra in vivo, which distinguishes the transgenic mouse liver from the livers of wild-type mice. Based on these findings, we hypothesized that ectopic expression of CK in donor hepatocytes could provide a specific marker enabling 31 P magnetic resonance spectroscopic imaging (MRSI) for assessment of hepatic repopulation by transplanted hepatocytes.…”

Myosin Va and myosin VI coordinate their steps while engaged in an in vitro tug of war during cargo transport

“…5F). Thus, myoVI can effectively anchor the cargo in the presence of micromolar ADP concentrations that are physiological (19,(35)(36)(37), which implies that the differential effects of ADP on myoV and myoVI (see Fig. 5 A and B) could serve as an in vivo regulator that links myoVI function to the metabolic state of the cell (19).…”

Tissue- and cell-specific distribution of creatine kinase B: A new and highly specific monoclonal antibody for use in immunohistochemistry

“…Because only very few reports have focussed on mice (Ikeda and Tomonaga 1987;Spicer and Schulte 1992), a detailed in ventory of B-CK distribution in this animal is lacking. This is unfortunate because the mouse has recently prov en to be particularly useful for the examination of the bi ological role of the CK system, through the generation of animal models overexpressing (Koretsky et al 1990;Brosnan et al 1993), or lacking (van Deursen et al 1993, 1994a, 1994b) CK-isoenzymes. For these reasons we studied the reactivity of MoAb CK-BYK /21E10 in various mouse tissues.…”