Cookies help us deliver our services. By using our services, you agree to our use of cookies. More information

Lanza 2009 Methods Enzymol

From Bioblast
Publications in the MiPMap
Lanza IR, Nair KS (2009) Functional assessment of isolated mitochondria in vitro. Methods in Enzymol. Mitochondrial Function, Part B 457: 349-372.

Β» PMID: 19426878

Lanza IR, Nair KS (2009) Methods Enzymol.

Abstract: Mitochondria play a pivotal role in cellular function, not only as a major site of ATP production, but also by regulating energy expenditure, apoptosis signaling, and production of reactive oxygen species. Altered mitochondrial function is reported to be a key underlying mechanism of many pathological states and in the aging process. Functional measurements of intact mitochondria isolated from fresh tissue provides distinct information regarding the function of these organelles that complements conventional mitochondrial assays using previously frozen tissue as well as in vivo assessment using techniques such as magnetic resonance and near-infrared spectroscopy. This chapter describes the process by which mitochondria are isolated from small amounts of human skeletal muscle obtained by needle biopsy and two approaches used to assess mitochondrial oxidative capacity and other key components of mitochondrial physiology. We first describe a bioluminescent approach for measuring the rates of mitochondrial ATP production. Firefly luciferase catalyzes a light-emitting reaction whereby the substrate luciferin is oxidized in an ATP-dependent manner. A luminometer is used to quantify the light signal, which is proportional to ATP concentration. We also review a method involving polarographic measurement of oxygen consumption. Measurements of oxygen consumption, which previously required large amounts of tissue, are now feasible with very small amounts of sample obtained by needle biopsy due to recent advances in the field of high-resolution respirometry. We illustrate how careful attention to substrate combinations and inhibitors allows an abundance of unique functional information to be obtained from isolated mitochondria, including function at various energetic states, oxidative capacity with electron flow through distinct complexes, coupling of oxygen consumption to ATP production, and membrane integrity. These measurements, together with studies of mitochondrial DNA abundance, mRNA levels, protein expression, and synthesis rates of mitochondrial proteins provide insightful mechanistic information about mitochondria in a variety of tissue types.


β€’ O2k-Network Lab: US MN Rochester Nair KS


Labels:


Organism: Human  Tissue;cell: Skeletal Muscle"Skeletal Muscle" is not in the list (Heart, Skeletal muscle, Nervous system, Liver, Kidney, Lung;gill, Islet cell;pancreas;thymus, Endothelial;epithelial;mesothelial cell, Blood cells, Fat, ...) of allowed values for the "Tissue and cell" property.  Preparation: Isolated Mitochondria"Isolated Mitochondria" is not in the list (Intact organism, Intact organ, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP, Chloroplasts, Enzyme, Oxidase;biochemical oxidation, ...) of allowed values for the "Preparation" property.  Enzyme: Marker Enzyme"Marker Enzyme" is not in the list (Adenine nucleotide translocase, Complex I, Complex II;succinate dehydrogenase, Complex III, Complex IV;cytochrome c oxidase, Complex V;ATP synthase, Inner mt-membrane transporter, Marker enzyme, Supercomplex, TCA cycle and matrix dehydrogenases, ...) of allowed values for the "Enzyme" property.  Regulation: Respiration; OXPHOS; ETS Capacity"Respiration; OXPHOS; ETS Capacity" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property. 


HRR: Oxygraph-2k