Preparation of Rat Gingival Mitochondria with an Improved Isolation Method

Noriaki Kaneko,Shigeyasu Mori,Hidehiro Kaya,Tetsuya Rikimaru,Tetsuyuki Fujimura,Saburo Hidaka

doi:10.1155/2010/275103

Abstract

In order to establish a method of obtaining rat gingival mitochondria (Mt), Mt fractions were prepared in various combinations of homogenizing time with collagenase concentration. Rat gingival tissues were excised, minced, treated with collagenase, homogenized, and subjected to differential centrifugation rates. Both the respiratory control ratio (RCR) and adenosine diphosphate/oxygen (ADP/O) ratio of the Mt fraction prepared in a combination of 40, 50, or 60 sec homogenization with collagenase in a concentration range of 0.115%–0.130% (w/v) were measured. The values for the RCR and ADP/O ratio of the Mt fraction obtained in an optimal condition was 1.80 ± 0.05 and 1.65 ± 0.03, respectively. These results suggest that Mt of fairly high quality can be obtained through this refined combination of the homogenizing time and collagenase concentration.

Highlights

Mitochondria play a central role in the energy metabolism of brain, heart, and muscle by controlling the production of adenosine triphosphate (ATP), and mitochondrial preparations are commonly used to evaluate the metabolic activities of these tissues in both the normal and diseased states [1]
To avoid the destruction of mitochondria or the loss of respiratory activity, chilled buffer solutions of an isotonic tension have generally been adopted. Those studies have yielded variable and generally low levels of respiration both in the presence and absence of substrate, and the quantitative assessment of mitochondrial ATP synthesis in oral tissues has been hindered by the extreme difficulty of homogenizing these tissues without damaging mitochondrial integrity and uncoupling oxidative phosphorylation [4]
We report here a procedure for the isolation of mitochondria from rat gingival tissues by an optimized combination between homogenizing time and collagenase concentration, which displayed a good respiratory control ratio (RCR) and adenosine diphosphate/oxygen (ADP/O) ratio using succinic acid as the substrate

Summary

Introduction

Mitochondria play a central role in the energy metabolism of brain, heart, and muscle by controlling the production of adenosine triphosphate (ATP), and mitochondrial preparations are commonly used to evaluate the metabolic activities of these tissues in both the normal and diseased states [1]. To avoid the destruction of mitochondria or the loss of respiratory activity, chilled buffer solutions of an isotonic tension have generally been adopted Those studies have yielded variable and generally low levels of respiration both in the presence and absence of substrate, and the quantitative assessment of mitochondrial ATP synthesis in oral tissues has been hindered by the extreme difficulty of homogenizing these tissues without damaging mitochondrial integrity and uncoupling oxidative phosphorylation [4]. We report here a procedure for the isolation of mitochondria from rat gingival tissues by an optimized combination between homogenizing time and collagenase concentration, which displayed a good respiratory control ratio (RCR) and adenosine diphosphate/oxygen (ADP/O) ratio using succinic acid as the substrate.

Materials and Methods

Results

Discussion