Quantitative isolation of liver mitochondria by zonal centrifugation

Wilson, Margaret A.; Cascarano, Joseph; Wooten, Wendy L.; Pickett, Cecil B.

doi:10.1016/0003-2697(78)90297-x

Cited by 25 publications

(4 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Differential centrifugation is not appropriate since large numbers of mitochondria are lost in this isolation procedure. Zonal centrifugation was utilized as developed by Wilson et al (1978), which makes possible the collection of over 90% of the mitochondria with minimal cosedimentation by other cellular organelles. If mitochondria are variable in their association with RER, zonal centrifugation might result in enrichment of some fractions with mitochondria having RER associations and others with mitochondria having no associations.…”

Section: Resultsmentioning

confidence: 99%

“…Rate zonal centrifugation utilizing a reorienting zonal rotor (SZ-14 Sorvall) was employed to fractionate liver homogenates (Sheeler et al, 1971). This procedure is described in detail in a previous publication (Wilson et al, 1978). The rotor contained a 150 ml cushion, 60% (wlw) sucrose, followed by a 1050 ml linear density gradient, 45% (wlw)-14.5% (wlw) sucrose.…”

Section: Isolation Of Mitochondriamentioning

confidence: 99%

See 1 more Smart Citation

Association between mitochondria and rough endoplasmic reticulum in rat liver

et al. 1982

Self Cite

View full text Add to dashboard Cite

Mitochondria were isolated from rat liver homogenate by both zonal and sedimentation equilibrium centrifugation. From electron microscopic examination of thin sections it was observed that 81% of the isolated mitochondria were in contact with rough endoplasmic reticulum (RER). Intact, non-sectioned mitochondria subjected to negative staining procedures appeared to show points of connection between RER and the outer mitochondrial membrane. After treatment of mitochondria with digitonin to remove outer membranes, some of the resulting mitoplasts (intact inner membranes) remained closely associated with RER. Serial section analysis of intact rat liver indicated that RER saccules fit over mitochondria like caps providing broad areas of contact between the two organelles. The RER saccules were also observed communicating with more than one mitochondrion.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Isolation Of Mitochondriamentioning

confidence: 99%

Association between mitochondria and rough endoplasmic reticulum in rat liver

et al. 1982

Self Cite

View full text Add to dashboard Cite

show abstract

“…[26]; mitochondrial protein content per g of liver, 27-45.5 mg/g wet wt. [27][28][29][30]. From these parameters, a calculated activity in the range 56.5-94.5 nmol/min per mg of DNA is obtained (depending on which value for mitochondrial protein content is taken).…”

Section: Characterization Of Cell Permeabilizationmentioning

confidence: 99%

Use of a selectively permeabilized isolated rat hepatocyte preparation to study changes in the properties of overt carnitine palmitoyltransferase activity in situ

Boon

Zammit

1988

Biochemical Journal

View full text Add to dashboard Cite

1. A permeabilized isolated rat liver cell preparation was developed to achieve selective permeabilization of the cell membrane to metabolites and to allow the assay of mitochondrial overt carnitine palmitoyltransferase (CPT I) activity in situ. By performing the digitonin-induced permeabilization in the presence of fluoride and bivalent-metal-cation sequestrants, it was possible to demonstrate that the activity of other enzymes, which are regulated by reversible phosphorylation, was preserved during the procedure and subsequent washing of cells before assay. 2. CPT activity at a sub-optimal palmitoyl-CoA concentration was almost totally (approximately 90%) inhibited by malonyl-CoA, indicating that mitochondrial CPT I was largely measured in this preparation. 3. The palmitoyl-CoA-saturation and malonyl-CoA-inhibition curves for CPT activity in permeabilized cells were very similar to those obtained previously for the enzyme in isolated liver mitochondria. Moreover, starvation and diabetes had the same effects on enzyme activity, affinity for palmitoyl-CoA and malonyl-CoA sensitivity of CPT I in isolated cells as found in isolated mitochondria. These physiologically induced changes persisted through the cell preparation and incubation period. 4. Neither incubation of cells with glucagon or insulin nor incubation with pyruvate and lactate before permeabilization resulted in alterations of these parameters of CPT I in isolated cells. 5. The results are discussed in relation to the temporal relationships of changes in the activity and properties of CPT I in vivo in relation to the effects of insulin and glucagon on fatty acid metabolism in vivo.

show abstract

“…Such an amount of tissue can only be obtained from brain dead donor hearts unsuitable for donation (similar to what we did in this study), or explanted failing hearts, which are known to be dysfunctional [8,34,37]. Moreover, the isolation procedure itself can lead to artificial damage of mitochondria during centrifugation [38], which only yields 20-40% of mitochondria in the sample [13][14][15][16]39], potentially inducing a selection bias [40]. Because isolation of mitochondria disrupts native environment of mitochondrial network and intracellular communication with other organelles [41], the generalizability of the results to in vivo environment have been questioned [29,42].…”

Section: Discussionmentioning

confidence: 91%