The isolation of mitochondria from dipteran flight muscle

Procedures for the isolation of mitochondria from dipteran flight muscle have been investigated in an attempt to determine the extent and to identify the causes of deterioration associated with isolation. In the light of the results obtained isolation procedures have been improved by minimising mechanical damage, avoiding the development of anoxic conditions, and by the use of an isolation medium of a more physiological nature, containing the potassium salt of an organic anion as the principal osmoeffector, phosphate as the principal buffer, and low concentrations of free Mg²⁺. The oxidative capacity of mitochondria isolated by the improved method is adequate to support the in vivo requirements of the flight system.     

Quantitative proteomic comparison of rat mitochondria from muscle, heart, and liver

Mitochondria, through oxidative phosphorylation, are the primary source of energy production in all tissues under aerobic conditions. Although critical to life, energy production is not the only function of mitochondria, and the composition of this organelle is tailored to meet the specific needs of each cell type. As an organelle, the mitochondrion has been a popular subject for proteomic analysis, but quantitative proteomic methods have yet to be applied to tease apart subtle differences among mitochondria from different tissues or muscle types. Here we used mass spectrometry-based proteomics to analyze mitochondrial proteins extracted from rat skeletal muscle, heart, and liver tissues. Based on 689 proteins identified with high confidence, mitochondria from the different tissues are qualitatively quite similar. However, striking differences emerged from the quantitative comparison of protein abundance between the tissues. Furthermore we applied similar methods to analyze mitochondrial matrix and intermembrane space proteins extracted from the same mitochondrial source, providing evidence for the submitochondrial localization of a number of proteins in skeletal muscle and liver. Several proteins not previously thought to reside in mitochondria were identified, and their presence in this organelle was confirmed by protein correlation profiling. Hierarchical clustering of microarray expression data provided further evidence that some of the novel mitochondrial candidates identified in the proteomic survey might be associated with mitochondria. These data reveal several important distinctions between mitochondrial and submitochondrial proteomes from skeletal muscle, heart, and liver tissue sources. Indeed approximately one-third of the proteins identified in the soluble fractions are associated predominantly to one of the three tissues, indicating a tissue-dependent regulation of mitochondrial proteins. Furthermore a small percentage of the mitochondrial proteome is unique to each tissue.     

Nuclear proteoglycans from mouse liver cells: isolation and identification]

Proteoglycans (PG) have been isolated from mouse liver nuclei and identified. Nuclear PG are represented by various classes: i) PG containing dermatan sulfate (DS) chains; ii) PG containing heparan sulfate (HS) chains and, apparently, iii) mixed PG whose protein core contains both DS and HS chains.     

Multiple species of methionyl-tRNA from mouse liver mitochondria

Methionyl tRNA acylated within mitochondria isolated from mouse liver, has been resolved into four species by RPC-5 chromatography. All four elute prior to the three cytoplasmic methionyl tRNA species. Of the four species, two are formylated. These results suggest that iso-accepting species of met-tRNA^met exist in mouse liver mitochondria.     

Surface glycosyltransferases on cultured mouse fibroblasts

Previous work has suggested the presence of galactosyltransferases on the outer surface of the plasma membrane of a malignant and a nonmalignant cell line. This paper summarizes data indicating that three other classes of glycosyltransfeases are similarly located on cell surfaces. In addition to the original two cell lines examined, BALB/c 3T3 and BALB/c 3T12, two other lines of BALB/c origin have been investigated. These are the SV40–transformed 3T3 line and one of the revertants of the virally infected cells that is no longer malignant but retains a viral genome.     

Trypanosoma cruzi: selective isolation of pure trypomastigotes from cultured muscle cells

Trypomastigote or trypomastigote-amastigote populations of Trypanosoma cruzi (Y strain, MERC 2C) entirely free of epimastigotes were obtained from infected muscle cell cultures, and separated from host-cell debris by passage through a DEAE-cellulose column. Approximately 75% of the parasites were recovered and mouse infectivity titrations with postcolumn trypomastigotes showed only a slight reduction in infectivity compared to starting material. Light and electron microscopic examination of material showed a high degree of purity had been achieved by the column procedure. No host-cell debris could be identified in the eluate and parasites were morphologically intact. Enumeration of trypomastigotes and amastigotes in mixed populations before and after column purification showed that there was no preferential loss of either stage and both had the same residence time. This procedure may be used to obtain clean, minimally altered, parasite material free of the invertebrate epimastigote stage and host-cell debris for studies which are sensitive to these contaminants.     

Organelle dynamics: ER embraces mitochondria for fission

The endoplasmic reticulum and mitochondria are engaged in an intimate relationship: they establish extensive contacts, exchange lipids and calcium, and coordinate their activities in cell life and death. Recent research has revealed a new role for the endoplasmic reticulum in promoting mitochondrial division.     

Human quadricepts muscle mitochondria: A functional characterization

Human quadriceps mitochondria were isolated from ca. 80 mg tissue in ca. 45% yield. The preparation is described with respect to content of mitochondrial markers and nine different respiratory activities. The specific state 3 activities were high in comparison with literature data, indicating high integrity and purity of the preparation. Examples of state 3 rates, in µmol O min^-1 g protein^-1 (25°C): pyruvate + malate, 400; succinate, 514; malate + glutamate, 444. The notion of high integrity was also supported by the reproducibility of the preparation and the magnitude of the respiratory control ratios and the P/O ratios. The mitochondria most likely had lost ca. 30% of their cytochrome c upon isolation, but it was substantiated that this loss had not influenced the state 3 rates. Functional assays of single reactions or groups of reactions could be based on respiration experiments. The respiratory chain activity, for instance, was measured as respiration of NADH in freeze-permeabilized mitochondria (1263 mol O min^-1 g protein^-1). Comparison of uncoupled rates of respiration and state 3 rates indicated that the ATP synthesis exerted major flux control over respiration of succinate + glutamate, malate + glutamate and pyruvate + malate. These reactions, showing very similar rates of ATP synthesis, could be used as a functional assay of ATP synthesis (1200 mol ATP min^-1 g protein^-1). Respiration of succinate, palmitoyl-carnitine + malate, or glutamate could not support the maximal rate of ATP synthesis and the upstream reactions probably exerted major flux control in these cases. The specific activities appeared very constant in this group of young men, only the respiratory activity with glutamate might show biological variation.     

L-iduronidase in cultured human fibroblasts and liver

Extracts of normal human livers and skin fibroblasts cultured from normal individuals and patients with the Hurler syndrome released L-iduronic acid when incubated with desulfated dermatan sulfate. Enzyme extracts of normal fibroblasts liberated larger amounts of L-induronic acid, as judged by paper chromatography, than did homogenates from Hurler fibroblasts. Preliminary studies with desulfated heparan sulfate using the same enzyme systems have also shown material with the R_f of iduronolactone on paper chromatography.     

Rapid isolation of plasma membranes in high yield from cultured fibroblasts.

1. A method was developed which allows the rapid preparation of pure plasma membranes in high yield from cultured fibroblasts. 2. Cells are lysed in hypo-osmotic borate/EDTA and, after differential centrifugation, the membranes collected by centrifugation on a sucrose barrier. 3. Electron microscopy of the isolated material shows large membrane vesicles essentially free from contaminating organelles. 4. There is no detectable activity of the endoplasmic-reticulum enzyme marker, NADH2--lipoamide oxidoreductase (EC 1.6.4.3), and that of succinate dehydrogenase (EC 1.3.99.1), a marker for mitochondria, is substantially decreased. Chemical compositions are in good agreement with previous observations. 5. This study confirms the usefulness of applied isotopic markers for isolating plasma membranes.     

Furosemide-sensitive potassium efflux in cultured mouse fibroblasts

Transfer of LM(TK-) cells from normal growth medium to medium lacking K+ leads to a rapid loss of intracellular K+, which is 50-70% inhibited by furosemide or bumetanide. The diuretic-sensitive component of K+ efflux requires both Na+ and Cl-, and is presumably mediated by a K+, Na+, Cl- cotransport system of the kind described in avian erythrocytes and Ehrlich ascites cells. It can be calculated that such a system should be near equilibrium under normal growth conditions but should mediate net efflux (as observed) when the driving force is altered by reducing extracellular K+. The diuretic-sensitive component of net K+ efflux is also sensitive to amiloride. This effect is probably indirect, however, with amiloride acting to block the Na+ influx that supplies Na+ to the cotransport system. At the low extracellular K+ concentrations employed in these studies, the diuretic-sensitive system is a physiologically important pathway of K+ loss. The rate of growth in low-K+ medium can be increased (or the rate of cell lysis decreased) by adding diuretic or by reducing external Na+ or Cl-.     

The enzymatic isolation of hepatocytes from the mouse liver

A modified version of the two-step enzymatic method for isolation of hepatocytes from the liver of adult mice is described. The method yields 6.10(7)-7.10(7) cells with viability of 70-80%. It is simple and economical, requiring no great amounts of collagenase.     

Purification and characterization of dicyclohexylcarbodiimide binding protein from mouse liver mitochondria

The DCCD-binding protein from mouse liver Mt has been purified by chloroform: methanol method. The DCCD binding is drastically reduced when lipids are extracted from the proteolipid. The proteolipid as well as the lipid extracted protein migrate as single component with 7.8 K daltons molecular weight. The protein fraction yields a single band (pI 5.8) on isoelectric focusing gels. The DCCD binding protein is a product of Mt translation and contains Val as the N-terminal residue.     

Quantitative isolation of liver mitochondria by zonal centrifugation

A method was developed using zonal centrifugation to recover liver mitochondria quantiatively and free of other cellular components from a sample of whole homogenate. The fractions containing mitochondria were identified by the distribution of cytochrome oxidase and these fractions contained over 90% of the total cytochrome oxidase recovered. The mitochondrial fractions were found to be only slightly contaminated by 5′-nucleotidase (plasma membranes), acid phosphatase (lysosomes), glucose-6-phosphatase (microsomes), and catalase (peroxisomes). There was no detectable contamination by nuclear DNA (nuclei). This method was used to quantitate total liver mitochondrial protein. The development of this procedure provides a means for following total changes in mitochondrial components during mitochondrial biogenesis.     

Growth stimulating activity from lysed cultured arterial smooth muscle cells and skin fibroblasts

Rabbit and bovine arterial smooth muscle cells (SMC) and human skin fibroblasts were lysed by freezing and thawing in the presence of protease inhibitors (PI). The supernatant was assayed for growth stimulating activity (GSA), and it stimulated the growth of SMC and fibroblasts, but not human and bovine endothelial cells. GSA was sensitive to heat and trypsin treatment, stimulated DNA synthesis after a lag time of 12-15 hours, and exhibited marked size and charge heterogeneity when subjected to gel chromatographies. GSA differed from many other known growth factors, mainly platelet derived growth factor (PDGF), through the behavior on ion exchange chromatography, the heat sensitivity and the lack of decline in activity in the presence of anti PDGF antibodies. The data suggests that several growth stimulating proteins can be obtained through the lysis of SMC or fibroblasts with possible implications for atherosclerosis and wound healing.     

Improved method for isolation of mitochondria from chick breast muscle using Nagarse.

An improved procedure to isolate mitochondria from chick pectoralis muscle is described. The muscle is digested with the proteinase Nagarse and homogenized using a Teflon pestle. Mitochondria are isolated by differential centrifugation. These organelles are able to utilize a variety of substrates with a higher degree of respiratory control than mitochondria isolated without Nagarse. Contrary to previous reports, mitochondria isolated by this method from chick pectoralis muscle (αW fibers) are able to oxidize the substrate β-hydroxybutyrate with good respiratory control.