Assessing enrichment of proteins in the mitochondrial fraction in HEK cells v1

protocols.io ◽  
2021 ◽  
Author(s):  
OLIVIA not provided HARDING
Keyword(s):  
Mitochondrial Fraction ◽  
Hek Cells

Glucose metabolism in cancer cells: immunogold localization of hexokinase to the outer mitochondrial membrane

1995 ◽  
Vol 53 ◽  
pp. 1044-1045
Author(s):  
Krishan K. Arora ◽  
Glenn L. Decker ◽  
Peter L. Pedersen
Keyword(s):  
Tumor Cells ◽  
Mitochondrial Membrane ◽  
Present Report ◽  
Large Fraction ◽  
Mitochondrial Fraction ◽  
Immunogold Labeling ◽  
Outer Mitochondrial Membrane ◽  
Immunogold Localization ◽  
Hexokinase Activity ◽  
Normal Tissues

Hexokinase (ATP: D-hexose 6-phophotransferase EC 2.7.1.1) is the first enzyme of the glycolytic pathway which commits glucose to catabolism by catalyzing the phosphorylation of glucose with ATP. Previous studies have shown diat hexokinase activity is markedly elevated in rapidly growing tumor cells exhibiting high glucose catabolic rates. A large fraction (50-80%) of this enzyme activity is bound to the mitochondrial fraction (1,2) where it has preferred access to ATP (3). In contrast,the hexokinase activity of normal tissues is quite low, with one exception being brain which is a glucose-utilizing tissue (4). Biochemical evidence involving rigorous subfractionation studies have revealed striking differences between the subcellular distribution of hexokinase in normal and tumor cells [See review by Arora et al (4)].In the present report, we have utilized immunogold labeling techniques to evaluate die subcellular localization of hexokinase in highly glycolytic AS-30D hepatoma cells and in the tissue of its origin, i.e., rat liver.

scholarly journals Isolation and characterization of minicircular DNAs found in mitochondrial fraction of Vicia faba

FEBS Letters ◽  
1982 ◽  
Vol 142 (1) ◽  
pp. 115-117 ◽  
Author(s):  
V.I. Negruk ◽  
D.I. Cherny ◽  
I.D. Nikiforova ◽  
A.A. Aleksandrov ◽  
R.G. Butenko
Keyword(s):  
Vicia Faba ◽  
Mitochondrial Fraction ◽  
Isolation And Characterization

Intracellular distribution of enzymes

1953 ◽  
Vol 141 (904) ◽  
pp. 420-433 ◽  
Keyword(s):  
Mitochondrial Fraction ◽  
Integrated System ◽  
Choline Oxidase ◽  
Optical Methods ◽  
Mitochondrial Activity ◽  
Permeability Barrier ◽  
Glutamic Dehydrogenase ◽  
Tissue Homogenates ◽  
Distribution Studies ◽  
Apparent Distribution

The localization of enzymes in cells may be studied by the differential centrifugation of tissue homogenates. This method has been used to study the distribution of L-malic and L-glutamic dehydrogenases, choline oxidase, adenosinetriphosphatase, and also of the nucleic acids and nitrogen between the fractions of the homogenate. L-Glutamic dehydrogenase is entirely m itochondrial, and malicdehydrogenase is shared almost equally, between the mitochondria and the supernatant although the true mitochondrial activity is not apparent unless unmasked, e. g. by water disruption. In the absence of this precaution intact mitochondria show only a small proportion of the activity of the whole homogenate. Choline oxidase is almost entirely mitochondrial, and adenosine triphosphatase has a large representation in all the particulate fractions. In our experiments we have found that the ribonucleic acid content of the mitochondria is higher than previously reported. Distribution studies of enzymes are misleading unless it can be shown that the methods employed are valid for all the fractions studied, and our evidence shows that serious errors of in terpretation may arise unless more than one method of determination is used. In particular, the physical state of the mitochondria affects their apparent enzyme content, as shown by the; investigation of malic and glutamic dehydrogenases by manometric and optical methods. This anomaly is due to an ‘accessibility barrier’ or ‘permeability barrier’ present in intact mitochondria, which hinders the entry of coenzyme I. In an integrated system composed of several enzymes, the rate of the whole reaction may be limited by the rate of any one of the intermediate steps. Thus intact mitochondria fail to develop their maximum oxygen up take with several substrates unless a continuous supply of phosphate acceptor is ensured. Otherwise the rate of transfer of phosphate limits the whole reaction, reducing the apparent activity of the mitochondrial fraction relative to that of the whole homogenate, and hence the apparent distribution of enzyme. Similarly, the activity of the choline oxidase of the mitochondrial fraction is more sensitive to pH changes than that of the whole homogenate so that at pH 6·8, 80% of the homogenate activity may be recovered in the mitochondria, whereas at pH 7·8, the recovery is only 50%. At pH 7·8 full activity of the mitochondria, and a recovery of over 80%, may be achieved by the addition of coenzyme I.

HORMONAL EFFECTS ON LIVER AND KIDNEY CYTOPLASM

1956 ◽  
Vol 13 (3) ◽  
pp. 319-329 ◽  
Author(s):  
E. REID
Keyword(s):  
Sucrose Solution ◽  
Kidney Tissue ◽  
Mitochondrial Fraction ◽  
Supernatant Fraction ◽  
Phospholipid Content ◽  
Hormonal Status ◽  
Hormonal Effects ◽  
Liver And Kidney ◽  
Hypophysectomized Rats ◽  
Microsome Fraction

SUMMARY 1. Differential centrifugation, in 0·25 m sucrose solution, has been performed with rat liver and kidney tissue to ascertain whether the yield and composition of the cytoplasmic fractions (mitochondrial, microsome and supernatant fractions) depend on the hormonal status of the animal. 2. After hypophysectomy the ribonucleic acid (RNA) of the mitochondrial fraction from liver underwent a decrease (in terms of body weight) which was sufficient to account for the fall in the RNA of the liver as a whole. There was also a decrease in the yield of the mitochondrial fraction. Administration of pituitary growth hormone (GH) to hypophysectomized rats not only restored to normal the amount of RNA in the mitochondrial fraction and the yield of that fraction, but also led to an apparent shift of RNA from the microsome fraction to the supernatant fraction. A further change observed after hypophysectomy, whether or not GH had been given, was a rise in the yield of the microsome fraction. Hypophysectomized rats given thyrotrophin (TSH) did not show significant cytoplasmic changes as found with untreated hypophysectomized rats, but it was not possible to conclude that TSH had actually reversed the effects of hypophysectomy. 3. Castrated rats showed no abnormalities in the yields of the liver cytoplasmic fractions or in the concentration of RNA in the fractions. Alloxan-diabetic rats showed a rise in the yield of the supernatant fraction from liver. 4. Untreated adrenalectomized rats showed a rise in liver deoxyribonucleic acid, a fall in the yield of the liver mitochondrial fraction, but not in the amount of RNA in that fraction, and a rise in the amount of RNA in the supernatant fraction. Replacement therapy with various adrenocorticoids was attempted, with only partial success. 5. In contrast with the RNA content, the phospholipid content of the liver cytoplasmic fractions was not, in general, dependent on hormonal status. 6. Determinations of the yield and composition (RNA and phospholipid) of the cytoplasmic fractions from kidney disclosed certain hormonal effects, differing from those observed with liver; for example, the kidneys from hypophysectomized rats furnished microsome fractions in lowered yield but with an increased concentration of RNA.

The ADP/ATP carrier is the 32-kilodalton receptor for an NH2-terminally myristylated src peptide but not for pp60src polypeptide

1993 ◽  
Vol 13 (5) ◽  
pp. 3084-3092
Author(s):  
C T Sigal ◽  
M D Resh
Keyword(s):  
Binding Capacity ◽  
Membrane Binding ◽  
Mitochondrial Fraction ◽  
Fatty Acyl ◽  
Affinity Tag ◽  
Peptide Probe ◽  
The Cross ◽  
Docking Protein

Membrane binding of pp60src is initiated via its myristylated NH2 terminus. To identify a candidate pp60src docking protein or receptor in the membrane, a radiolabelled peptide corresponding to the pp60src NH2-terminal membrane binding domain was cross-linked to fibroblast membranes and found to specifically label a 32-kDa protein. This protein was purified by appending an affinity tag to the peptide probe so that the cross-linked complex could be isolated via affinity chromatography. Microsequencing indicated that the 32-kDa protein was the mitochondrial ADP/ATP carrier (AAC). This result was further confirmed by the ability of an antibody to the AAC to immunoprecipitate the cross-linked complex, by the ability of certain inhibitors of the AAC to block cross-linking, and by membrane fractionation to show that complex formation occurred essentially exclusively in the mitochondrial fraction. While the AAC bound the myristyl-src peptide in a specific manner both in vitro and in vivo, its localization to the inner membrane of the mitochondrion precludes its being a pp60src binding protein. An analysis of pp60v-src binding in vitro was consistent with this expectation. Thus, use of a myristyl-src peptide revealed an unexpected and previously unidentified binding capacity of the AAC, most likely related to the ability of long-chain fatty acyl coenzyme As to serve as AAC inhibitors. The amphipathic nature of the pp60src NH2 terminus suggests alternative strategies for uncovering pp60src membrane binding species.

Abstract 70: Protein Phosphatase 1 Contributes to Atrial Stunning in Atrial Fibrillation

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Srikanth Perike ◽  
Xander Wehrens ◽  
Dawood Darbar ◽  
Mark McCauley
Keyword(s):  
Atrial Fibrillation ◽  
Light Chain ◽  
Protein Phosphatase ◽  
Myosin Light Chain ◽  
Stroke Risk ◽  
Protein Phosphatase 1 ◽  
Regulatory Subunit ◽  
Future Studies ◽  
Hek Cells

Background: Atrial fibrillation (AF) is the most common cardiac arrhythmia, and increases a patient’s stroke risk five-fold. Reduced atrial contractility (stunning) is observed in AF and contributes to stroke risk; however, the mechanisms responsible for atrial stunning in AF are unknown. Recent data from our laboratory indicate that protein phosphatase 1 (PP1) dephosphorylation of myosin light chain 2a (MLC2a) may contribute to atrial stunning in AF. Objective: To determine how the PP1 regulatory subunit 12C (PPP1R12C) and catalytic (PPP1c) subunits modify atrial sarcomere phosphorylation in AF. Methods: We evaluated the protein expression, binding and phosphorylation among PPP1R12C, PPP1c, and MLC2a in transfected HL-1 cells, murine atrial tissue (Pitx2null +/– mice, with a genetic predisposition AF), and in HEK cells. An inhibitor of PPP1R12C phosphorylation, BDP5290, was used to enhance the PPP1R12C-PPP1C interaction. Results: In Pitx2 null +/– mice, PPP1R12C was increased by 2-fold ( P <0.01) and associated with a 40% reduction in S-19-MLC2a phosphorylation versus WT mice ( P <0.058). BDP5290 increased PPP1R12C-PPP1C binding by >3-fold in HL-1 cells ( P <0.01). BDP5290 reduced MLC2a phosphorylation by 40% through an enhanced interaction with PPP1R12C by >3-fold in HEK cells ( P <0.01). Conclusion: In Pitx2 null+/- mice, increased expression of PPP1R12C is associated with PP1 holoenzyme targeting to sarcomeric MLC2a, and is associated with reduced S19-MLC2a phosphorylation. Additionally, BDP5290 enhances the PPP1R12C-PPP1C interaction and models PP1 activity in AF. Future studies will examine the effects of both AF and BDP5290 upon atrial contractility in vitro.

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