Peroxisomes preparation

Figure 1. Anti-DNP antibody reproducibly detects only one DNP-ctomoxiryl-CoA-label led protein (88kDa) in mitochondria, microsomes and peroxisomes prepared from rat livers previously perfused with DNP-etomoxir.

Density gradients are widely used in separating and purifying biological samples. In addition to this preparative application, measurements of s can be made. Gradient techniques have been used to isolate and purify the sub-cellular components, microsomes, ribosomes, lysosomes, mitochondria, peroxisomes, chloroplasts, and others. After isolation, they have been biochemically characterized as to their protein, lipid, nucleic acid, and enzyme contents. 

Phorbol esters were first detected in oil prepared from seeds of Croton tiglium, and are the most widely studied skin tumor promoters however, many other chemical compounds have been shown to possess skin tumor-promoting properties, for example, phenobarbitol, DDT, and the peroxisomal prolifera-tors. Within a few hours after application of a single effective dose of phorbol 12-myristate 13-acetate (also known as TPA and 12-O-tetradecanoyTphor-boTl3-acetate, CAS 16561-29-8) to mouse skin, localized edema and erythema characteristic of inflammation and irritation are evident, and within 24 h there is leukocytic infiltration of the dermis. Within 1 or 2 days after a single promoter treatment, stimulation of mitotic activity in the basal cell layer of the epidermis is evident and continues for several days. This results in an increased number of... 

POase activity in tissue or cell preparations is usually eliminated prior to the test. In some cases, endogenous POase is confined to peroxisomes and does not interfere with the test. Fixation in methanol is often accompanied by a decrease in peroxidatic activity. The method of Straus (1971) using 1% sodium nitroferricyanide and 1% acetic acid in methanol, is effective to abolish endogenous POase, but also weakens the immunohistochemical reaction. Streefkerk (1972)... 

Highly purified mitochondria and peroxisomes were prepared as described previously. The liver tissue was homogenized in a solution containing 0.25 M sucrose, 1 mM EDTA, 0.1 % ethanol, 5 mM HEPES-KOH (pH 7.4), and 0.2 mM phenylmethylsulfonyl fluoride. The light mitochondrial fraction obtained by differential centrifugation of the homogenate was layered on top of a 12-mL Nycodenz linear gradient (density, 1.15 to 1.25 g/mL) with a 1-mL cushion of... 

Because each organelle has unique morphological features, the purity of organelle preparations can be assessed by examination In an electron microscope. Alternatively, organelle-specific marker molecules can be quantified. For example, the protein cytochrome c Is present only In mitochondria so the presence of this protein in a fraction of lysosomes would Indicate Its contamination by mitochondria. Similarly, catalase Is present only In peroxisomes acid phosphatase, only In lysosomes and ribosomes, only In the rough endoplasmic reticulum or the cyd osol. 

The earliest electrophoretic techniques concentrated mainly on the preparation of mitochondria " and lysosomes, but further research has made possible the purification of secretory vesicles clathrin-coated vesicles " endoplasmic reticulum early, middle-, and late-stage endosomes peroxisomes " microsomes " and phagosomes." These preparatory techniques have functioned as the proof-of-principle for analytical separations of intact organelles using CE, by demonstrating that isolated organelles are amenable to eleetrophoretic separation techniques. 

Kinetic data are available for many aminotransferases, though in most cases the enzyme preparations have consisted either of crude extracts or incompletely purified proteins. Examples of such data are shown in Table 1. The An, value for the keto acid substrate is usually lower than the for the amino acid substrate, but there are exceptions to this generalization. Observed An, values may depend on the conditions of assay, and especially on the ionic composition of the buffer employed. For animal enzymes the anionic components of the buffer are said to be of particular importance in affecting kinetic parameters (cf. Braunstein, 1973). Plant enzymes have not been very systematically investigated in this respect, but phosphate inhibition has been observed for a peroxisomal enzyme transaminating with glyoxylate (Rehfeld and Tolbert, 1972). 

Therefore, in the present study we have measured CPT, activity in mitochondrial, microsomal, peroxisomal and high-speed supernatant fractions prepared from rat liver on a quantitativle basis. We have also quantified the relative expression of proteins that bind dinitrophenyl (DNP)-etomoxiryl-CoA (an oxirane ring-containing inhibitor of hepatic CPT I") in each of these fractions in the intact liver under conditions in which all three activities were optimally inhibited. Our results indicate that the overt CPT activity of microsomes and peroxisomes is associated with a protein that is very similar, if not identical, to mitochondrial CPT I. In microsomes too, the CPT, activity is associated with a protein of Mr 88,(X)0, but in this membrane fraction, there is evidence that the N-terminal domain has different properties from those of mitochondrial and peroxisomal CPT I. 

Subcellular fractions were prepared by standard differential and density gradient centrifugation protocols. Purified fractions of mitochondria and peroxisomes were prepared by Percoll and Nycodenz centrifugation, respectively. The following marker... 

Purified preparations of COT from beef Uver peroxisomes and CPT-Il from the iimer membrane of beef liver mitochondria, the two enzymes that can be isolated in stable form, were used for the kinetic studies. Based on the kinetic patterns obtained when both substrates were varied and on product inhibition studies, we concluded that the mecha-... 

We also checked whether CD 16 2 was formed in humans in vivo. In a pilot trial where subjects with type 2 diabetes mellitus were supplemented with 6 g/day of a CLA mixture, we detected CD 16 2 from both isomers, and again CD 16 2 from rl0,fl2 was more abundant reflecting the data obtained in vitro with skin fibroblasts (2) and in vivo in rats (manuscript in preparation). It appears therefore that both isomers are partially P-oxidized in peroxisomes in both experimental animals and humans, in vivo and in vitro. More studies are needed to find out whether the metabolism of CLA is directly linked to its activity as a ligand of PPAR a. It has actually been found that CLA is able to upregulate acyl CoA oxidase, the key enzyme of peroxisomal P oxidation (8). If this link is definitively proven, CD 16 2 could be used as a marker of PPAR a activation. 

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