CPT activity

Decreased production or mutation of TOPO-1 can cause resistance to the cytotoxic effects of topotecan and other CPTs active efflux of TPT by P-glycoprotein-mediated transport might also contribute to resistance. 

I. Effects of Ceramide Treatment on Lung Microsomal CPT Activity.258... 

FIGURE 12.7 Time-dependent effects of 0.5 mg/kg body weight. CEES treatment on the mitochondrial and microsomal CPT activity. N=3. 

The time-dependent effects of CEES treatment showed a biphasic effect on CPT activity in both mitochondria and microsomes. The time-dependent studies indicated that a single infusion of CEES (0.5 mg/kg body weight) caused an increase in the activity for a short time after CEES exposure (up to 4 h), followed by a decrease (6 h onward) (Figure 12.7). The dose-dependent smdies indicated that CEES treatment caused an initial increase in the CPT activity at low doses (0-2 mg/kg body weight), followed by a decrease at higher doses (4 and 6 mg/kg body weight) at incubation times of 1 and 4 h. This decrease was more acute in microsomes than in the mitochondria (Figure 12.8). 

We have previously demonstrated that in addition to its predominant localization in the microsomes, CPT also exists in the mitochondria (Stith and Das, 1982 Sikpi and Das, 1987). Thus, it is possible that during the early stage of lung injury as observed in this smdy, cells try to repair the membrane damage by stimulating PC synthesis. Therefore, with increased CPT activity... 

When the lung microsomal fraction from control animals was incubated with C2 ceramide at different concentrations (50, 100, and 200 xM) and time periods (0, 0.5, 1, and 6 h) before the assay for CPT activity, CPT activity decreased significantly in a time- and dose-dependent manner (Figure 12.9). 

FIGURE 12.9 Effect of C2 ceramide treatment on lung microsomal CPT activity. N—3. 

However, the effect was more pronounced when the microsomal fraction was preincubated with the ceramide before assay of the CPT activity. The degree of inhibition was increased with the increase in incubation time (0.5, 1, and 6 h). The highest inhibition (50%) was achieved after 6 h of incubation. However, only 20% inhibition was observed when ceramide was directly added into the assay mixture. 

It has been shown that a 30% inhibition could be obtained in CPT activity when ceramides were directly added to the assay mixture at 50 pM concentrations (Bladergroen et al., 1999). Since this inhibition of 30% was less than the 64% obtained when cells were incubated directly, it would indicate competitive inhibition was not the only mechanism. In the present work, we found similar results with lung microsomal fraction, that is, with an increase in the incubation time with ceramides, the inhibition of the enzyme activity increases. Therefore, we support the observations by Bladergroen et al. (1999) that ceramide inhibition of CPT activity may be only partially through direct competitive inhibition with DAG ceramide may act through interaction with other CPT enzyme inhibitors present in the microsomal fraction. 

The effect of CLA on P-oxidation of fatty acid was assessed by measuring carnitine palmitoyltransferase (CPT) activity, a... 

There are several ways in which CLA may cause an increase in CPT activity. First, CLA can modify the fatty acid composition of the cell membrane, thus altering the receptor-enzyme coupling that may enhance fatty acid oxidation by stimulating CPT activity. Second, CLA may activate peroxisome proliferator-activated receptors (PPAR), which in turn may enhance fatty acid oxidation (21-23). 

Y (37), we speculate that the relative potency of activation of a vs. Y could be responsible for the different actions of these isomers. These results demonstrate that the hypolipidemic effect of CLA can be attributed to the effect of the 10f,12c-CLA isomer. In addition, it is possible that the flO,cl2-CLA isomer enhanced CPT activity and reduced FAS activity through its action at the transcriptional levels of these lipogenic genes. 

CPT I of the liver mitochondrial outer membrane (L-CPT I). However, whereas reaction with anti-L and anti-C antipeptide antibodies were proportional to the respective overt CPT activities and DNP-etomoxir labelling in all three membrane fractions, reaction with anti-N peptide antibody was much stronger for microsomal CPT. We conclude that in all three membrane systems overt CPT activity is associated with the same or highly similar molecular species to mitochondrial outer membrane CPT 1, but that the protein expressed in microsomes has a modified N-terminal domain, which gives the microsomal enzyme its higher malonyl-CoA sensitivity and may target the protein to its microsomal location. 

The overt CPT activities (CPT,) of mitochondria, microsomes and peroxisomes are inhibited by malonyl-CoA, in contrast to the latent CPT isoforms. Of all the hepatic overt CPT activities, only the protein responsible for the activity in the mitochondrial outer membrane (L-CPT I) is well-characterised." Results published in abstract form have indicated that a peroxisomal protein resembling L-CPT 1 may be responsible for the overt CPT activity of these organelles, but the authors suggested that it is immunologi-cally distinct from L-CPT I. A microsomal protein of Mr 47,000 has previously been postulated to represent the microsomal CPT, on the basis that, when microsomes are incubated with [ H]-etomoxir, CoA and ATP, a protein of this molecular size is labelled most prominently. Similar observations were made by who, however, also observed that under these in vitro conditions at least one other protein of Mr 87,000 was also prominently labelled. More recently, it has been confirmed that microsomes express a malonyl-CoA-sensitive overt CPT which has an apparent molecular size of 300 kDa in detergent extracts of the membranes. On the basis of its different kinetic behaviour, ease of solubilisation and stability of catalytic activity when solubilised, it was concluded that the microsomal enzyme is distinct from the mitochondrial outer membrane CPT 1. So, current opinion is that, apart from the common sensitivity of all the overt CPT enzymes to malonyl-CoA inhibition, the proteins responsible for this activity in the mitochondrial outer membrane, microsomes and peroxisomes are distinct molecular entities. 

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. 

The overt carnitine palmitoyltransferase activities in each fraction were measured with an optimal concentration of palmitoyl-CoA (above). A previous report suggested that palmitoyl-CoA is not a good substrate for overt CPT in microsomes. However, we have not been able to confirm this, as the palmitoyl-CoA requirement for the enzymes in all three membranous fractions was very similar to that described previously for mitochondrial outer membrane CPT Peroxisomes displayed the highest specific activity of overt CPT (see below) and their overall contribution, computed on a per gram liver basis was appreciable and of the same order of that in microsomes, which had a relatively low specifie aetivity, but which occurred at much higher protein densities within the cell. Mitochondria accoimted for about 65% of total CPT activity. These estimates are very similar to those reported by. It is evident, therefore, that the peroxisomal and microsomal forms of overt CPT constitute an important component of the overall CPT activity with access to the cytosolic pools of acyl-CoA and malonyl-CoA. 

A representative Western blot is shown in Figure 1. In all three membrane fractions only a protein that migrates with an apparent mass of 88kDa was reproducibly labelled by DNP-etomoxiryl-CoA when quantitative CPT inhibition had been achieved. A smaller protein was sometimes labelled in peroxisomes, but its extent of labelling was very variable and not proportional to CPT activity. While for mitochondria and peroxisomes the labelling of the 88kDa protein was as expected from the studies the result... 

Table I. Effects of Protease T reatment of Intact Mitochondria on C arnitine Palmitoyltransferase Activity and its Inhibition by Malonyl-CoA. Intact mitochondria were incubated willi proteases at the concentrations indicated and then assayed for outer CPT activity using 40pM palmitoyl-CoA and ().5mM carnitine. Results are means S.E.M. of 3 10 difl erent preparations. Where no S.E.M. is indicated, results arc means of 2 separate experiments with different preparations of mitochondria. Percentage inhibition by malonyl-CoA is indicated in parentheses. Abbreviation n.d., not determined. Data are reproduced with...

Table 2. Inhibition of Carnitine Palmitoyltransferase by Several Known Inhibitors in Control and Protease-Treated Mitochondria. Intact mitochondria were assayed for outer CPT activity in presence and absence of different inhibitors in control and protease-treated mitochondria. All inhibitors were present throughout a 5min preincubation with palmitoyl-CoA and a 5min assay that was initiated by adding carnitine. Results are means S.E.M. of. 3 different preparations, where no standard error is indicated, results are means of 2 separate experiments with different mitochondrial preparations. For specific activity of CPT see Table I. Data are reproduced with...

KIgure 2. EtVcct of Nagarse on CPT Activity (A) and Maloiiyl-CoA inhibition (B) as a Function of Time. 

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