scholarly journals Effects of the mode of addition of acyl-CoA on the initial rate of formation of acylcarnitine in the presence of carnitine by intact rat liver mitochondria in vitro

1985 ◽  
Vol 229 (1) ◽  
pp. 273-275 ◽  
Author(s):  
V A Zammit
Keyword(s):  
Rat Liver ◽  
Initial Rate ◽  
Liver Mitochondria ◽  
Final Concentration ◽  
Rat Liver Mitochondria ◽  
Molar Ratios ◽  
Time Courses ◽  
Concentrated Solution ◽  
Cpt I

Time courses for the formation of palmitoylcarnitine from palmitoyl-CoA and carnitine, catalysed by the overt activity of carnitine palmitoyltransferase (CPT I) in rat liver mitochondria, were obtained. Significant initial non-linearity was observed only when reactions were started by addition of a concentrated solution of palmitoyl-CoA (4mM, to give a final concentration of 100 microM) uncomplexed to albumin. Minimal effects were observed when the reactions were started by addition of palmitoyl-CoA-albumin mixtures, even though the final palmitoyl-CoA/albumin molar ratios in the assay medium were identical in the two sets of experiments.

scholarly journals Time-dependence of inhibition of carnitine palmitoyltransferase I by malonyl-CoA in mitochondria isolated from livers of fed or starved rats. Evidence for transition of the enzyme between states of low and high affinity for malonyl-CoA

1984 ◽  
Vol 218 (2) ◽  
pp. 379-386 ◽  
Author(s):  
V A Zammit
Keyword(s):  
Rat Liver ◽  
Initial Rate ◽  
Liver Mitochondria ◽  
Time Dependent ◽  
Rat Liver Mitochondria ◽  
High Affinity ◽  
Malonyl Coa ◽  
Rate Of Increase ◽  
Cpt I ◽  
Zero Time

The degree of inhibition of CPT I (carnitine palmitoyltransferase, EC 2.3.1.21) in isolated rat liver mitochondria by malonyl-CoA was studied by measuring the activity of the enzyme over a short period (15s) after exposure of the mitochondria to malonyl-CoA for different lengths of time. Inhibition of CPT I by malonyl-CoA was markedly time-dependent, and the increase occurred at the same rate in the presence or absence of palmitoyl-CoA (80 microM), and in the presence of carnitine, such that the time-course of acylcarnitine formation deviated markedly from linearity when CPT I activity was measured in the presence of malonyl-CoA over several minutes. The initial rate of increase in degree of inhibition with time was independent of malonyl-CoA concentration. CPT I in mitochondria from 48 h-starved rats had a lower degree of inhibition by malonyl-CoA at zero time, but was equally capable of being sensitized to malonyl-CoA, as judged by an initial rate of increase of inhibition identical with that of the enzyme in mitochondria from fed rats. Double-reciprocal plots for the degree of inhibition produced by different malonyl-CoA concentrations at zero time for the enzyme in mitochondria from fed or starved animals indicated that the enzyme in the latter mitochondria was predominantly in a state with low affinity for malonyl-CoA (concentration required to give 50% inhibition, I0.5 congruent to 10 microM), whereas that in mitochondria from fed rats displayed two distinct sets of affinities: low (congruent to 10 microM) and high (less than 0.3 microM). Plots for mitochondria after incubation for 0.5 or 1 min with malonyl-CoA indicated that the increased sensitivity observed with time was due to a gradual increase in the high-affinity state in both types of mitochondria. These results suggest that the sensitivity of CPT I in rat liver mitochondria in vitro had two components: (i) an instantaneous sensitivity inherent to the enzyme which depends on the nutritional state of the animal from which the mitochondria are isolated, and (ii) a slow, malonyl-CoA-induced, time-dependent increase in sensitivity. It is suggested that the rate of malonyl-CoA-induced sensitization of the enzyme to malonyl-CoA inhibition is limited by a slow first-order process, which occurs after the primary event of interaction of malonyl-CoA with the mitochondria.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Changes in the ability of malonyl-CoA to inhibit carnitine palmitoyltransferase I activity and to bind to rat liver mitochondria during incubation in vitro. Differences in binding at 0°C and 37°C with a fixed concentration of malonyl-CoA

1984 ◽  
Vol 222 (2) ◽  
pp. 335-342 ◽  
Author(s):  
V A Zammit ◽  
C G Corstorphine ◽  
S R Gray
Keyword(s):  
Liver Mitochondria ◽  
Carnitine Palmitoyltransferase ◽  
Time Dependent ◽  
Rat Liver Mitochondria ◽  
Temperature Dependent ◽  
Fixed Concentration ◽  
Malonyl Coa ◽  
Time Courses ◽  
Cpt I

Time courses for inhibition of carnitine palmitoyltransferase (CPT) I activity in, and [14C]malonyl-CoA binding to, liver mitochondria from fed or 48 h-starved rats were obtained at 37 degrees C by using identical incubation conditions and a fixed concentration of malonyl-CoA (3.5 microM), which represents the middle of the physiological range observed previously [Zammit (1981) Biochem. J. 198, 75-83] Incubation of mitochondria in the absence of malonyl-CoA resulted in a time-dependent decrease in the ability of the metabolite instantaneously to inhibit CPT I and to bind to the mitochondria. Both degree of inhibition and binding were restored in parallel over a period of 6-8 min on subsequent addition of malonyl-CoA to the incubation medium. However, the increased inhibition of CPT I activity on addition of mitochondria directly to malonyl-CoA-containing medium was not accompanied by an increase in the amount of [14C]malonyl-CoA bound to mitochondria at 37 degrees C. Time courses for binding of [14C]malonyl-CoA performed at 0 degree C were different from those obtained at 37 degrees C. There was little loss of ability of [14C]malonyl-CoA to bind to mitochondria on incubation in the absence of the metabolite, but there was a time-dependent increase in binding on addition of mitochondria to malonyl-CoA-containing medium. It is suggested that these temperature-dependent differences between the time courses obtained may be due to the occurrence of different changes at 37 degrees C and at 0 degree C in the relative contributions of different components (with different affinities) to the binding observed at 3.5 microM-malonyl-CoA. Evidence for multi-component binding was obtained in the form of strongly curvilinear Scatchard plots for instantaneous (5s) binding of malonyl-CoA to mitochondria. Such multi-component binding would be expected from previous results on the different affinities of CPT I for malonyl-CoA with respect to inhibition [Zammit (1984) Biochem. J. 218, 379-386]. Mitochondria obtained from starved rats showed qualitatively the same time courses as those described above, with notable quantitative differences with respect both to the absolute extents of CPT I inhibition and [14C]malonyl-CoA binding achieved as well as to the time taken to attain them.

scholarly journals Protein-mediated uptake of vitamin B12 by isolated mitochondria

Blood ◽  
1976 ◽  
Vol 47 (6) ◽  
pp. 923-930 ◽  
Author(s):  
RA Gams ◽  
EM Ryel ◽  
F Ostroy
Keyword(s):  
Vitamin B12 ◽  
Rat Liver ◽  
Mitochondrial Respiration ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Isolated Rat Liver ◽  
Isolated Mitochondria ◽  
Isolated Rat

Abstract Protein-mediated B12 uptake by isolated rat liver mitochondria has been shown to be enhanced by plasma transcobalamin (TC-II) but not by salivary R binder in vitro. The process is enhanced by calcium and depends on active mitochondrial respiration. Following uptake, cyanocobalamin is converted to adenosyl and methylcobalamins and released from the mitochondria. TC-II appears to be required for both cellular and mitochondrial uptake of vitamin B12.

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