Problem The citric acid cycle

The key experiments that led to elucidation of the citric acid cycle (tricarboxylic acid cycle) were described by Krebs and Johnson in 1937. They measured the consumption of oxygen by a preparation of minced pigeon breast muscle when incubated with various additions. The results in Table 5.8 show the volume of oxygen consumed during the incubation by 460 mg wet weight of tissue (the complete oxidation of 1 mmol of citrate to CO2 and H2O consumes 100 ^L of O2).

Previous studies had shown a similar effect of adding fumarate, oxaloacetate or succinate. What conclusions can you draw from these results?

Isolated hepatocytes were incubated for 40 minutes in a phosphate—bicarbonate— CO2 buffer system, with [U-14C]palmitate (i.e. palmitate labelled with 14C in all 16 carbon atoms), at a specific radioactivity of 103 dpm (radioactive disintegrations per minute) per micromole, with and without the addition of 60 mmol/L malonate and/ or oxaloacetate.

One set of incubations was set up to act as a control, in which the perchloric acid was added at the beginning of the experiment.

After collection of the CO2 for measurement of the radioactivity incorporated, the denatured incubation mixture was extracted with a chloroform—methanol mixture to separate unmetabolized palmitate (in the organic phase) from water-soluble metabolites

Table 5.8 Oxygen consumption by pigeon breast muscle with and without added citrate

Incubation time (min)

Oxygen consumed (^L)

No citrate

Plus citrate

Difference

30

645

682

+37

60

1055

1520

+465

90

1132

1938

+806

Source: From data reported by Krebs HA and Johnson WA, Enzymologia 4: 148—156, 1937.

Source: From data reported by Krebs HA and Johnson WA, Enzymologia 4: 148—156, 1937.

Table 5.9 Recovery of radioactivity from {U-14C}palmitate in isolated hepatocytes Radioactivity (103 dpm/min/g cells) found in

CO2 Organic phase Aqueous phase

Table 5.9 Recovery of radioactivity from {U-14C}palmitate in isolated hepatocytes Radioactivity (103 dpm/min/g cells) found in

CO2 Organic phase Aqueous phase

Control

0

10.0

0

(unincubated)

No addition

2.3

7.5

0.2

Plus malonate

0

9.8

0.2

Plus oxaloacetate

4.8

5.0

0.2

Plus malonate +

0

5.0

5.0

oxaloacetate

Table 5.10 Metabolism of palmitate, lactate and glutamate in

isolated hepatocytes

Change (^mol/min/g cells)

Substrate

Lactate

Palmitate

Glutamate Glucose

Lactate

-4.11

+0.21

0 +0.60

Palmitate

0

-0.35

00

Palmitate + lactate

-2.4

-0.59

0 +1.20

Glutamate

0

0

-3.42 +0.81

Table 5.11 Recovery of radioactivity from {14C-2'}lactate and {U-14C}palmitate in isolated hepatocytes

Radioactivity (103

dpm/min/g cells) found in

Substrate

CO2

Glucose

[l4C-2]Lactate

3.71

1.20

[U-l4C]Palmitate

3.71

0

[U-l4C]Palmitate + :

glutamate

7.75

0.51

(which remained in the aqueous layer). The radioactivity in both phases was determined, and the results are shown in Table 5.9.

Can you account for these observations?

What is the water-soluble compound that accumulates in the presence of malonate?

Isolated hepatocytes were incubated with lactate, glutamate and/or palmitate as substrates. At the end of the experiment lactate, palmitate, glutamate, glucose (after acid hydrolysis of glycogen) and total ketone bodies (acetoacetate plus P-hydroxybutyrate) were determined. The results have been expressed as change during the incubation, compared with similar incubations which were stopped with perchloric acid at the beginning of the experiment, and are shown in Table 5.10.

What conclusions can you draw from these results?

Isolated hepatocytes were incubated with lactate labelled with 14C in carbon-2 ([14C-2]lactate) or palmitate labelled with 14C in all carbon atoms ([U-14C]palmitate); in each case the specific radioactivity of the labelled substrate in the incubation medium was 103 dpm/^mol. In a further series of incubations with [U-14C]palmitate, nonradioactive glutamate was also added. The results are shown in Table 5.11.

What conclusions can you draw from these results?

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