Liver tissue was homogenised in 50mL. 0.1M citrate buffer, pH6.5; and then 40mL of the homogenate were subjected to differential centrifugation. The nuclear, mitochondrial and microsomal fractions were each resuspended in 20mL citrate buffer. The protein content of each fraction (and the original

Each fraction was assayed to measure glucose-6-phosphatase (G6Pase) activity as described below:

1. 1.5mL 2mM glucose-6-phosphate was added to 0.5mL sample of each fraction

2. Samples were mixed and incubated at 25 C for 10 min

3.Reactions were stopped by the addition of 8mL 5%TCA

4. All tubes were centrifuged for 5mins

5. For each fraction, zero time controls were prepared by adding TCA before adding glucose-6 4. 5.

6.A no enzyme control was also prepared

7. The amount of inorganic phosphate in 5mL supernatant was determined using a colorimetric method (Fiske and Subbarow) and Aes recorded for all samples and controls

8. The amount of inorganic phosphate in 5ml standards of known phosphate concentration was determined using the Fiske and Subbarow method and a standard curve constructed.

Table 1: Protein content and enzyme assay for experimental samples

Volume 0.4mM inorganic phosphate ml Volume	Volume (ml)	Protein concentration (mg/ml)	Aese for zero time control	Ase aftere 10 min incubnation
Homogenate	40	3.26	0.16	0.56
Nuclear	20	1.62	0.14	0.32
Nuclearmithochondrial	20	1.58	0.15	0.26
Microsomal	20	1.51	0.16	0.69
Final supermanent	40	0.82	0.18	0.24
No enzyme control	NA	NA	0.02	0.24

Table 2: Aese for standard solutions of phosphate using the Fiske and Subbarow method

Volume 0.4mM inorganic phosphate ml Volume water (mL)	Volume water (mL)	Aese
0	5	0(blank)
1	4	0.21
2	3	0.42
3	2	0.60
4	1	0.80
5	0	0.97