Fróðskaparrit - 01.01.1975, Síða 3

Amylo-l,6-glucosidase deficiency
11
into the tube which was then closed at the other end with a rubber
stopper, inverted 10 times and placed vertically in a rack for 75 min
at room temperature1. After sedimentation of the erythrocytes the tube was
pierced just above the layer of red cells with a cannula and the plasma
containing the white cells and the platelets was tapped. The plasma was
centrifuged at a speed of 150 g for 4 min and decanted. The sedimented
leucocytes were washed once by resuspension in 0.9 °/o NaCl and centri-
fuged as before. They were then resuspended in 2 ml 0.1 M NaF, kept
for 15 min in an icebath, centrifuged (2000 g) for 5 min, decanted and
stored at —20° C. During transport the cells were kept on dry ice. For
cnzyme assay the cells were thawed, resuspended in 700 /A 0.1 M NaF,
sonicated and centrifuged (2000 g) for 5 min at 4° C. The supernatant was
used as enzyme source.
The erythrocytes were washed 5 times in cold 0.9 °/o NaCl using centri-
fugation at 1500 g. After the last centrifugation one volume of destilled
water was added to the sediment, the mixture was inverted a couple of
times and stored at —20° C. For enzyme assay the cells were thawed, shaken
vigorously by hand and centrifuged at a speed of 2000 g for 15 min. at
4° C. The supernatant was used as enzyme source.
Analytical procedures. Amylo-l,6-glucosidase activity was measured
by its ability to incorporate glycosyl residues into glycogen by a modi-
fication of the method described by Nelson and Larner8. The reaction
mixture contained in a total volume of 90 «1: 9 mg rabbit liver glycogen
(Sigma type III), 135 nanomoles glucose-U-C14 (specific activity 25,000
cpm/nanomole), 3 nanomoles maleate buffer pH 6.5 and 30 /A enzyme
solution. After incubation for 1 h at 30° C, 75 [A of the reaction mixture
was withdrawn, spotted on filter paper and immersed in icecold 66 °/o
ethanol as described by Nelson and Larner. Each assay was accompanied
by a zero time incubation. This method was applied to both leucocytes
and erythrocytes. The filter paper bits from the leucocyte assay could,
after washing and drying, be counted directly in 10 ml 0.5 °/o PPO in
toluene. In the assay of enzyme frorn erythrocytes, the haemoglobin pre-
cipitated on the paper bits gave such heavy colour-quenching that reprodu-
cible results could not be obtained. The paper bits were therefore placed
in a disposable plastic counting vial, bleached with 300 /íl 30 °/o hydrogen
peroxide and treated with 3 ml of a 1:1 mixture of Soluene (Packard)
and isopropanol for 30 min at 60° C to extract the radioactive glycogen.
The radioactivity was then counted in 15 ml of a 1:9 mixture of 0.1 N
HCl and Instagel (Packard).
Protein was determined by a Lowry method and leucocyte glycogen
was measured with the method of Kristmann6. The ratio between tha
extinctions at 460 nm an 390 nm was also determined in this method.