Abstract
In the present paper a model system is described utilizing suspensions of peripheral blood leukocytes in which glycogen synthesis and degradation can be studied.
Leukocyte suspensions containing 72-94% granulocytes were prepared essentially free of platelets and erythrocytes and consisted almost entirely of neutrophile granulocytes. Initial glycogen content averaged 7.36 ± 2.05 mg/109 neutrophiles. In a glucose-free medium glycogenolysis took place with glycogen losses averaging 38% in 2 hr. When adequate glucose was added to the medium, glycogen was resynthesized to the original level.
Glycogen resynthesis was studied with varying glucose “loads” to determine (a) the glucose level which was adequate for cell maintenance without utilization of glycogen stores, and (b) the glucose level which provided maximal glycogen resynthesis. With cell densities of 20-50 × 106/ml the minimum glucose load which allowed maintenance of glycogen stores was 2 mg and 5.3 mg/109 neutrophiles for 30 and 60 min, respectively.
During resynthesis with glucose-14C, as much as 88.9% of the intracellular radioactivity could be found in glycogen. Leukocyte glycogen was made radioactive by a “pulse” of glucose-14C followed by a “chase” with nonradioactive glucose. Specific activity changes in glycogen isolated during the “chase” showed that glycogen was in constant turnover.
When glycogen was made radioactive by a “pulse” of glucose-14C and the cells placed in glucose-free medium, the specific activity of isolated glycogen fell rapidly. Thus, the most recently added glucose units of the molecule were also the first to be removed when conditions favoring synthesis were changed to conditions favoring degradation.
Even though glycogen is constantly turning over, the enzymatic “machinery” for its synthesis is relatively stable and not dependent on continuous protein or RNA synthesis, as shown by experiments with puromycin and actinomycin.
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