Release of vitamin B12—binding protein by human leukocytes in vitro

Abstract

Human granulocytes (G) contain a vitamin B₁₂-binding protein (B₁₂BP). There is evidence that chronic myelogenous leukemia leukocytes (CMLL) may synthesize B₁₂BP. Our prior studies suggested that intact, living intravascular G synthesize and release such protein into extracellular compartments in vivo. In the present study, CMLL were incubated in Trisbuffered Hank's basal salt solution (pH 7.2) containing 0.1% human serum albumin to study release of B₁₂BP into the medium. B₁₂BP was released continuously and in increasing amounts over a 5 hr period at 37°C; this release was inhibited almost completely when the cells were incubated at 4°C and by about half as much in the presence of N-ethylmaleimide (1 mmole/liter). Cycloheximide (50 μg/ml) had no effect on the release of B₁₂BP but significantly inhibited incorporation of leucine-³H into leukocyte protein. G incubated with 20 mg/ml of compound 48/80, an experimental histamine-releasing agent, had a 6-fold increase in release of B₁₂BP over a 2 hr period. Subcellular fractionation studies of human granulocytes demonstrate that most of the B₁₂BP is associated with the granular (20,000 g) layer with an excellent correlation observed between its subcellular distribution and that of acid phosphatase. These findings suggest that the release of B₁₂BP from G is mediated by an active process and provide further evidence that granulocytes are secretory as well as phagocytic cells.