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Characterization of insect calmodulin during oogenesis and embryogenesis of Blattella germanica
Abstract
A Ca$\sp{2+}$-binding protein has been purified and characterized from Blattella germanica eggs. This protein has biochemical features in common with calmodulin. These common features include a relatively low molecular weight of $\sim$19 KDa, thermal stability, an acidic pI of 4.0, a low specific absorbance (E$\sb{\rm 277nm}\sp{1\%}$ = 2.8), an altered electrophoretic mobility in SDS-polyacrylamide gels in the presence of 1 mM Ca$\sp{2+},$ and calcium-dependent binding to the calmodulin antagonist W-7. These features, considered together with activation of calmodulin-dependent phosphodiesterase in a Ca$\sp{2+}$-dependent manner and cross-reactivity with anti-bovine brain calmodulin antibody, are sufficient to define this protein as bone fide calmodulin. A rabbit antibody specific for the B. germanica calmodulin cross-reacts with bovine brain calmodulin. The calmodulin levels during oogenesis and embryogenesis were estimated by densitometric analysis of immunoblots using anti-Blattella germanica egg calmodulin antibody as a probe. A high concentration of calmodulin is present in vitellogenic follicles and early embryonic eggs (about 15 ng calmodulin/$\mu$g protein). During oogenesis calmodulin accumulates in the oocyte throughout the yolk deposition phase. During embryogenesis calmodulin is present at uniformly high levels until vitellin utilization starts, then it is undetectable until the pharate larval stage at the end of embryogenesis. $\sp{14}$C-labeled egg calmodulin in a gel-overlay technique binds to vitellin, the major yolk protein of B. germanica eggs. The calmodulin-binding site of vitellin is located on the 95 KDa subunit before degradation, and on the 53 KDa fragment after 95 KDa subunit breakdown. There is sufficient calmodulin to bind stoichiometrically (1:1) with the vitellin trimer. Circumstantial evidence suggests that this CaM is derived from outside the oocyte. In vitro experiments with ($\sp{35}$S) -methionine showed that the highly abundant calmodulin accumulating in vitellogenic follicles was not synthesized by the oocyte. On the other hand, isolated follicle cells rapidly synthesize large amounts of calmodulin. No calmodulin is detectable in serum. These facts suggest that calmodulin produced by follicle cells is most likely the source of calmodulin in the vitellogenic oocyte. Indirect immunofluorescent staining with anti-egg calmodulin demonstrated that in early- and mid-vitellogenic follicles calmodulin is localized in the cytoplasm of follicle cells and the cytoplasmic compartment surrounding yolk granules but not inside yolk granules. Immunofluorescence was most intense in the cortex of the oocyte and outside the membranes of yolk granules. Transport of calmodulin into the cytoplasmic compartment of the oocyte is not via binding to vitellogenenin.
Subject Area
Cellular biology|Zoology|Entomology
Recommended Citation
Zhang, Yujun, "Characterization of insect calmodulin during oogenesis and embryogenesis of Blattella germanica" (1992). Doctoral Dissertations Available from Proquest. AAI9219518.
https://scholarworks.umass.edu/dissertations/AAI9219518