Development of the gastrointestinal mucosal barrier V. Comparative effect of calcium binding on microvillus membrane structure in newborn and adult rats.

Abstract

Electron spin resonance (ESR) and the spin label method, with 5-doxyl stearic acid as a probe, were used to investigate the structure of microvillus membrane (MVM) from small intestine of adult and newborn rats. It was shown that the spin label in MVM of newborn was maintained in a more disordered environment than the spin label in adult animals. Calcium ion was used as an external stimulus to study the structural response and organization of these two membrane preparations. Ca++ enhanced the order of 5-doxyl stearic acid labeled MVM from mature and immature rats in a concentration-dependent saturable process, but Ca++ exerted a greater ordering effect on MVM from immature than MVM from the mature rat. Ca++ binding to MVM was also a concentration-dependent, saturable process. MVM from immature rat bound significantly more Ca++ in CaCl2 concentration ranges from 12.5 micron to 4mM. Scatchard analysis of the binding data showed two classes of binding sites with a high affinity constant of 3.1 X 10(4) M-1 and a low affinity constant of 9.1 X 10(3) M-1, with corresponding maximum binding capacities for each class site of 129.8 nmole of calcium/mg protein and 252.7 nmole calcium/mg of protein in newborn and 13-day-old MVM. Only one high affinity constant of 2.6 X 10(4)M-1 with a corresponding maximum binding capacity of 106.4 nmole/mg of protein was observed in adult MVM. Proteolytic hydrolysis of the membranes by trypsin produced an increase in Ca++ binding in adult MVM and a decrease in Ca++ binding in newborn MVM. Neuraminidase and phospholipase C reduced the amount of bound Ca++ in both adult and newborn MVM. These results indicate a more disordered structure of newborn MVM and a differential effect of Ca++ on MVM during development.