HORNE, F. R.; Texas State University, San Marcos: Carbonic Anhydrase In The Alligator Eggshell

The reptilian eggshell varies from being poorly calcified and leathery to being rather highly calcified and hard like an avian egg. To study reptilian eggshell calcification, alligator (Alligator mississippiensis) eggshells from both hatched and unhatched eggs were used. Alligator egg shells were commercial Louisiana farm eggs provided by Louisiana Department of Wildlife and Fisheries. The crux of the study was to show that an extracellular enzyme, carbonic anhydrase (CA), occurs in the eggshell matrix and may play a role in providing bicarbonate ion for in the calcified shell. Proteins of the eggshell matrix were isolated, molecular weights estimated (SDS/PAGE), Western Blots run, and antibodies to bovine RBC carbonic anhydrase II used to identify the CA. The alligator eggshells were found to contain one protein, an extracellular CA, that had a molecular weight of about 31,000 and that cross reacted with antibodies to bovine RBC CA II. Unlike the avian eggshell CA, the alligator CA did not have more than one gel band and thus may not dimerize. The calcareous portion of the eggshell was composed of 97% mineral, 1.9% organic matrix, and 0.026% water soluble protein (WSP). Alligator eggshell carbonic anhydrase was isolated via affinity chromatography and estimated spectrophotometrically to consist of 0.3 to 0.75% of the eggshell WSP fraction. Data suggest that not only does the cellular CA of the oviduct supply bicarbonate for eggshell mineralization, but that an extracellular CA in the shell matrix probably also plays a role in crocodilian shell calcification as it does in birds. By maintaining the availability of bicarbonate ion via the action of CA, more carbonate ions would be available for calcification. Eggshell CA could also assist in the disposal of protons formed as shell calcification occurs and/or be important in calcium mobilization during embryonic development.