The binding constants, K(1) and K(2), and the number of Ca(2+) ions in each of the two high affinity sites of Ca(2+)-regenerated bacteriorhodopsin (bR) are determined potentiometrically at different pH values in the range of pH 3.5-4.5 by using the Scatchard plot method. From the pH dependence of K(1) and K(2), it was found that two hydrogen ions are released for each Ca(2+) bound to each of the two high affinity sites. Furthermore, we have measured by a direct spectroscopic method the association constant, K(s), for the binding of Ca(2+) to deionized bR, which is responsible for producing the blue to purple color change. Comparing the value of K(s) and its pH dependence with those of K(1) and K(2) showed that the site corresponding to K(s) is to be identified with that of K(2). This is in agreement with the conclusion reached previously, using a different approach, which showed that it is the second Ca(2+) that causes the blue to purple color change.Our studies also show that in addition to the two distinct high affinity sites, there are about four to six sites with lower binding constants. These are attributed to the nonspecific binding in bR.
We studied a six-month-old infant with severe megaloblastic anemia, coma and hyperpigmentation of the extremities. He was found to have methylmalonic aciduria (79 mumol per milligram of creatinine) and homocystinuria (0.85 mumol per milligram of creatinine). Additional biochemical abnormalities included cystathioninuria, glycinuria, methylcitric aciduria, 3-hydroxypropionic aciduria and formic aciduria. The concentration of vitamin B12 in the serum was 20 pg per milliliter. This severe nutritional deficiency was a consequence of inadequate intake, for the infant was exclusively breast-fed by a strictly vegetarian mother who manifested methylmalonic aciduria. Our observations emphasize the importance of educating strict vegetarians about the deficiency of vitamin B12 in their diets and the importance of vitamin B12 supplementation.
Prenatal diagnosis for the genetic counselling of families at risk for having children with the life-threatening organic acidurias is advancing rapidly. The two major approaches to prenatal diagnosis are the assay for deficient activity of the enzymes in cultured amniocytes and the measurement of increased concentrations of the organic acids in the amniotic fluid. The latter, when done by stable isotope dilution analysis, is rapid, relatively inexpensive and very reliable.
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