No abstract

Duodenal alkaline phosphatase of juvenile (11-day-old) mice, like other non-specific alkaline phosphatases, has the ability to hydrolyse PP(i). When a constant Mg(2+)/PP(i) concentration ratio is maintained, plots of velocity as a function of PP(i) concentration are consistent with Michaelis-Menten kinetics. Mg(2+) activates pyrophosphate hydrolysis and maximal activity is obtained at a constant Mg(2+)/PP(i) concentration ratio of 0.66. At higher ratios there is strong inhibition. At constant concentrations of Mg(2+) and increasing concentrations of PP(i), the velocity-substrate (PP(i)) concentration plots show sigmoidal dependence. By assuming that the true substrate is MgP(2)O(7) (2-) complex, and using complexity constants, the concentrations of free Mg(2+), Mg(2)P(2)O(7) and MgP(2)O(7) (2-) were calculated in assay mixtures ranging in PP(i) concentration from 0.1 to 2.5mm and in total Mg(2+) concentration from 0.6 to 2.6mm. From these data, the concentrations of added Mg(2+) and PP(i) in the assay mixtures were selected so that the velocity could be measured (1) at three fixed concentrations of free Mg(2+) ions with varied concentrations of MgP(2)O(7) (2-) and (2) at four fixed concentrations of Mg(2)P(2)O(7) with varied concentrations of MgP(2)O(7) (2-). Lineweaver-Burk and Hill plots from these data showed that the inhibition is caused by free Mg(2+) ions, of a mixed type and consistent with Michaelis-Menten kinetics. The sigmoidal dependence observed between velocity and PP(i) concentration at constant concentration of total Mg(2+) is therefore not due to allosteric inhibition. It is due to a combined effect of (1) inhibition by free Mg(2+) ions, (2) depletion of the true substrate, MgP(2)O(7) (2-), owing to the formation of Mg(2)P(2)O(7) and (3) the manner in which the concentrations of these three molecular or ionic species change when PP(i) concentration is increased maintaining the total Mg(2+) concentration constant.

No abstract

Mouse duodenal microvillus membrane protein metabolism was measured using radioactive labelling techniques. Labelled amino acids were introduced into the lumen of ligatured duodena. Following exposure to label, brush border membranes were isolated and analyzed. Experiments measuring the specific activity of protein labelled with a single amino acid revealed that total membrane protein appeared to turnover in about 14 hr. Protein in the mucosal homogenate had a faster turnover rate. Turnover rates of individual proteins were measured with single and dual isotope experiments. Membrane protein was solubilized with sodium dodecyl sulphate (SDS) buffer. Single isotope experiments showed that all polypeptides separated on SDS-gels were maximally labelled at 6 hr after injection. Bands did not incorporate label linearly. Rates of loss (degradation) of label from membrane proteins in the seventeen bands appeared to be related to the estimated molecular size of the proteins. Rates were highest for larger polypeptides. A double isotope technique, in which proteins were allowed to incorporate the same amino acid in two isotopic forms, delivered with a set time interval intervening, revealed that the ratios of the second label to the first in the SDS-separated polypeptides were highest for larger proteins and lowest for smaller polypeptides. Certain assumptions were outlined and the ratios taken as measures of turnover of proteins. Loss of label due to cell sloughing is discussed. A mixture of labelled amino acids (excluding leucine) was used to show that differences in leucine contents of different proteins was not an explanation for the variation in level of leucine radioactivity in different bands. For specific activity measurements throughout, protein in gels was quantitated with reference to the uptake of Coomassie stain. The use of this stain was validated by the finding that, at low protein concentration, the amount of stain taken up was proportional to the amount of bovine serum albumin or membrane protein loaded.

Polyacrylamide-gel electrophoresis and Bio-Gel P-300 molecular-sieve chromatography of mouse duodenal alkaline phosphatase demonstrates its molecular heterogeneity, which, in a kinetic sense, is manifest also in the differential relative velocities of the heterogeneous forms of the enzyme with two substrates, phenylphosphate and beta-glycerophosphate. Different treatments that eliminate most of the electrophoretic and chromatographic variability of the enzyme also decrease the velocities with both substrates so that the molar ratio of hydrolysis of one substrate relative to the other is also altered to a low but stable value. Concomitant with these changes, lipids and peptides are dissociated from the enzyme. The lipids are tentatively identified as a sterol and phospholipids. The peptides have an average composition of four to six amino acids and appear to be strongly electropositive. The conditions of dissociation suggest that their binding to the enzyme is non-covalent and predominantly based on hydrophobic and ionic bonding. The concept of lipid and peptide association would suggest prima facie differential molecular weights as a factor in the observed electrophoretic and chromatographic heterogeneity. However, the molecular forms of the enzyme with differences in elution volume equivalent to more than one-half the void volume of the Bio-Gel P-300 column, or even enzyme fractions dissociated from the lipids and peptides compared with undissociated portions, do not show any differences in sedimentation on sucrose-density-gradient centrifugation. This may be because the alterations in molecular weight owing to binding of small molecules are too small to be detected by this method. Alternatively, since lipids are involved, the binding may alter the partial specific volume in such a way that the buoyant density is not significantly altered.