title: The effects of enzyme concentration on the kinetic behavior of adrenal 21-hydroxylase and 11beta-hydroxylase in the pregnant rat.
abstract: Experiments were designed to study the kinetic behavior of 21-hydroxylase and 11beta-hydroxylase as a function of enzyme concentration (Et) during proestrus, dasy 5 (D5), 12 (D12), and 22 (D22) of pregnancy, and within 24 h post-partum. The enzymes were prepared from rat adrenal microsomes and mitochondria, respectively. The experiments consisted of measuring the initial velocity of each reaction for a series of substrate concentrations at three fixed Et. Double reciprocal plots were constructed and the slope (Km/Vmax) of each line estimated. Variation in the value of the slope as a function of enzyme dilution would predict the presence of an endogenous effector. The kinetic behavior of 21-hydroxylase was not altered throughout the range of Et (10-100 microgram protein) at any of the reproductive stages. In contrast, kinetic behavior of 11beta-hydroxylase was clearly dependent upon Et. Dilution of the enzyme preparation (25-200 microgram of protein) increased the slope of the double reciprocal plot at all reproductive stages, thus suggesting that an activator substance may be present within the mitochondrial preparation. A secondary plot of the slope (Km/Vmax) versus Et described a power function (Km/Vmax = a [Et]b) with the greatest rate of change in Km/Vmax occurring at low values of Et. The rate of change in Km/Vmax per mg rise in mitochondrial protein at all dilutions of enzyme was greatest for proestrus and post-partum, followed by D22 greater than D12  greater than D5. In addition, repeated washing of the enzyme preparation at 4 degrees C increased Km/Vmax to a greater extent at all Et than did the control preparation. These findings suggest the presence of a diffusible endogenous activator of 11beta-hydroxylase whose influence decreases markedly at D5 and D12. On the other hand, there is no evidence to suggest the presence of a diffusible endogenous effector for 21-hydroxylase.
mesh_terms: D000311:Adrenal Glands / Q000201:enzymology* / Q000648:ultrastructure; D000818:Animals; D005260:Female; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D009994:Osmolar Concentration; D049590:Postpartum Period; D011247:Pregnancy; D011270:Pregnancy, Animal; D011359:Proestrus; D051381:Rats; D013252:Steroid 11-beta-Hydroxylase / Q000378:metabolism*; D013255:Steroid 21-Hydroxylase / Q000378:metabolism*; D013250:Steroid Hydroxylases / Q000378:metabolism*
chemical_list: D013250:Steroid Hydroxylases; D013255:Steroid 21-Hydroxylase; D013252:Steroid 11-beta-Hydroxylase