Substrate analogue induced changes of the CO-stretching mode in the cytochrome P450cam-carbon monoxide complex.

The CO-stretching mode of the carbon monoxide ligand in reduced cytochrome P450cam, in the absence or presence of camphor and in the presence of nine different camphor analogues, was measured at room temperature using Fourier transform infrared spectroscopy. Substrate-free cytochrome P450cam--CO reveals a broad, slightly structured band resulting from an overlap of several stretching mode signals. The multitude of the signals indicates that cytochrome P450 exists in a dynamic equilibrium of several conformational substates. Binding of camphor or camphor analogues strongly influences this equilibrium. For substrate analogues which are not able to form a hydrogen bond to the hydroxyl group of tyrosine 96, the CO-stretching band is rather broad and asymmetric. In contrast, substrate analogues with one quinone group which form a hydrogen bond to the Tyr96 OH induce a shift and a sharpening of the CO-stretching mode band. For substrate analogues with two hetero groups, the infrared spectrum is slightly asymmetric or a minor band appears. Sterical hindrance, substrate mobility, and protein flexibility finally determine the position and width of the CO-stretching mode signals.     

An anti-insulin serum, but not a glucagon antagonist, alters glycemia in fed chickens.

Attempts at altering plasma glucose and, as a consequence, food intake were performed in fed broiler chickens by single i.v. injection of des-His1(Glu9) glucagon amide (a glucagon antagonist) or a non-stimulating anti-insulin serum. Plasma glucose level was not altered by des-His1(Glu9) glucagon amide but was rapidly and largely increased (for at least 2 h) by the injection of the insulin-immune serum. Hour and cumulative food intake were unaltered up to 10 h post injection. These results strongly suggest that in fed chickens, plasma glucose is mainly, if not exclusively, controlled by plasma insulin, and that the transient and heavy hyperglycemia evoked by inhibiting insulin action does not alter food intake.     

Studies on the Tissue Distribution of the Puromycin-Sensitive Enkephalin-Degrading Aminopeptidases

An antiserum generated to the soluble form of the rat brain puromycin-sensitive enkephalin-degrading aminopeptidase was used to determine the tissue distribution of the soluble and membrane-associated forms of this enzyme. All tissues examined contained significant levels of the soluble enzyme form, with this enzyme accounting for greater than 90% of the arylamidase activity in brain, heart, and skeletal muscle. Native gel electrophoresis coupled with activity staining as well as inhibition studies were used to confirm the presence of this enzyme in various tissues. Serum was found not to contain this particular aminopeptidase. In contrast to the results obtained with the soluble enzyme form, brain was the only tissue found to contain the membrane-associated enzyme form. Although all tissues contained membrane-associated aminopeptidase activity only the brain enzyme could be maintained in solution in the absence of detergent. In addition, the brain membrane-associated enzyme could be distinguished from the membrane-associated aminopeptidase activity in other tissues on the basis of its sensitivity to inhibition by puromycin.     

The effect of iron binding on the conformation of transferrin. A small angle x-ray scattering study.

Distance distribution functions, p(r), radii of gyration, Rg, and radii of gyration of cross section, Rq, of apotransferrin, monoferric transferrin, and diferric transferrin have been compared. The alteration of Rg and Rq upon iron binding has been determined by a difference method. An unusual feature of the stepwise structural changes of transferrin upon iron saturation is that binding of the first ferric ion is responsible for more than half of the whole change in Rq, whereas Rg alters significantly only after the binding of the second ferric ion.     

Spectroscopic studies of protein-heme interactions accompanying the allosteric transition in methemoglobins

Resonance Raman, optical absorption, and circular dichroism spectroscopic techniques have been used to examine the effect of the addition of inositol hexaphosphate (IHP) to a series of carp and human methemoglobin derivatives. Markers of spin equilibrium in the high-frequency region (1450-1650 cm-1) of the resonance Raman spectrum yield high/low-spin ratios consistent with direct magnetic susceptibility measurements. Changes in the low-frequency region (100-600 cm-1) of the resonance Raman spectrum appear to correlate with the quaternary structure transition. Changes in the ultraviolet absorption spectra and the circular dichroism spectra also appear to be related to the quaternary structure change. By using the resonance Raman spin markers, we find that those derivatives of carp methemoglobin which are in spin equilibrium have a larger ratio of high-spin to low-spin populations than the corresponding derivatives of human methemoglobin. Upon the addition of IHP to the methemoglobins the spin equilibrium is shifted toward a larger high-spin population. This change in equilibrium is larger for the carp protein than for the human protein. We obtain an IHP-induced change in the free energy difference between the high-spin and low-spin states of 300 cal/mol for those human methemoglobins in which a quaternary structure change occurs and 600 cal/mol for carp methemoglobins. Our data are consistent with a quaternary structure change induced by IHP in all the carp methemoglobins studied (F-, H2O, SCN-, NO2-, N3-, and CN-) and in the F-, H2O, and SCN- derivatives of the human protein but not in the NO2-, N3-, and CN- derivatives. The Fe-CN stretching mode has been identified by isotopic substitution and found to be unchanged in frequency in carp CN- metHb when the quaternary structure is changed. On the basis of our results we conclude that the protein forces at the heme due to the addition of IHP do not significantly affect the position of the iron atom with respect to the heme plane. Rather, the changes in spin equilibrium may be caused by protein-induced changes in the orientation of the proximal histidine or tertiary structure changes in the heme pocket which affect the porphyrin macrocycle. Either of these changes, or a combination thereof, leads to changes in the iron d orbital energies and concomitant changes in the spin equilibrium.     

Immobilization of lactate dehydrogenase on polyacrylamide beads

Pig muscle lactate dehydrogenase (L-lactate:NAD oxidoreductase, EC 1.1.1.27) was covalently immobilized on polyacrylamide beads containing carboxylic functional groups activated by water-soluble carbodiimide. The effects of immobilization on the catalytic properties and stability of the lactate dehydrogenase were studied. There was no shift in the pH optimum of the immobilized enzyme compared to that of the soluble one. The apparent optimum temperature of the soluble enzyme was 65 degrees C, while that of the immobilized enzyme was between 50 and 65 degrees C. The apparent Km values of the immobilized enzyme with pyruvate and NADH substrates were higher than those of the soluble enzyme. As a result of immobilization, enhanced stabilities were found against heat treatment, changes in pH, and urea denaturation.     

Conversion of exogenous insulin into high molecular weight forms in vivo

[¹²⁵I]-insulin, injected in rats, was converted into high molecular weight forms as judged by gel filtration of blood serum samples collected at various intervals. These forms represented 26% (10 min. after injection) to 81% (240 min. after injection) of the total immunoprecipitable radioactivity. Their molecular weights were not affected by rechromatography in 0.1 M borate buffer (pH 8) or in 8 M urea-1 M acetic acid (pH 2.4). On incubation of [¹²⁵I]-insulin with blood serum $\text{in}\text{vitro}$, no high molecular weight forms could be observed.