Bile acids. XLIX. Allocholic acid, the major bile acid of Uromastix hardwickii.

Tauroallocholate is the major bile salt of the lizard, Uromastix hardwickii. Alkaline hydrolysis of bile from 25 gallbladders provided 1.21 g of acidic material, about 90% of which was allocholic acid. Analyses by gas-liquid chromatography, and mass spectrometry verified the presence of almost 10% of deoxycholic acid and smaller amounts of other 5alpha and 5beta-bile acids.     

Sialic acid interference with the thiobarbituric acid method for measuirng 3-deoxyoctulosonic acid.

Sialic acid produced an interfering color in an assay designed for measuring 3-deoxyoctulosonic acid. The color yield from sialic acid was not as high as that from 3-deoxyoctulosonic acid but was high enough to cause concern about problems arising when sialic acid-containing substances are present in preparations of bacterial lipopolysaccharides. The instabilityof 3-deoxyoctulosonic acid in acidic media led to reduced color yields. In general, the results suggested that data generated by the application of the thiobarbituric acid method to the measurement of 3-deoxyoctulosonic acid should be approached with caution.     

The structure and function of acid proteases. IV. Inactivation of the acid protease from Mucor pusillus by acid protease-specific inhibitors.

Mucor pusillus acid protease was rapidly inactivated with 1 : 1 stoichiometry by reaction with diazoacetyl-DL-norleucine methyl ester (DAN) in the presence of cupric ions. Cupric ions were essential for this inactivation. The rate of inactivation was maximal at around pH 6 when the enzyme was mixed with DAN and cupric ions without prior mixing of the reagents, and at pH 5.3 when DAN and cupric ions were mixed and incubated before addition to the enzyme solution. In both cases, the rate of inactivation decreased as the pH was either increased or decreased. The amino acid composition of an acid hydrolysate of the DAN-Modified enzyme was indistinguishable from that of the native enzyme except for the incorporation of about one norleucine residue per molecule of protein. The enzyme was also inactivated by reaction with 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP). At the stage of about 90% inactivation, 1.50 residues of EPNP were incorporated per molecule of protein and the rate of inactivation followed pseudo-first order kinetics. The optimal pH for the inactivation was pH 3.0 and the rate of inactivation decreased as the pH was either increased or decreased. Furthermore, the enzyme was strongly inhibited by pepstatin, and the reactions of DAN and of EPNP was also inhibited significantly by prior treatment of the enzyme with pepstatin. These results suggest that the enzyme may have two essential carboxyl groups at the active site, one reactive with DAN in the presence of cupric ions and the other with EPNP, and that pepstatin binds part of the active site to inhibit the reactions with DAN and EPNP as well as the enzyme activity.     

Protonated polynucleotide structures. 16. Thermodynamics of the melting of the acid form of polycytidylic acid

A phase diagram (pH, ionic strength, temperature) for the double helical form of poly(C) is presented. The thermo-dynamic analysis of these data shows that poly(C) behaves essentially as cytidine, if the electrostatic (ionic strength) contributions and the free energy of double helix formation are considered and taken into account.     

The structure and function of acid proteases. VI. Effects of acid protease-specific inhibitors on the acid proteases from Aspergillus niger var. macrosporus.

1. The Type B acid protease from Aspergillus niger var. macrosporus was inactivated by reaction with diazoacetyl-DL-norleucine methyl ester (DAN), DL-1-diazo-3-tosylamido-2-heptanone (DTH), and L-1-diazo-3-tosylamido-4-phenyl-2-butanone (DTPB) in the presence of cupric ions. The reaction with DAN took place with 1:1 stoichiometry. The enzyme was also inactivated by reaction with 1, 2-epoxy-3-(p-nitrophenoxy)-propane (EPNP) with concomitant incorporation of approximately two EPNP molecules per molecule of protein. Moreover, these reactions of DAN and of EPNP were markedly inhibited by pepstatin. These results seem to indicate that, as in the case of porcine pepsin [EC 3.4.23.1] and related acid proteases, the enzyme has two essential carboxyl groups at the active site, one reactive with DAN and related diazo reagents in the presence of cupric ions and the other reactive with EPNP, and that pepstatin binds in the vicinity of these residues. 2. The Type A acid protease from the same mold, on the other hand, was found to be markedly less sensitive to these specific inhibitors. Under conditions where the Type B enzyme was completely inactivated by DAN and related diazo reagents, only partial inactivation of this enzyme occurred. The effect of prior mixing of DAN and cupric ions on the pH profile of inactivation was also different from that for the Type B enzyme. Moreover, the Type A enzyme was not inactivated by EPNP. These results thus indicate that the nature of the active site of the Type A enzyme is rather different from that of the Type B enzyme and hence that the Type A enzyme belongs to a different class of acid proteases from the Type B enzyme.     

Preparation of the 3-monosulphates of cholic acid, chenodeoxycholic acid and deoxycholic acid.

Extraction with butan-1-ol of freeze-dried microsomal fractions from livers of 3-methyl-cholarthrene-pre-treated hamsters removed about 90% of the total lipid content, but the lipid remaining proved sufficient for the cytochrome P-450 enzyme system to retain about 15-40% of its original catalytic activity for dimethylnitrosamine demethylation. Addition of butan-1-ol-extracted total phospholipid or phosphatidylcholine could not restore any activity, whereas the addition of the synthetic phospholipid dilauroyl phosphatidylcholine was able to restore almost complete activity. Synthetic dipalmitoyl or distearoyl phosphatidylcholine was ineffective in restoring the activity in this reconstituted system.