Mouse mammary carcinoma porphobilinogenase and hydroxymethylbilane synthetase.

1. Porphobilinogenase (PBGase) and hydroxymethylbilane synthetase (HMB-S) were investigated in crude extracts from mouse mammary carcinoma, normal mouse liver and tumor bearing mouse liver. 2. A Michaelis-Menten kinetics for both enzymes in either source was observed. Km values of 87 to 108 microM, Vmax of 1.57-1.83 nmol porphyrins/2 hr and a Hill coefficient of n = 1 were obtained for PBGase and Km values of 13 to 19 microM and Vmax of 2.6-4.8 were obtained for HMB-S. 3. Porphyrin synthesis was linear up to 180 min of incubation in all cases for PBGase and HMB-S, and greatly increased with higher incubation temperature being maximal at 60 degrees C and nil at 70 degrees C, optimal temperature was 37 degrees C for either enzyme in either source. 4. Uroporphyrinogen III synthetase was heat inactivated while HMB-S was a heat stable enzyme. Optimum pH was 8.2 for PBGase and HMB-S in either tissue.

Mouse mammary carcinoma delta-aminolevulinate dehydratase.

1. Aminolevulinate dehydratase (ALA-D) was studied in crude extract from mouse mammary carcinoma, normal mouse liver and tumour bearing mouse liver. 2. A Michaelis-Menten behaviour and Km values between 0.24 and 0.31 mM were obtained for the enzyme in either source. 3. In all three tissues there was a linear relationship between porphobilinogen formation and incubation time, up to 120 min, ALA-D was thermostable and optimum pH was at 6.8. 4. There seems to be no structural alterations in tumoural ALA-D as compared with the enzyme from liver of both normal and tumour bearing mice.

Heme biosynthesis in human breast cancer--mimetic "in vitro" studies and some heme enzymic activity levels.

1. Porphyrin biosynthesis from delta-aminolevulinic acid (ALA) was investigated using the technique of tissue explant cultures, in both human breast cancer and its original normal tissue. 2. The activity of ALA-dehydratase, porphobilinogenase and uroporphyrinogen decarboxylase was directly determined in both tumor and normal mammary tissues. 3. Porphyrin synthesis capacity of human breast carcinoma was 20-fold enhanced, as compared with normal tissue, at least between the stages of porphobilinogen and coproporphyrinogen formation. 4. The activity of the three enzymes examined was always lower in normal tissue than in tumoral tissue. 5. Present findings show that porphyrin biosynthesis is increased in breast cancer tissue.

Factors influencing fluorescence spectra of free porphyrins.

We recorded fluorescence excitation and emission spectra of uro- and coproporphyrin under different experimental conditions, to see how these conditions influence quantifications based on measurement of fluorescence intensity. We found that, for bands alpha and beta of the emission spectra and the main peak of the excitation spectra, fluorescence depends on pH and is minimal near pH 5 and near pH 7-7.5 for copro- and uroporphyrin, respectively. For band gamma of the emission spectra there was a constant decrease of fluorescence with increasing alkalinity of the solution. The intensity of porphyrin fluorescence also depends on ionic strength, reaching sharp maxima at 0.1 mol/L (for uroporphyrin) and 1 mol/L (for coproporphyrin). The organic mixture ethyl acetate:acetic acid (4:1 by vol), commonly used to extract porphyrins from biological samples, markedly diminishes the fluorescence of both porphyrins as compared with the same concentration of each porphyrin in aqueous acidic solvent. Furthermore, when we measured different ratios of uro:copro mixture at three distinct pHs and buffers, we found that at pH 10.5 (in carbonate buffer) the measured units of fluorescence depend only on total porphyrin concentration and not on the composition of the mixture.