Animal highly purified, preparation

Lethal dose determined from a single study using a highly purified preparation of ricin the postexposure observation interval was 3 days for goat and unspecified for other animals (Field, 1910 Hunt et al., 1918). 

A pyrimidine phosphoribosyltransferase activity with a broader specificity than the yeast enzyme has been demonstrated in animal tissues. Highly purified preparations from calf thymus (15) and beef erythrocytes (16) accepted orotate and 5-fluorouracil as substrates. Uracil phosphoribosyltransferase activity has also been demonstrated in extracts from mouse leukemia cells. Fluorouracil is a better substrate for this enzyme than uracil at pH 7.5, possibly because the acid dissociation constant for the analogue (pif 8.15) is higher than that of uracil (pK, 9.45) (17). This reasoning would suggest that the anionic form of the substrate might be the species required by the enzyme. This enzyme has been implicated in the... 

The purine nucleoside phosphorylase activity of animal tissues cleaves both ribo- and deoxyribonucleosides of 6-oxypurines. For example, the the purine nucleoside phosphorylase of human erythrocytes has been highly purified and crystallized by Parks and co-workers 2) this enzyme will cleave the ribo- and deoxyribonucleosides of guanine and hypoxan-thine. Zimmerman et al. (3) have shown that purine nucleoside phosphorylase of several animal tissues has a low intrinsic activity toward adenine in the presence of ribose 1-phosphate. The cleavage of deoxyadenosine by highly purified preparations of the animal enzyme has not been reported but by analogy with adenosine, one might expect it also to be a poor substrate. The specificities of the purine nucleoside phosphorylases of E. coli and S. typhimurium differ from that of the animal enzyme in that adenosine and deoxyadenosine are readily phosphorolyzed H-6). 

The precise biological significance of polynucleotide phosphorylase is still unknown. It has been detected in several bacteria besides Azotobacter vinelandii, and in plant tissues, but not so far in animal tissues. It is likely to have a degradative rather than biosynthetic role. Highly purified preparations of the enzyme require an oligonucleotide to prime the reaction but the enzyme does not require or copy a template. After the isolation in 1960 of an E. coli enzyme (RNA polymerase) which synthesizes RNA upon directions from a DNA template, Ochoa isolated a similar enzyme from Azotobacter vinelandii and showed it to be distinct from polynucleotide phosphorylase. 

This long quest was marked by milestones. As already discussed, the obtention of highly purified preparations of infectious scrapie particles was necessary to identify the PrP protein. Circular dichroism studies of PrP and PrP demonstrated the existence of two totally distinct conformations of PrP, one corresponding to the physiologically expressed brain protein and the other to the infectious protein. However, the masterly demonstration of the mechanism of prion replication is that PRNP° mice, which do not express the PrP protein, failed to propagate prion infectivity. Hence, without the brain reservoir of normal PrP proteins, infectious PrP proteins are harmless and unable to cause any disease. If we link this information with the respective structures of PrP and PrP , then we have a molecular mechanism accounting for the replication, by force, of prions invaders in the brain of healthy animals (Fig. 9.5). 

Natural Product hGH. In 1944 the preparation of a highly purified growth hormone from bovine pituitary glands was reported (37). Subsequendy, growth hormones derived from animal pituitaries were found to be ineffective in humans the existence of specificity among species for growth hormone was thus estabUshed. 

Proteins have been studied for a long time. Beccari published an account of his experiments to isolate gluten in 1747 In 1805 Einhof discovered that a fraction of wheat gluten was soluble, while in 1858 Denis showed that many proteins of both plant and animal origin were soluble in saline solutions. In 1859 Ritthausen started to prepare highly purified proteins, only to be criticised by Weyl for using alkali to extract the proteins. Weyl in his work used the Denis method of extraction with neutral salts. 

Recently Hansson and Wikvall studied 12 -hydroxylation in a reconstituted system consisting of highly purified cytochrome P-450 LM4 from rabbits [101]. The cytochrome P-450 fractions used were electrophoretically homogenous, but the cytochrome P-450 from starved rabbits had up to 4 times higher capacity to catalyse 12a-hydroxylation than had cytochrome P-450 from untreated, phenobarbital-treated, or -naphthoflavone-treated rabbits. It should be mentioned that treatment with /8-naphthoflavone increases the amount of cytochrome P-450 LM4 in the liver. Amino acid analyses, peptide-mapping experiments as well as absorption spectral and circular dichroism spectral analyses revealed physical differences between cytochrome P-450 LM4 preparations from starved and phenobarbital-treated animals. It was concluded that the cytochrome P-450 LM4 fraction was heterogenous and contained a species of cytochrome P-450 specific for 12a-hydroxylation. 

Male rats of the Wistar strain were purchased from Depre (Saint-Doulchard, France). Animals had free access to tap water and food containing 5% lipids (pellets A03-type from U.A.R., Villemoisson/Orge, France). Rats used for preparing highly purified mitochondrial fractions were starved for 20 h before sacrifice, while rats used for GPAT measurements had constant access to food and drinking water. 

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