FORMATION OF PURINE AND PYRIMIDINE NUCLEOSIDES, DEOXYNUCLEOSIDES, AND THE CORRESPONDING MONONUCLEOTIDES BY SALMON MILT EXTRACT NUCLEOSIDE PHOSPHORYLASE AND NUCLEOSIDE KINASE ENZYMES

1964 ◽  
Vol 42 (11) ◽  
pp. 1535-1545 ◽  
Author(s):  
H. L. A. Tarr

A cell-free extract of immature Salmo gairdneri milts possessed both nucleoside and deoxynucleoside phosphorylase activities. Formation of guanosine, deoxyguanosine, uridine, deoxyuridine, thymidine, thymine riboside, inosine, and deoxycytidine from the corresponding purine or pyrimidine bases was very marked, and the formation of deoxyinosine and cytidine generally was less pronounced, under the experimental conditions. The extract also possessed nucleoside phosphokinase and deoxynucleoside phosphokinase activities, although these were considerably less marked than the former. Formation of the mononucleotides of adenosine, uridine, thymidine, deoxyuridine, guanosine, inosine, and deoxyinosine in the presence of adenosinetriphosphate was recorded. When orotic acid was employed as enzyme substrate, uridine, deoxyuridine, and uridylic acid were formed. Comparatively feeble formation of cytidylic and deoxycytidylic acids from the corresponding nucleosides was found. The possible significance of these findings, in relation to known routes of biosynthesis of mononucleotides, is discussed.

1964 ◽  
Vol 42 (1) ◽  
pp. 51-57 ◽  
Author(s):  
H. L. A. Tarr

C14-labeled adenine, guanine and cytosine, and tritiated thymidine were incorporated into the deoxyribonucleic acid of salmon milts, either by injection into the milts of live fish or into excised milts. The amount incorporated was very small. Under the experimental conditions radioactive nucleosides, deoxyuridine, adenosine 5′-mono- and tri-phosphates, orotic acid, uracil, ribose 1-phosphate, and ribose 5-phosphate were not incorporated. It is suggested that these results may be due to the comparative impermeability of the cells to the various compounds.


1971 ◽  
Vol 125 (3) ◽  
pp. 743-749 ◽  
Author(s):  
Asru K. Sinha ◽  
J. K. Bhattacharjee

The reduction of α-aminoadipate to α-aminoadipic δ-semialdehyde by a cell-free extract of Saccharomyces is shown to be a three-step process. First the amino acid reacts with ATP to form an adenylyl derivative. Then the adenylyl derivative of α-aminoadipate is reduced in the presence of NADPH. In the third step the reduced adenylyl derivative of the amino acid is cleaved to form α-aminoadipic δ-semialdehyde. The presence of Mg2+is necessary for the first and second steps. The third step does not need any cofactors. The product of the first step was isolated by chromatography after incubating the cell-free extract of Saccharomyces with α-aminoadipate, ATP and Mg2+. The isolated product was identified as an adenylyl derivative of α-aminoadipate and could be converted into α-aminoadipic δ-semialdehyde under the stated experimental conditions. The product of the second step was too unstable to be identified.


1993 ◽  
Vol 2 (6) ◽  
pp. 489-496 ◽  
Author(s):  
Anne Pihlanto-Leppälä ◽  
Eero Pahkala ◽  
Veijo Antila

The aim of this study was to examine the enzymatic hydrolysis of κ-casein by isolating and identifying the released peptides. The enzymes employed in the study were chymosin, plasmin and trypsin, as well as a cell-free extract from three Lactobacillus helveticus and nine Lactobacillus casei strains. The findings showed that the bond most sensitive to the proteolytic activity of chymosin was the Phe 105-Met 106. After 24 hours of hydrolysis a few other bonds in the casein macropeptide were also cleaved. Plasmin was found to have weak proteolytic activity under the conditions of this study. When the enzyme-substrate ratio was raised from 1:200 to 1:50, a few peptides were released from the N-terminal region. Trypsin was found to hydrolyze several κ-casein bonds, and peptides were released from almost all regions of the protein. The proteases of Lactobacillus had less effect than chymosin, plasmin or trypsin. The strains could be divided into three categories. L. helveticus strains had activity on bonds in the mid-section and C-terminal region, L. casei strains EB, P3, P8 and A 1 had activity on bonds in the N- and C-terminal regions, while L. casei A5 and M9 had activity only on bonds in the C-terminal region.


2020 ◽  
Author(s):  
Felix Kaspar ◽  
Peter Neubauer ◽  
Anke Kurreck

The poor solubility of many nucleoside and nucleobases in aqueous solution demands harsh reaction conditions (base, heat, cosolvent) in nucleoside phosphorylase-catalyzed processes to facilitate substrate loading beyond the low millimolar range. This, in turn, requires enzymes which withstand these conditions. Herein we report that the pyrimidine nucleoside phosphorylase from <i>Thermus thermophilus</i> is active over an exceptionally broad pH (4-10), temperature (up to 100 °C) and cosolvent space (up to 80% (v/v) non-aqueous medium) and displays tremendous stability under harsh reaction conditions with predicted total turnover numbers of more than 10<sup>6</sup> for various pyrimidine nucleosides. However, its use as a biocatalyst for preparative applications is critically limited due to its inhibition by nucleoside substrates at low concentrations, which is unprecedented among non-specific pyrimidine nucleoside phosphorylases.<br>


2012 ◽  
Vol 17 (10) ◽  
pp. 1348-1361 ◽  
Author(s):  
Sayali S. Kukday ◽  
Surya P. Manandhar ◽  
Marissa C. Ludley ◽  
Mary E. Burriss ◽  
Benjamin J. Alper ◽  
...  

The insulin-degrading enzyme (IDE) cleaves numerous small peptides, including biologically active hormones and disease-related peptides. The propensity of IDE to degrade neurotoxic Aβ peptides marks IDE as a potential therapeutic target for Alzheimer disease. Using a synthetic reporter based on the yeast a-factor mating pheromone precursor, which is cleaved by multiple IDE orthologs, we identified seven small molecules that stimulate rat IDE activity in vitro. Half-maximal activation of IDE by the compounds is observed in vitro in the range of 43 to 198 µM. All compounds decrease the Km of IDE. Four compounds activate IDE in the presence of the competing substrate insulin, which disproportionately inhibits IDE activity. Two compounds stimulate rat IDE activity in a cell-based assay, indicating that they are cell permeable. The compounds demonstrate specificity for rat IDE since they do not enhance the activities of IDE orthologs, including human IDE, and they appear specific for a-factor–based reporters since they do not enhance rat IDE-mediated cleavage of Aβ-based reporters. Our results suggest that IDE activators function in the context of specific enzyme-substrate pairs, indicating that the choice of substrate must be considered in addition to target validation in IDE activator screens.


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