Spleen exonuclease

After the initial and fundamental work of Heppel and Hilmoe, and Razzell and Khorana, already reviewed in the previous edition of The Enzymes (5) and in two other articles (3, 9), the major advances have been the preparation of spleen exonuclease in a very highly purified form (10, 11), and the recognition that the enzyme has no endonucleolytic activity and that it can attack oligonucleotides carrying a terminal phosphate in the 5 position (12) this represents, however, a strong rate-limiting step. 

A method for the partial purification of spleen exonuclease was described by Heppel and Hilmoe in 1955 (13) and by Hilmoe in 1960 (14) this was later improved by Razzell and Khorana (15) and Richardson and Kornberg (16). In 1966, we described a novel purification procedure (10) leading to an enzyme preparation with a specific activity comparable to that of the best preparation of Razzell and Khorana (15). Enzyme yields were, however, low the method was therefore modified and satisfactory results were obtained (11). The new method involves the preparation of a crude enzyme obtained essentially as in the case of acid deoxyribonuclease (5, 17). The main differences are that acidification to pH 2.5 is avoided and (NH4)2S04 fractionation is done between 35 and 60%> saturation. The crude enzyme is then purified by chroma-... 

The sedimentation coefficient of spleen exonuclease, measured by centrifugation in a sucrose density gradient, using cytochrome c as the reference protein, was 4.6 S 11). The enzyme is eluted from Sephadex G-100 between acid phosphomonoesterase (s = 5.6 S) and acid DNase (s = 3.4 S). 

Spleen exonuclease is active on the 5 -OH oligonucleotides of both the ribo and the deoxyribo series. These are sequentially split from the 5 -OH end with formation of 3 -mononucleotides. It has been suggested that an enzyme-product intermediate may exist in the form of nucleoside-3 -phosphoryl-enzyme complex (3) since transfer of nucleoside-3 -phosphate to available 5 -hydroxyl functions (or other alcoholic functions) occurs at high substrate (or acceptor) concentrations 15, 18). 

The mechanism of action of spleen exonuclease is similar to that seen for venom exonuclease (19-21) but different from the processive type of attack exhibited by E. coli RNase II, sheep kidney exonuclease, and polynucleotide phosphorylase (22, 23), in which cases each polynucleotide molecule is completely degraded before the enzymes attack a new molecule. The results of Bernardi and Cantoni (12) contradict the previous beliefs that the enzyme has an intrinsic, though weak, endonucleolytic activity (5) and that a phosphate group in a terminal 5 position makes a polynucleotide chain completely resistant to the enzyme (15, 24, 25). 

The enzyme is very sensitive to the secondary structure of the substrate. Native calf thymus DNA is quite resistant to enzymic attack by spleen exonuclease, being split at less than 4% the rate at which alkali-denatured DNA is split (11). Long deoxyribonucleotides (average chain length 20-50), which still have complementary double-stranded structure, are rather resistant to the enzyme (26). Some limited results obtained with synthetic polyribonucleotides (11) are rather puzzling since poly C was found to be completely resistant, whereas poly A, poly I, and poly U were degraded at comparable rates. In the solvent used (0.15 M acetate buffer-0.01 M EDTA, pH 5.0), poly A and poly C are believed to have... 

Glucosylated oligonucleotides obtained from T4 phage DNA by acid DNase digestion are resistant to spleen exonuclease (28). It has been reported that acetylation of the 2 -OH groups of tRNA completely inhibits the action of the enzyme, whereas venom exonuclease is not affected (29). The naturally occurring methylation of sugars and bases in tRNA does not seem to hinder the action of spleen exonuclease. 

Other substrates for spleen exonuclease are the p-nitrophenyl esters of nucleoside-3 -phosphates and bis(p-nitrophenyl) phosphate, which is split only very slowly. These substrates are also split by enzymes having quite different natural substrates (Table I) (80-87). In fact, not only phosphodiesterases, in a broad sense, such as acid DNase, micrococcal nuclease, spleen and venom exonucleases, and cyclic phosphodiesterase but also enzymes such as nucleoside phosphoacyl hydrolase and nucleoside polyphosphatase split these substrates. As pointed out by Spahr and Gesteland (86), this may be explained by the fact that these substrates are not true diesters but rather mixed phosphoanhydrides because of the acidic character of the phenolic OH. It is evident that the use of the synthetic substrates, advocated by Razzell (3) as specific substrates for exonucleases, may be very misleading. Table II shows the distinctive characters of three spleen enzymes active on bis(p-nitrophenyl) phosphate which are present in the crude extracts from which acid exonuclease is prepared. 

It should be pointed out that the successful purification of spleen exonuclease (11) was greatly helped by use of a DNA hydrolyzate produced by spleen acid DNase as the substrate, since the synthetic substrates are nonspecific, and RNA core (the water-undialyzable ribooligonucleo-tides obtained by exhaustive digestion of RNA with pancreatic RNase) is also hydrolyzed by both acid and basic spleen ribonucleases (38, 39). Spleen exonuclease is unable to hydrolyze cyclic phosphates (14). 

Bernardi A. and Bernardi G. (1968). Studies on acid hydrolases. IV. Isolation and properties of spleen exonuclease. Biochim. Biophys. Acta 155 360-370. 

Spleen plio.splioclie.stera.se a specific exonuclease C G A... 

Hog spleen acid DNase, as obtained by the above procedure, is completely free of contaminating phosphatase, exonuclease, and adenosine deaminase activities. The enzyme has a weak intrinsic hydrolytic activity on bis(p-nitrophenyl) phosphate and the p-nitrophenyl derivatives of deoxyribonucleoside 3 -phosphates (see Section III,D,3). 

Venom exonuclease has been widely used for the determination of the a terminus in oligonucleotides. The normal procedure was to divide the dephosphorylated sample into two parts and digest one part with venom exonuclease. The a terminus appeared as a nucleoside the rest of the chain was degraded to 5 -mononucleotides. The other part of the dephosphorylated chain was degraded with spleen a-exonuclease. In this case the w terminus appeared as a nucleoside, the rest of the chain as 3 -mononucleotides. 

Spleen acid exonuclease is an enzyme particularly useful in sequence studies of oligonucleotides, derived from both ribonucleic acid and deoxyribonucleic acid, since it splits off, in a sequential way, nucleoside-3 -phosphates starting from the 5 OH end. 

Determination of the stereochemical course of the reactions catalyzed by the exonucleases from snake venom and bovine spleen and by Staphylococcal nuclease is in progress. 

This final acid phosphatase preparation had a specific activity of 468 and represented an approximately 1900-fold purification of the acid phosphatase in the starting crude spleen nuclease II. It contained no acid deoxyribonuclease, acid ribonuclease, exonuclease, and phosphodiesterase activities that could be detected in a 0.1-ml sample after 2 hours of incubation with the appropriate substrate. The relative rates of hydrolysis of various substrates were as follows p-nitrophenyl phosphate, 100 5 -AMP, 63 j8-glycerophosphate, 60 ATP, 0. With p-nitrophenyl phosphate as substrate, the pH optimum was broad and lay between pH 3.0 and pH 4.8. The Michaelis constant at 37°C was 7.25 X 10" mM. Phosphate and chloride ions acted as competitive inhibitors. 

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