Hans Klenow

Further studies on the effect of deoxyadenosine on the accumulation of deoxyadenosine triphosphate and inhibition of deoxyribonucleic acid synthesis in Ehrlich ascites tumor cells in vitro.

Abstract 1. 1. The accumulation of deATP in ascites tumor cells incubated with deoxyadenosine is prevented by adenosine. At the same time the inhibition of DNA synthesis is abolished. 2. 2. Deoxycytidine and thymidine do not affect the accumulation of deATP from deoxyadenosine. The inhibiting effect of deoxyadenosine on DNA synthesis is not completely reversed by deoxycytidine or deoxyguanosine separately but only by the two together. 3. 3. The rate of accumulation of deATP in the tumor cells increases with increasing concentration of deoxyadenosine. The concentration of deATP may exceed that of ATP in the control cells. 4. 4. In the absence of available deoxyadenosine deATP is rapidly degraded in the cells. The products of its degradation have been determined. 5. 5. The findings are discussed in relation to possible mechanisms of regulation of DNA synthesis.

On the effect of some adenine derivatives on the incorporation in vitro of isotopically labelled compounds into the nucleic acids of ehrlich ascites tumor cells

Abstract 1. 1. The incorporation of [ 14 C]formate into DNA thymidylic acid of Ehrlich ascites tumor cells in vitro is slightly stimulated by addition of adenine or adenosine. Deoxyadenosine has, however, a pronounced inhibitory effect on this process. All 3 adenine compounds reduce the incorporation of [ 14 C]formate into DNA purine deoxyribotides to very low levels. 2. 2. The specific radioactivity of pooled acid-soluble thymine compounds is not reduced by the addition of either adenine or deoxyadenosine to cell suspensions. The total radioactivity of acid-soluble thymine compounds is reduced in the presence of adenine, but in the presence of deoxyadenosine this activity is doubled. Addition of adenine, adenosine or deoxyadenosine reduces the total radioactivity of the purines of the acid-soluble fraction. 3. 3. The incorporation in vitro of [ 32 P]orthophosphate into DNA of Ehrlich ascites tumor cells is slightly activated by adenine or adenosine but is markedly reduced by deoxyadenosine. A 50% inhibition of the rate is obtained at about 1 μmole of deoxyadenosine/ml cell suspension. The effect of deoxyadenosine is unchanged after 3 recrystallizations and is abolished by weak acid hydrolysis. At concns. up to about 2 μmoles/ml the 3 adenine compounds have little effect on [ 32 P]labelling of RNA, but all inhibit it at high concns. (6.6 μmoles/ml). 4. 4. It is postulated that deoxyadenosine inhibits the DNA synthesis in Ehrlich ascites tumor cells in vitro .

Formation of the mono-, DI- and triphosphate of cordycepin in ehrlich ascites-tumor cells in vitro

Abstract Three adenine nucleotides resistant to periodate treatment have been isolated from Ehrlich ascites-tumor cells incubated with cordycepin in vitro . Chemical and enzymatic analyses have strongly suggested that the three compounds are the mono-, di- and triphosphates of cordycepin with a phosphate esterified at the hydroxymethyl group. The di- and the triphosphates contain one and two pyrophosphate bonds, respectively.

The enzymatic reaction between ribose 1-phosphate and glucose 1, 6-diphosphate.

Abstract The properties of the reaction between ribose 1-phosphate and glucose 1,6-diphosphate as catalyzed by crystalline phosphoglucomutase have been studied with a spectrophotometrical assay, which can be used for a specific quantitative determination of glucose 1,6-diphosphate. It was found that the rate of the reaction depends on the sequence of addition of some of the components. The reaction required both cysteine and magnesium ions. High concentrations of magnesium ions were inhibitory, whereas no such effect was found for high cysteine concentrations. The stoichiometry of the reactions catalyzed by phosphoglucomutase in the presence of ribose 1-phosphate and glucose 1,6-diphosphate has been worked out, and the optimum conditions for formation of ribose 1,5-diphosphate have been determined. Under the conditions used here also deoxyribose 1-phosphate, galactose 1-phosphate, and ribose 5-phosphate react with glucose 1,6-diphosphate.

Proteolytic cleavage of DNA polymerase from Escherichia Coli B into an exonuclease unit and a polymerase unit

DNA polymerase from E. coli consists of a single polypeptide chain with a molecular weight of 109,000 [I] . The ezyme c.ttalyzes the extension of polydeoxyribonucleotides from the 3’OH end group, the exchange of inorganic pyrophosphate with the two terminal phosphate groups in deoxyribonucleoside triphosphates, and an exonucleolytic degradation of polydeoxyribotides from both the 3’-hydroxyl and the 5’-phosphate end groups [2,3]. It has recently been shown in this laboratory that treatment of DNA polymerase from E. coli B with the proteolytic enzyme subtilisin (type Carslberg) under the assay conditions used results in an increase of the polymerase activity and a concomitant decrease of the exonuclease activity to a level of few percent of the initial value. The resulting DNA polymerase has been isolated and a molecular weight of about 70,000 has been estimated from gel filtration experiments. This enzyme has been found to be almost completely devoid of exonuclease activity, assayed with 3H-polyd(A-T) [4]. Experiments leading to very similar results have recently been independently performed by Brutlag et al. [5]. We wish to report on the treatment of native DNA polymerase with subtilisin under conditions which lead to the cleavage of the enzyme into two separate subunits, one which is associated with exonuclease activity and the other with polymerase activity. 2. Experimental

INHIBITION BY CORDYCEPIN AND 2-DEOXYGLUCOSE OF THE INCORPORATION OF (32P)ORTHOPHOSPHATE INTO THE NUCLEIC ACIDS OF EHRLICH ASCITES-TUMOR CELLS IN VITRO.

Abstract 1. 1. When tumor cells are incubated with low concentrations of cordycepin, cordycepin triphosphate accumulates in the cells, and incorporation of [ 2 P]P i into DNA is not inhibited. 2. 2. With higher concentrations of cordycepin also cordycepin monophosphate and cordycepin diphosphate accumulate besides cordycepin triphosphate. This event is accompanied by both a rapid decrease in the pool of ribotides of the cells and a pronounced inhibition of incorporation of [ 32 P]P i into the nucleic acid. 3. 3. The effect of cordycepin on both accumulation of cordycepin phosphates, decrease in ribotide pool, and inhibition of incorporation of [ 32 P]P i into the nucleic acids may be prevented by simultaneous addition of adenosine. 4. 4. 2 -Deoxyglucose has an effect similar to that of cordycepin with respect to inhibition of [ 32 P]P i incorporation into the nucleic acids and decrease of the ribotide pool. 5. 5. The previously demonstrated inhibiting effect of DNA synthesis by deoxy-adenosine cannot be explained by an effect on the intracellular pool of ribotides. 6. 6. The findings are discussed in relation to the possible mechanism of effect of cordycepin on the intracellular ribotide pool and the biosynthesis of nucle1c acids.

On the enzymic formation and the isolation of polyphosphates of adenine deoxyribosides.

Abstract By means of paper chromatography it has been shown that extracts of red bone marrow and preparations of myokinase contain enzymes which catalyze the formation of two additional deoxyribose compounds from mixtures of either deoxy-AMP and ATP or deoxy-GMP and ATP. Two of the products of the reaction between deoxy-AMP and ATP have been prepared in mg quantities by column chromatography. The analysis of the compounds obtained suggest that they are the deoxyribose analogs of ATP and ADP.

Some properties of the phosphoribomutase reaction

Abstract A number of properties of the phosphoribomutase activity in extracts from muscle suggests it as being identical with the phosphoglucomutase. Glucose 1,6-diphosphate (GDP) was found to be an activator for the phosphoribomutase reaction catalyzed by enzymes from muscle or yeast. From an incubation mixture consisting of enzyme, R 1-P 32 and GDP, a new labeled phosphate ester has been isolated by column chromatography. The properties of this ester suggest it to be ribose 1,5-diphosphate, the possible biological significance of which is discussed.

2-Amino-4-hydroxy-6-formylpteridine, an inhibitor of purine and pterine oxidases☆

Abstract Most folic preparations are found to exert an inhibitory effect on xanthopterin and xanthine oxidase. This seems to be due to the presence of an impurity in the preparations. The inhibitor is supposed to be 2-amino-4-hydroxy-6-formylpteridine. The inhibitory activity is destroyed by irradiation with light, reduction with metallic zinc or by binding of the aldehyde group with 2,4-dinitrophenylhydrazine. A colorimetric method for the estimation of the aldehyde is given. By incubation of the aldehyde with xanthine oxidase the inhibitory activity almost completely disappers. The enzymatic conversion of the aldehyde can be followed both bythe changes in the fluorescence and in the absorption spectrum of the compound.

3-aminobenzamide stimulates unscheduled dna synthesis and rejoining of strand breaks in human lymphocytes

Summary A comparative study of the effect of 3-aminobenzamide on the activity of DNA repair in human lymphocytes and in L 1210 cells has been made. Damage of DNA has been performed by treatment with UV light or with dimethylsulphate. DNA repair has been measured both as unscheduled DNA synthesis and by the rejoining of strand breaks. Both types of measurement have shown that 3-aminobenzamide stimulates DNA repair in human lymphocytes while inhibiting it in L 1210 cells.