Hydrolysis of serum albumin by proteinase K was strongly (> 7-fold) stimulated by urea and dodecylsulfate in a dose-dependent manner. With an oligopeptide as substrate, however, proteinase K was inactivated by dodecylsulfate. This indicates that the apparent activation of proteinase K by urea and dodecylsulfate is caused primarily by denaturation of the protein substrates.Although dodecylsulfate inhibited ribonuclease activity in the test-tube completely, it could not prevent RNA degradation during isolation of polysomal RNA, to which ribonuclease had been added, because of the reversible nature of the dodecylsulfate inhibition. Complete protection of RNA, however, was achieved by a combination of dodecylsulfate and proteinase K.The combined action of the detergent and proteinase K was also effective in degrading "masked" proteins in a poly(adenosine diphosphoribose) preparation which could not be attacked by the proteinase alone.Dodecylsulfate and other protein-denaturing agents are often used with the intention of inhibiting enzymic degradation of nucleic acids during isolation. Although it is generally assumed that these agents inactivate nucleolytic enzymes irreversibly, we were unable to prevent degradation of RNA during isolation from ribonuclease-containing samples by sodium dodecylsulfate. Therefore, we had introduced proteinase K (a mold enzyme from Tritirachium album Limber [l]) for the isolation of undegraded mRNA and ribosomal RNA [2 -41. The procedure was based on the simultaneous proteolytic digestion of (endogenous) ribonuclease and of ribosomal proteins. In the meantime the method has also been successfully applied to the isolation of various mRNA species and the preparation of superlong DNA [5-71. In one of these papers [7] a slight stimulation of proteinase K by sodium dodecylsulfate was reported. This paper demonstrates that a dose-dependent stimulation of proteinase K by dodecylsulfate and urea is only

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Kinetic analyses of mRNA and 28-S RNA labeling by [3H]uridine revealed distinctly different steady-state specific radioactivities finally reached for uridine in mRNA and 28-S RNA when exogenous [3H]uridine was kept constant for several cell doubling times. While the steady-state label of (total) UTP and of uridine in mRNA responded to the same extent to a suppression of pyrimidine synthesis de novo by high uridine concentrations in the culture medium, uridine in 28-S RNA was scarcely influenced. Similar findings were obtained with respect to labeling of cytidine in the various RNA species due to an equilibration of UTP with CTP. [5-3H]Uridine is also incorporated into deoxycytidine of DNA, presumably via dCTP. The specific radioactivity of this nucleoside attained the same steady-state value as UTP, uridine in mRNA and cytidine in mRNA.The data indicate the existence of two pyrimidine nucleotide pools. One is a large, general UTP pool comprising the bulk of the cellular UTP and serving nucleoplasmic nucleic acid formation (uridine and cytidine in mRNA, deoxycytidine in DNA). Its replenishment by de novo synthesis can be suppressed completely by exogenous uridine above 100 pM concentrations. A second, very small UTP (and CTP) pool with a high turnover provides most of the precursors for nucleolar RNA formation (rRNA). This pool is not subject to feedback inhibition by extracellular uridine to an appreciable extent. Determinations of (total) UTP turnover also show that the bulk of cellular RNA (rRNA) cannot be derived from the large UTP pool.Incorporation of labeled uridine into RNA has often been used as a measure of RNA synthesis. It also forms the basis of new half-life determinations of mRNA [I -41. In all these determinations it was assumed that one UTP pool served all the different RNA species. When we applied newly developed methods for the direct determination of specific radioactivities of individual RNA species [5, 61 and of UTP [7, 81 indications of rather different steady-state specific radioactivities for 28-S RNA zierms mRNA and (total) UTP were obtained [9, 101. These data pointed to the existence of different precursor pools for the various RNA species. This means that the turnover determinations mentioned above are inaccurate, as well as the conclusions drawn from labeling experiments with pyrimidine nucleotide precursors. Therefore, a study on steady-state specific radioDedicated to Feodor Lynen on the occasion of his 65th birthday. activities of uridine and cytidine in several RNA species under carefully controlled conditions of logarithmic growth and constant exogenous [3H]uridine concentrations over several generation times was started. The data obtained provide strong evidence that ribosomal and messenger RNA formation depend on separate pyrimidine nucleotide precursor pools.

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