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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

The apparent NAD:protein ADP-ribosyl transferase activity of mitochondria and submitochondrial particles from beef heart and rat liver is simulated by a reaction sequence that consists of an enzymic hydrolysis of NAD to ADP-ribose (ADP-Rib) by NAD glycohydrolase(s) and a nonenzymic ADP-ribosylation of acceptor proteins by the free ADP-Rib formed. The nonenzymic ADP-ribosylation of mitochondrial proteins showed two pH optima and exhibited the same remarkable selectivity as the reaction with NAD. The predominant acceptor in beef heart mitochondria was a 30-kDa protein, whereas in mitochondrial extracts of rat liver a 50-55 kDa polypeptide served as an acceptor. No authentic ADP-Rib transferase activity could be detected even when free ADP-Rib was trapped by NH2OH. Once formed, the mitochondrial ADP-Rib conjugates were resistant to hydroxylamine. NH2OH-resistant mono(ADP-Rib)-protein conjugates as found in most cells may also be products of nonenzymic ADP-ribosylation. In mouse tissues, their amounts relate to protein and NAD contents, and they increase specifically and reversibly in the hypothyroid status. Furthermore, intact rat liver mitochondria contain a mono(ADP-Rib)-polypeptide (50-55 kDa) that appeared to be identical with the polypeptide reacting with ADP-Rib in vitro.Various phage enzymes and bacterial toxins catalyze the transfer of single ADP-ribose (ADP-Rib) residues from NAD to one or several protein acceptors (for reviews, see refs. 1-4). Eukaryotic cells also contain endogenous proteins that are modified by single ADP-Rib residues (cf. ref. 5). In rat liver, most of these mono(ADP-Rib)-protein conjugates are located in the cytoplasm, whereas poly(ADP-Rib)-protein conjugates appear to be confined to the nucleus (6). Two types of endogenous mono(ADP-Rib)-conjugates can be distinguished by their sensitivity towards neutral hydroxylamine. They show independent changes and they are distributed unevenly in various organs of the mouse (5, 7).Interest in cytoplasmic ADP-ribosylation was greatly stimulated when diphtheria toxin-and cholera toxin-catalyzed ADP-ribosylation was detected (7-12) and when membrane-bound (12, 13), cytosolic (14), and mitochondrial (15, 16) ADP-ribosyl transferase activities were described. It was also reported that free ADP-Rib can form Schiff bases with amino groups of proteins, especially of the histones, when incubated at slightly alkaline conditions (17), while true ADP-Rib transferase activity in mitochondria was said to be operating at pH values <7 (18). However, the use of various inhibitors in mitochondrial preparations indicated to us that nonenzymic ADP-ribosylation was occurring at lower pH values as well.This paper describes a reaction sequence found in mitochondria (and plasma membranes) that involves the hydrolysis of NAD by NAD glycohydrolases and the nonezymic ADP-ribosylation of specific acceptors to form acid-stable conjugates. This reaction sequence simulated ADP-Rib transferase activity, and the similarity of acceptors in vivo and in vitro provides sugg...

ADP-ribosylated histone H1 was isolated from intact HeLa cells grown for 24 h with [3H]-adenosine and compared with ADP-ribosylated histone H1 synthesized from [3H]NAD by isolated HeLa nuclei. Most (ADP-ribose),-histone H1 conjugates formed in vivo carried single ADP-ribose units, less than one fourth of the total ADP-ribose residues being in the form of oligomeric or polymeric chains. (ADP-ribose), linked to H1 in vivo was not released by neutral NH2OH to a significant extent. Alkali treatment (pH 10.5) liberated most but not all of the ADP-ribose residues which may indicate the existence of a new type of linkage so far found only in conjugates isolated from intact tissue. No ADP-ribosylated histone H1 complex of higher molecular weight ('HI dimer') could be detected in intact cells.By contrast, isolated HeLa nuclei formed ADP-ribosylated histone H1 which contained predominantly polymeric ADP-ribose residues. The (ADP-ribose), residues were linked by NH20H-sensitive and by NH20H-resistant, alkali (pH 10.5) labile bonds, the majority of the conjugates appearing in the form of the higher-molecular-weight complex.A comparison with the ADP-ribosylated non-histone proteins indicated that histone H1 formed in vivo carried less than 2.5 % of the total protein-bound ADP-ribose residues and less than 1 % of the protein-bound ADP-ribose synthesized in vitro.Poly(ADP-ribose) is formed in cell nuclei by the enzymic transfer of ADP-ribose residues from NAD with the concomitant release of nicotinamide [l -31. Most, if not all (ADP-ribose), residues formed, appear to be linked covalently to nuclear proteins [4,5]. Nishizuka et al. [4] and Otake et al. [5] reported that the histones function as the main acceptor proteins. Others [6,7] showed association of the bulk of protein-bound ADP-ribose residues with the non-histone fraction (for a review see [8,9]), The linkage region of the (ADP-ribose),-protein conjugates is not known. Hayaishi and coworkers postulated an ester glycoside type of linkage because of the high lability of the conjugates formed in vitro towards neutral hydroxylamine and towards alkaline conditions [4]. Supporting evidence for an ester bond involving glutamic acid resi-