An affinity matrix for the purification of poly(ADP-ribose) glycohydrolase.

The preparation of quantities of poly(ADP-ribose) glycohydrolase sufficient for detailed structural and enzymatic characterizations has been difficult due to the very low tissue content of the enzyme and its lability in late stages of purification. To date, the only purification of this enzyme to apparent homogeneity has involved a procedure requiring 6 column chromatographic steps. Described here is the preparation of an affinity matrix which consists of ADP-ribose polymers bound to dihydroxyboronyl sepharose. An application is described for the purification of poly(ADP-ribose) glycohydrolase from calf thymus in which a single rapid affinity step was used to replace 3 column chromatographic steps yielding enzyme of greater than 90% purity with a 3 fold increase in yield. This matrix should also prove useful for other studies of ADP-ribose polymer metabolism and related clinical conditions.     

Adenosine diphosphate ribosyltransferase and protein acceptors associated with cytoplasmic free messenger ribonucleoprotein particles

ADP-ribosyltransferase activity has been characterized in free messenger ribonucleoprotein particles (mRNP) from mouse plasmacytoma cells. This enzymatic activity appears to be associated with the free mRNP and not due to nuclear contamination. The enzyme activity is not stimulated by added DNA or histone H1 and represents 34 per cent of the total cellular ADP-ribosyltransferase activity while the DNA contamination in free mRNP is less than 4 per cent of the total cellular DNA. Moreover, the ADP-ribosyltransferase specific activity per mg of DNA is about 75-fold higher in free mRNP than in the nuclei. During CsCl gradient centrifugation of the cytoplasmic fraction, the ADP-ribosylated material separates out at a buoyant density similar to that of free mRNP.This ADP-ribosyltransferase activity is inhibited by thymidine, nicotinamide and 3-aminobenzamide, while it is highly stimulated by exogenous pancreatic RNase. The in vitro synthesized acid insoluble material is rendered partly soluble by treatment by a proteolytic enzyme or by snake venom phosphodiesterase resulting in phosphoribosyl-AMP formation: the pancreatic RNase does not solubilize this material. Several ADP-ribosylated proteins are detected by lithium dodecylsulfate gel electrophoresis.Such an ADP-ribosyltransferase activity has also been detected in free mRNP from rat liver. It is suggested that this ADP-ribosylation of specific free mRNP proteins may be associated with free mRNP structure and/or with some chemical covalent type of modification rendering mRNA available for translation.     

Neonatal glutaric aciduria type II: An X-linked recessive inherited disorder

Summary A new case of neonatal glutaric aciduria type II is reported. Neonatal acidosis, hypoglycemia, and hyperammonemia were characteristic. The baby died at four days of age. Organic acid analysis revealed massive glutaric aciduria with elevated concentrations of butyric, isobutyric, n-butyric, and isovaleric acid in his urine. The baby's pedigree suggested strongly an X-linked recessive mode of inheritance. Clinically, biochemically, and genetically glutaric aciduria type II is an heterogeneous disorder. The neonatal form is an X-linked inherited disorder which presents early in life, and is associated with metabolic acidosis, hypoglycemia, and hyperammonemia, and leads to death in the neonatal period. The mild form is an autosomal recessive inherited disease which may present even in adults, and is associated with recurrent hypoglycemia without ketosis and usually improves. Nevertheless the same unusual organic acid pattern is observed in both forms. The basic biochemical defect must be distinct and has not been elucidated.     

Poly(ADP-ribosyl)ation of chromatin in an in-vitro poly(ADP-ribose)-turnover system.

This paper describes the effect of an in-vitro poly(ADP-ribose) turnover system on the poly(ADP-ribosyl)ation of chromatin. Both poly(ADP-ribose)polymerase and poly(ADP-ribose)glycohydrolase were highly purified and used in 4 different turnover systems: non-turnover, slow, medium and fast turnover. These turnover systems were designed to reflect possible turnover conditions in intact cells. The major protein acceptors for poly(ADP-ribose) are histones and the polymerase itself, a process referred to as automodification. The level of poly(ADP-ribose) modification of polymerase, histone H1 and core histones has been measured. The size of the polymer for each of the 3 groups of acceptor proteins has been determined by gel electrophoresis. After many turnover cycles at medium and fast turnover, the histones (H1 and core) become the main poly(ADP-ribose) acceptor proteins. The rate at which steady-state polymer levels are reached and the total accumulation of polymer in a given turnover system are both inversely proportional to the amount of glycohydrolase present. Furthermore, increasing amounts of glycohydrolase in the turnover systems reduces average polymer size. The polymer synthesized in the medium and fast turnover systems is degraded by glycohydrolase in a biphasic fashion and in these systems the half-life of polymer agreed with results found in intact cells. Our results show that the relative levels of polymerase and glycohydrolase activities can regulate the proportional poly(ADP-ribose) distribution on chromatin-associated acceptor proteins during steady-state turnover conditions. The patterns of modification of polymerase and histones under turnover conditions agree with in vivo observations.     

An antisense RNA regulates the bidirectional silencing property of the Kcnq1 imprinting control region

The Kcnq1 imprinting control region (ICR) located in intron 10 of the Kcnq1 gene is unmethylated on the paternal chromosome and methylated on the maternal chromosome and has been implicated in the manifestation of parent-of-origin-specific expression of six neighboring genes. The unmethylated Kcnq1 ICR harbors bidirectional silencer activity and drives expression of an antisense RNA, Kcnq1ot1, which overlaps the Kcnq1 coding region. To elucidate whether the Kcnq1ot1 RNA plays a role in the bidirectional silencing activity of the Kcnq1 ICR, we have characterized factor binding sites by genomic footprinting and tested the functional consequence of various deletions of these binding sites in an episome-based system. Deletion of the elements necessary for Kcnq1ot1 promoter function resulted in the loss of silencing activity. Furthermore, interruption of Kcnq1ot1 RNA production by the insertion of a polyadenylation sequence downstream of the promoter also caused a loss of both silencing activity and methylation spreading. Thus, the antisense RNA plays a key role in the silencing function of the ICR. Double-stranded RNA (dsRNA)-mediated RNA interference is unlikely to be involved, as the ICR is active irrespective of the simultaneous production of dsRNA from the genes it silences.     

Solute complexation degree with human serum albumin: biochromatographic approach

A mathematical model was developed for the study of the D,L-dansylamino acid retention mechanism in reversed-phase liquid chromatography using a C18 column as a stationary phase and human serum albumin (HSA) as an eluent modifier. The solute retention factor is dependent on the HSA concentration in the eluent as well as the binding constant of the guest-HSA complex. A determination of the degree of complexation n(c) (the percent of the complexed guest) could be carried out. Different Van 't Hoff plot shapes of the degree of complexation were observed with different eluent pH, confirming a change in the solute complexation mechanism for physiological pH (between 7-7.5). Enthalpy-entropy compensation was also analysed in relation to this mathematical model to confirm the solute complexation behavior with HSA. These results finally confirmed that at physiological pH and temperature (approximately 35 degrees C) values the HSA was in a favorable structural conformation for its binding with a great majority of drugs.