Phosphorylation of high-mobility-group chromatin proteins by protein kinase C from rat brain

Chromosomal high-mobility-group (HMG) proteins have been examined as substrates for calcium/phospholipid-dependent protein kinase C. Protein kinase C from rat brain phosphorylated efficiently both HMG 14 and HMG 17 derived from calf thymus and the reactions were calcium/phospholipid-dependent. About 1 mol of ³²P was incorporated per mol of HMG 14 and HMG 17. Phosphopeptide mapping suggested that the same major site was phosphorylated in both proteins at serine. The apparent K_m values for HMG 14 and HMG 17 were about 5 μM. HMG 14, HMG 17 and the five histone H1 subtypes prepared from rat thymus, liver and spleen were phosphorylated by the kinase. HMG 14 and HMG 17 from transformed human lymphoblasts (Wi-L2) were also phosphorylated in a calcium/phospholipid-dependent manner. HMG 1 and HMG 2 from the tissues examined were found to be poor substrates for the kinase.     

The screening of expression and purification conditions for replicative DNA polymerase associated B-subunits, assignment of the exonuclease activity to the C-terminus of archaeal pol D DP1 subunit

The B-subunits of replicative DNA polymerases belong to the superfamily of calcineurin-like phosphoesterases and are conserved from Archaea to humans. Recently we and others have shown that the B-subunit (DP1) of the archaeal family D DNA polymerase is responsible for proofreading 3'-5' exonuclease activity. The similarity of B-subunit sequences implies a common fold, but since the key catalytic and metal binding residues of the phosphoesterase domain are disrupted in the eukaryotic B-subunits, their common function has not been identified. To study the structure and activities of B-subunits in more detail, we expressed 13 different recombinant B-subunits in Escherichia coli. We found that the solubility of a protein could be predicted from the calculated GRAVY score. These scores were useful for the selection of proteins for successful expression. We optimized the expression and purification of Methanocaldococcus (Methanococcus) jannaschii DP1 of DNA polymerase D (MjaDP1) and show that the protein co-purifies with a thermostable nuclease activity. Truncation of the protein indicates that the N-terminus (aa 1-134) is not needed for catalysis. The C-terminal part of the protein containing both the calcineurin-like phosphoesterase domain and the OB-fold is sufficient for the nuclease activity.     

Characterization of the 3' exonuclease subunit DP1 of Methanococcus jannaschii replicative DNA polymerase D

The B-subunits associated with the replicative DNA polymerases are conserved from Archaea to humans, whereas the corresponding catalytic subunits are not related. The latter belong to the B and D DNA polymerase families in eukaryotes and archaea, respectively. Sequence analysis places the B-subunits within the calcineurin-like phosphoesterase superfamily. Since residues implicated in metal binding and catalysis are well conserved in archaeal family D DNA polymerases, it has been hypothesized that the B-subunit could be responsible for the 3′-5′ proofreading exonuclease activity of these enzymes. To test this hypothesis we expressed Methanococcus jannaschii DP1 (MjaDP1), the B-subunit of DNA polymerase D, in Escherichia coli, and demonstrate that MjaDP1 functions alone as a moderately active, thermostable, Mn²⁺-dependent 3′-5′ exonuclease. The putative polymerase subunit DP2 is not required. The nuclease activity is strongly reduced by single amino acid mutations in the phosphoesterase domain indicating the requirement of this domain for the activity. MjaDP1 acts as a unidirectional, non-processive exonuclease preferring mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreading exonuclease of archaeal family D DNA polymerase.     

Metabolic Consequences of Inhibition of Cerebral Adenosylmethionine Decarboxylase by Methylglyoxal bis(Guanylhydrazone)

Abstract: Effects of intracerebroventricularly injected methylglyoxal bis(guanylhydrazone) on polyamine metabolism in mouse brain were recorded during 180 h after a single dose of 3.4 μmol/kg body weight. Cerebral concentrations of 31 other amino compounds were also asayed during the experiment. The drug caused a significant inhibition of adenosylmethionine decarboxylase that lasted for 50 h, with the maximal decrease, about 70%, occurring between 5 and 10 h after injection. Significant decreases of brain spermidine and spermine concentrations were observed in three phases. Two transient decreases occurred at 10 and at 35 h, and a longer-lasting one between 60 and 100 h. Ornithine decarboxylase was stimulated within 5 h after the injection, reaching a maximal level about 30-fold above normal at 60 h, and returned to control level at 140 h. This stimulation was accompanied by significant accumulation of the reaction product, putrescine, in the brain. It was maximally > 10-fold above normal at 160 h, and was still significantly above control at the end of the observation period. The time course of changes in the parameters of polyamine metabolism was regarded as support of a previously presented hypothesis that limiting putrescine concentration may play a role in the regulation of cerebral polyamine metabolism. In addition, the present results emphasize the possibility that changes in the activities of catabolic reactions may also contribute to the regulation of cerebral polyamine concentrations. Of the 31 amino compounds analyzed, only the concentrations of ornithine, urea, glutamine, and glutamate showed significant changes from normal. Ornithine concentration first was significantly increased at 25 h, whereafter it decreased and was somewhat below normal for most of the period between 60 and 180 h. The urea concentration showed a tendency to increase throughout the experiment, being significantly elevated at the end. These changes were regarded as suggesting that the increased need for ornithine in putrescine synthesis is satisfied mainly by increased arginine uptake and degradation. The magnitude of urea accumulation suggested that metabolism of ornithine to glutamate was also accelerated. An unexpected shift toward glutamine in the glutamine/glutamate relationship was observed during the first 100 h. However, the total concentration of these two compounds was quite constant throughout the experiment. Inhibition of ornithine decarboxylase by intraventricular injection of 2-difluoromethylornithine was tried during the study, but sufficient doses could not be used without induction of acute side effects.     

Possible Involvement of Humoral Regulation in the Effects of Elevated Cerebral 4-Aminobutyric Acid Levels on the Polyamine Metabolism in Brain

Abstract: It has been reported in several recent studies that the manipulation of cerebral 4-aminobutyric acid (GABA) level results in unexpected changes in the cerebral polyamine metabolism in vivo . The mechanisms behind these interactions have remained unknown. The present results show that the changes in polyamine metabolism are not limited to the brain, but are observable also in the liver, which served as a peripheral reference tissue. Different types of responses in the activities of the poiyamine-synthesizing enzymes, ornithine decarboxylase and adenosylmethionine decarboxylase, were observed after increasing the cerebral GABA concentration of mice with varying doses of two GABA transaminase inhibitors, gabaculine and ethanolamine- O- sulphate. The time course of the significant changes in the enzyme activities showed significant correlation between the brain and liver. The possibility of direct effects of the drugs on liver was excluded by injecting them intracerebroventricularly, and by performing control experiments with equal doses given peripherally. It is concluded that the observed changes in the polyamine metabolism of liver are produced through centrally mediated humoral regulation, and that the corresponding changes in the brain are obviously due to the same factor or factors, since they are significantly correlated to the changes in liver.     

Mice Deficient in Transmembrane Prostatic Acid Phosphatase Display Increased GABAergic Transmission and Neurological Alterations

Prostatic acid phosphatase (PAP), the first diagnostic marker and present therapeutic target for prostate cancer, modulates nociception at the dorsal root ganglia (DRG), but its function in the central nervous system has remained unknown. We studied expression and function of TMPAP (the transmembrane isoform of PAP) in the brain by utilizing mice deficient in TMPAP (PAP^−/− mice). Here we report that TMPAP is expressed in a subpopulation of cerebral GABAergic neurons, and mice deficient in TMPAP show multiple behavioral and neurochemical features linked to hyperdopaminergic dysregulation and altered GABAergic transmission. In addition to increased anxiety, disturbed prepulse inhibition, increased synthesis of striatal dopamine, and augmented response to amphetamine, PAP-deficient mice have enlarged lateral ventricles, reduced diazepam-induced loss of righting reflex, and increased GABAergic tone in the hippocampus. TMPAP in the mouse brain is localized presynaptically, and colocalized with SNARE-associated protein snapin, a protein involved in synaptic vesicle docking and fusion, and PAP-deficient mice display altered subcellular distribution of snapin. We have previously shown TMPAP to reside in prostatic exosomes and we propose that TMPAP is involved in the control of GABAergic tone in the brain also through exocytosis, and that PAP deficiency produces a distinct neurological phenotype.