Purification, molecular cloning, and immunohistochemical localization of dipeptidyl peptidase II from the rat kidney and its identity with quiescent cell proline dipeptidase

We purified dipeptidyl peptidase II (DPP II) to homogeneity from rat kidney and determined its physicochemical properties, including its molecular weight, substrate specificity, and partial amino acid sequence. Furthermore, we screened a rat kidney cDNA library, isolated the DPP II cDNA and determined its structure. The cDNA was composed of 1,720 base pairs of nucleotides, and 500 amino acid residues were predicted from the coding region of cDNA. Human quiescent cell proline dipeptidase (QPP) cloned from T-cells is a 58-kDa glycoprotein existing as a homodimer formed with a leucine zipper motif. The levels of amino acid homology were 92.8% (rat DPP II vs. mouse QPP) and 78.9% (rat DPP II vs. human QPP), while those of nucleotide homology were 93.5% (rat DPP II vs. mouse QPP) and 79.4% (rat DPP II vs. human QPP). The predicted amino acid sequences of rat DPP II and human and mouse QPP possess eight cysteine residues and a leucine zipper motif at the same positions. The purified DPP II showed similar substrate specificity and optimal pH to those of QPP. Consequently, it was thought that DPP II is identical to QPP. Northern blot analysis with rat DPP II cDNA revealed prominent expression of DPP II mRNA in the kidney, and the order for expression was kidney > testis > or = heart > brain > or = lung > spleen > skeletal muscle > or = liver. In parallel with Northern blot analysis, the DPP II antigen was detected by immunohistochemical staining in the cytosol of epithelial cells in the kidney, testis, uterus, and cerebrum.     

Dietary linolenic acid-mediated increase in vascular prostacyclin formation

To define vascular effects of an enhanced dietary -linolenic acid intake, 28 spontaneously hypertensive rats were fed a 3% sunflowerseed oil (44% linoleic acid) diet; in 3 groups (7 rats each), the diet was supplemented with 1, 2.5 or 5% linseed oil containing 62% -linolenic acid. -Linolenic acid was incorporated up to 12% in the aorta of the 5% linseed oil group. The eicosapentaenoic acid content was not significantly increased. The content of arachidonic acid and docosatetraenoic acid was moderately reduced in rats fed 5% linseed oil. The generation of 6-keto-PGF₁ (degradation product of prostacyclin) assessed by HPLC/electrochemical detection was, however, markedly increased (p < 0.05) in rats fed 2.5 and 5% linseed oil. The minor prostanoids TXB₂, PGE₂ and PGF₂ were not significantly altered. The high systolic and diastolic blood pressure of SHR monitored by radio telemetry was more effectively reduced (p < 0.05) in the light, i.e. sleep, cycle. An increased prostacyclin formation and lowered vascular arachidonic acid content associated with enhanced dietary -linolenic acid intake would thus be expected to prove beneficial in the prevention of vascular disorders.     

Cloning of a full-length cDNA encoding bovine thymus poly(ADP-ribose) synthetase: evolutionarily conserved segments and their potential functions

The primary structure of bovine thymus poly(ADP-ribose) synthetase, as deduced from the nucleotide sequence of a cloned cDNA, indicated that this enzyme is composed of 1016 amino acids (aa) with an M_r of 113481. An abundance of Lys and Arg residues was in accord with the known basic nature of this protein. A comparison with reported sequences of human counterparts revealed: (1) three functional domains separated by partial proteolysis, i.e., DNA-binding (N-terminal), auto-modification (central), and NAD-binding (C-terminal) domains, have, in this order, increasing degrees of homology; (2) the DNA-binding domain is composed of two distinct regions: one, less conserved, containing zinc-binding fingers and the other, more conserved, containing helix-turn-helix motifs; (3) all Glu and Asp residues in the automodification domain are conserved; and (4) a 78-aa stretch encompassing the nucleotide-binding fold in the NAD-binding domain is completely conserved. These results are compatible with specific features of each domain, i.e., complex DNA-enzyme interactions, multiple automodification at acidic aa residues, and a stringent specificity for the substrate, NAD.     

The ribosomal protein gene cluster of Mycoplasma capricolum

Summary The DNA sequence of the part of the Mycoplasma capricolum genome that contains the genes for 20 ribosomal proteins and two other proteins has been determined. The organization of the gene cluster is essentially the same as that in the S10 and spc operons of Escherichia coli. The deduced amino acid sequence of each protein is also well conserved in the two bacteria. The G+C content of the M. capricolum genes is 29%, which is much lower than that of E. coli (51%). The codon usage pattern of M. capricolum is different from that of E. coli and extremely biased to use of A and U(T): about 91% of codons have A or U in the third position. UGA, which is a stop codon in the universal code, is used more abundantly than UGG to dictate tryptophan.     

The structure of glutaraldehyde in aqueous solution determined by ultraviolet absorption and light scattering.

The structure of glutaraldehyde (GA) in aqueous solutions has been the subject of much debate. Since there were fundamental problems in the experiments in the preceding studies, in this article, the structure of GA was investigated with uv absorption and light scattering to avoid those problems. It was discovered that 70% glutaraldehyde solution contains a large quantity of polymeric species with cyclic hemiacetal structure. On dilution, the polymerized glutaraldehyde slowly converted to monomers. In dilute solution, glutaraldehyde is almost monomeric at pH 3-8, the major portion taking the cyclic hemiacetal structure. The structure of GA in 20% solution is similar to that in more dilute solution. alpha, beta-Unsaturated structure does not exist in aqueous solution regardless of the concentration of glutaraldehyde.     

Structural study of phosphomannan of yeast-form cells of Candida albicans J-1012 strain with special reference to application of mild acetolysis

Structural analysis of the phosphomannan isolated from yeast-form cells of a pathogenic yeast, Candida albicans J-1012 strain, was conducted. Treatment of this phosphomannan (Fr. J) with 10 mM HCl at 100 degrees C for 60 min gave a mixture of beta-1,2-linked manno-oligosaccharides, from tetraose to biose plus mannose, and an acid-stable mannan moiety (Fr. J-a), which was then acetolyzed by means of an acetolysis medium, 100:100:1 (v/v) mixture of (CH3CO)2O, CH3COOH, and H2SO4, at 40 degrees C for 36 h in order to avoid cleavage of the beta-1,2 linkage. The resultant manno-oligosaccharide mixture was fractionated on a column of Bio-Gel P-2 to yield insufficiently resolved manno-oligosaccharide fractions higher than pentaose and lower manno-oligosaccharides ranging from tetraose to biose plus mannose. The higher manno-oligosaccharide fraction was then digested with the Arthrobacter GJM-1 alpha-mannosidase in order to cleave the enzyme-susceptible alpha-1,2 and alpha-1,3 linkages, leaving manno-oligosaccharides containing the beta-1,2 linkage at their nonreducing terminal sites, Manp beta 1----2Manp alpha 1----2Manp alpha 1----2Manp alpha 1----2Man, Manp beta 1----2Manp beta 1----2Manp alpha 1----2Manp alpha 1---- 2Manp alpha 1----2Man, and Manp beta 1----2Manp beta 1----2Manp beta 1----2Manp alpha 1---- 2Manp alpha 1----2Manp alpha 1----2Man. However, the result of acetolysis of Fr. J-a by means of a 10:10:1 (v/v) mixture of (CH3CO)2O, CH3COOH, and H2SO4 at 40 degrees C for 13 h was significantly different from that obtained by the mild acetolysis method; i.e., the amount of mannose was apparently larger than that formed by the mild acetolysis method. In summary, a chemical structure for Fr. J as a highly branched mannan containing 14 different branching moieties was proposed.     

'Protected DNA Probes' capable of strong hybridization without removal of base protecting groups

We propose a new strategy called the ‘Protected DNA Probes (PDP) method’ in which appropriately protected bases selectively bind to the complementary bases without the removal of their base protecting groups. Previously, we reported that 4-N-acetylcytosine oligonucleotides (ac⁴C) exhibited a higher hybridization affinity for ssDNA than the unmodified oligonucleotides. For the PDP strategy, we created a modified adenine base and synthesized an N-acylated deoxyadenosine mimic having 6-N-acetyl-8-aza-7-deazaadenine (ac⁶az⁸c⁷A). It was found that PDP containing ac⁴C and ac⁶az⁸c⁷A exhibited higher affinity for the complementary ssDNA than the corresponding unmodified DNA probes and showed similar base recognition ability. Moreover, it should be noted that this PDP strategy could guarantee highly efficient synthesis of DNA probes on controlled pore glass (CPG) with high purity and thereby could eliminate the time-consuming procedures for isolating DNA probes. This strategy could also avoid undesired base-mediated elimination of DNA probes from CPG under basic conditions such as concentrated ammonia solution prescribed for removal of base protecting groups in the previous standard approach. Here, several successful applications of this strategy to single nucleotide polymorphism detection are also described in detail using PDPs immobilized on glass plates and those prepared on CPG plates, suggesting its potential usefulness.     

Alteration of channel activities and gating by mutations of slow ISK potassium channel

ISK is a small membrane protein consisting of 129-130 amino acid residues with a single putative transmembrane domain and induces a very slow voltage-dependent K+ channel activity in the Xenopus oocyte expression system. We investigated the nature and structure-function relation of ISK by examining the effects of various mutations of ISK on the K+ channel activities measured in Xenopus oocytes. Deletion and truncation of the ISK protein indicated that the 63-amino acid sequence covering a transmembrane domain is sufficient for eliciting a K+ channel activity characteristic of ISK. Amino acid substitutions at a total of 31 positions within and surrounding the transmembrane domain caused different effects on the channel activity. A channel activity was enhanced by substitution of leucine with isoleucine at position 52 within the transmembrane domain, and the kinetic analysis of this mutation indicated that the enhancement of the channel activity is due to an alteration of a gating property of the ISK protein and thus supported the view that ISK forms an integral part of the K+ channel itself. The substitutions at many positions of the membrane-following region produced drastic reduction of the channel activity, and this is in marked contrast to the lack of effects of amino acid substitutions at the membrane-preceding region. Thus, the cytoplasmic portion immediately following the transmembrane domain plays a crucial role in inducing the channel activity of ISK.     

Structure of lithocholic acid binding to the N-terminal 8-kDa domain of DNA polymerase beta

The purpose of this study was to investigate the molecular action of lithocholic acid (LCA), known as a selective inhibitor of DNA polymerase beta (pol beta). The 39-kDa pol beta was separated proteolytically into two fragments of the template-primer binding domain (8 kDa) and the catalytic domain (31 kDa). LCA bound tightly to the 8-kDa fragment but not to the 31-kDa fragment. We examined the structural interaction with the 8-kDa domain using LCA. On (1)H-(15)N HMQC NMR analysis of pol beta with LCA, the 8-kDa domain bound to LCA as a 1:1 complex with a dissociation constant (K(D)) of 1.56 mM. The chemical shifts were observed only in residues mainly in helix-3, helix-4, and the 79-87 turn of the same face. No significant shifts were observed for helix-1, helix-2, and other loops of the 8-kDa domain. This region was composed mainly of three amino acid residues (Lys60, Leu77, and Thr79) of pol beta on the LCA interaction interface. The inhibition mechanism and the structure-function relationship between pol beta and LCA is discussed.     

Mode analysis of a fatty acid molecule binding to the N-terminal 8-kDa domain of DNA polymerase beta. A 1:1 complex and binding surface.

We reported previously that long-chain fatty acids are potent inhibitors of mammalian DNA polymerase beta. At present, based on information available from the NMR structure of the N-terminal 8-kDa domain, we examined the structural interaction with the 8-kDa domain using two species, C(18)-linoleic acid (LA) or C(24)-nervonic acid (NA). In the 8-kDa domain with LA or NA, the structure that forms the interaction interface included helix-1, helix-2, helix-4, the three turns (residues 1-13, 48-51, and 79-87) and residues adjacent to an Omega-type loop connecting helix-1 and helix-2 of the same face. No significant shifts were observed for any of the residues on the opposite side of the 8-kDa domain. The NA interaction interface on the amino acid residues of the 8-kDa domain fragment was mostly the same as that of LA, except that the shifted cross-peaks of Leu-11 and Thr-79 were significantly changed between LA and NA. The 8-kDa domain bound to LA or NA as a 1:1 complex with a dissociation constant (K(D)) of 1.02 or 2.64 mM, respectively.