Effect of amino acid at the beta 6 position on surface hydrophobicity, stability, solubility, and the kinetics of polymerization of hemoglobin. Comparisons among Hb A (Glu beta 6), Hb C (Lys beta 6), Hb Machida (Gln beta 6), and Hb S (Val beta 6)

Surface hydrophobicity, stability, solubility, and kinetics of polymerization were studied using hemoglobins with four different amino acids at the beta 6 position: Hb A (Glu beta 6), Hb C (Lys beta 6), Hb Machida (Gln beta 6), and Hb S (Val beta 6). The surface hydrophobicity increased in the order of Hb C, Hb A, Hb Machida, and Hb S, coinciding with the hydrophobicity of the amino acid at the beta 6 position. Solubility of the oxy-form of these hemoglobins decreased in relation to increases in their surface hydrophobicity, suggesting that the solubility is controlled by the strength of hydrophobicity of the amino acid at the beta 6 position. The solubility of the oxy-form of these hemoglobins is always higher than that of the deoxy-form. There is a similar linear relationship between the solubility and surface hydrophobicity among deoxyhemoglobins A, C, and Machida. However, the solubility of deoxy-Hb S deviated significantly from the expected value, indicating that the extremely low solubility of deoxy-Hb S is not directly related to the hydrophobicity of the beta 6 valine. Kinetic studies on the polymerization of deoxy-Hb Machida revealed a distinct delay time prior to polymerization. This confirms our previous hypothesis that beta 6 valine is not responsible for the delay time prior to gelation. The kinetics of the polymerization of 1:1 mixtures of sickle and non-sickle hemoglobins were similar to those of pure Hb S, suggesting that only one of the two beta 6 valines is involved in an intermolecular contact. In mixtures of equal amounts of Hb S and Hb A, Hb C, or Hb Machida, half of the asymmetrical AS, SC, and S-Machida hybrid hemoglobins behaved like Hb S during nucleation, while the other half behaved like the non-sickle hemoglobin.     

Identification of membrane anchoring site of human renal dipeptidase and construction and expression of a cDNA for its secretory form

The chemical properties of human renal dipeptidase (hrDP) purified from the membrane fraction of kidney have been characterized. When treated with phosphatidylinositol-specific phospholipase C, hrDP was released from renal membrane fractions. After digestion with trypsin, carboxyl-terminal peptide was isolated employing anhydrotrypsin-agarose column chromatography and reversed-phase high performance liquid chromatography. The amino acid sequence of the peptide was identified at positions 363-369 in the primary structure deduced from the cDNA sequence (Adachi, H., Tawaragi, Y., Inuzuka, C., Kubota, I., Tsujimoto, M., Nishihara, T., And Nakazato, H. (1990) J. Biol. Chem. 265, 3992-3995). Further examination of the chemical composion of the peptide showed that it contained, respectively, 2, 1, 5, 1, and 1 mol of ethanolamine, glucosamine, mannose, inositol, and phosphate in addition to amino acids. These results suggest that the mature hrDP molecule lacks the carboxyl-terminal hydrophobic peptide extension predicted from the cDNA sequence and is anchored at Ser369 via glycosylphosphatidylinositol to the membrane. To characterize further the action of the enzyme, we have established expression systems for both secretory and membrane anchored forms of hrDP using COS-1 cells and found that both recombinant forms were as active as natural enzyme. Our expression system made it possible to prepare large amounts of soluble enzyme, and will contribute toward elucidation of the physiological roles of the enzyme.     

Intact carboxysomes in a cyanobacterial cell visualized by hilbert differential contrast transmission electron microscopy

Carboxysomes in rapidly frozen ice-embedded whole cells of the cyanobacterium Synechococcus sp. strain PCC 7942 were visualized by the recently developed Hilbert differential contrast transmission electron microscope. Structural details of carboxysomes were especially clearly visualized in the ruptured cells. The novel electron microscopy exhibited the paracrystalline arrays of molecules of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase in the carboxysomes in much better contrast than conventional transmission electron microscopy with ultrathin sections of cells. The carboxysome was surrounded by a 5- to 6-nm-thick monolayer shell which consisted of orderly arrays of globular particles.     

Purification of nitric oxide synthase from bovine brain: immunological characterization and tissue distribution.

Nitric oxide (NO) synthase (EC 1.14.23) was purified to homogeneity from bovine cerebrum. The molecular weight of NO synthase was estimated to be 150 kDa by both SDS/PAGE and gel filtration at high salt concentration. For activity, the enzyme required NADPH, Ca2+, calmodulin and tetrahydrobiopterin as cofactors. Rabbit polyclonal antibody to bovine brain NO synthase reacted with 150 kDa NO synthase in various bovine and rat organs, including the brain, pituitary and adrenal glands, but not with that in stimulated macrophages, indicating that there are at least two immunologically distinct NO synthases.     

Active-site residues of the transpeptidase domain of penicillin-binding protein 2 from Escherichia coli: similarity in catalytic mechanism to class A beta-lactamases.

By means of amino acid sequence alignment with class A beta-lactamases, the residues essential for the catalytic activity of the peptidoglycan transpeptidase of penicillin-binding protein 2 (PBP2) have been predicted to be Lys333, Asp447, and Lys544, in addition to the acylation site residue for the acyl-enzyme mechanism, Ser330. Accordingly, these residues were replaced by site-directed mutagenesis, and the resultant mutants were examined as to penicillin-binding activity and genetic complementation, which represent only the acylation step and the total reaction during transpeptidation, respectively. All the mutants at position 333 showed the complete loss of both the binding and complementation activities. Most of the mutants at position 447 retained the binding activity but lost the complementation activity, the exception being the D447E mutant, which retained both. The binding rates for various penicillins of the D447N mutant, which had lost the complementation activity, were almost identical to those of the wild type. The binding of the mutants at position 544 tended to require a higher penicillin concentration, and that of the K544H mutant required a lower pH. When the roles of the counterpart residues, Lys73, Glu166, and Lys234, in class A beta-lactamases were considered, the results suggested that Lys333 and Asp447 are essential for the acylation and acyl-transfer steps, respectively, and that Lys544 stabilizes the Michaelis complex through its side-chain positive charge.     

Wild‐type and specific mutant androgen receptor mediates transcription via 17β‐estradiol in sex hormone‐sensitive cancer cells

We previously encountered regulatory processes wherein dihydrotestosterone (DHT) exerted its inhibitory effect on parathyroid hormone‐related protein (PTHrP) gene repression through the estrogen receptor (ER)α, but not the androgen receptor (AR), in breast cancer MCF‐7 cells. Here, we investigated whether such aberrant ligand‐nuclear receptor (NR) interaction is present in prostate cancer LNCaP cells. First, we confirmed that LNCaP cells expressed large amounts of AR at negligible levels of ERα/β or progesterone receptor. Both suppression of PTHrP and activation of prostate‐specific antigen genes were observed after independent administration of 17β‐estradiol (E2), DHT, or R5020. Consistent with the notion that the LNCaP AR lost its ligand specificity due to a mutation (Thr‐Ala877), experiments with siRNA targeting the respective NR revealed that the AR monopolized the role of the mediator of shared hormone‐dependent regulation, which was invariably associated with nuclear translocation of this mutant AR. Microarray analysis of gene regulation by DHT, E2, or R5020 disclosed that more than half of the genes downstream of the AR (Thr‐Ala877) overlapped in the LNCaP cells. Of particular interest, we realized that the AR (wild‐type [wt]) and AR (Thr‐Ala877) were equally responsible for the E2‐AR interactions. Fluorescence microscopy experiments demonstrated that both EGFP‐AR (wt) and EGFP‐AR (Thr‐Ala877) were exclusively localized within the nucleus after E2 or DHT treatment. Furthermore, reporter assays revealed that some other cancer cells exhibited aberrant E2‐AR (wt) signaling similar to that in the LNCaP cells. We herein postulate the presence of entangled interactions between wt AR and E2 in certain hormone‐sensitive cancer cells. J. Cell. Physiol. 230: 1594–1606, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Identification of a docking groove on ERK and p38 MAP kinases that regulates the specificity of docking interactions

MAP kinases (MAPKs) form a complex with MAPK kinases (MAPKKs), MAPK-specific phosphatases (MKPs) and various targets including MAPKAPKs. These docking interactions contribute to regulation of the specificity and efficiency of the enzymatic reactions. We have previously identified a docking site on MAPKs, termed the CD (common docking) domain, which is utilized commonly for docking interactions with MAPKKs, MKPs and MAPKAPKs. However, the CD domain alone does not determine the docking specificity. Here we have identified a novel site on p38 and ERK2 MAPKs that regulates the docking specificity towards MAPKAPKs. Remarkably, exchange of two amino acids in this site of ERK2 for corresponding residues of p38 converted the docking specificity for MAPKAPK-3/3pk, which is a dominant target of p38, from the ERK2 type to the p38 type, and vice versa. Furthermore, our detailed analyses with a number of MAPKAPKs and MKPs suggest that a groove in the steric structure of MAPKs, which comprises the CD domain and the site identified here, serves as a common docking region for various MAPK-interacting molecules.     

Cerebrospinal Fluid Nitrite/Nitrate Levels in Neurologic Diseases

Abstract: Nitric oxide has been proposed to mediate cytotoxic effects in inflammatory diseases. To investigate the possibility that overproduction of nitric oxide might play a role in the neuropathology of inflammatory and noninflammatory neurological diseases, we compared levels of the markers of nitric oxide, nitrite plus nitrate, in the CSF of controls with those in patients with various neurologic diseases, including Huntington's and Alzheimer's disease, amyotrophic lateral sclerosis, and HIV infection. We found that there were no significant increases in the CSF levels of these nitric oxide metabolites, even in patients infected with HIV or in monkeys infected with poliovirus, both of which have significantly elevated levels of the neurotoxin quinolinic acid and the marker of macrophage activation, neopterin. However, CSF quinolinic acid, neopterin, and nitrite/nitrate levels were significantly increased in a small group of patients with bacterial and viral meningitis.