Copper binding to plant ozone-inducible proteins (OI2-2 and OI14-3)

Ozone-inducible proteins (OI2-2 and OI14-3) from Atriplex canescens whose structure and function are unknown are rich in glycine intercepted with histidine and tyrosine with putative signal peptides at the N-terminus. OI2-2 and OI14-3 contain 8 and 10 tandem repeats of YGHGGG, respectively. In order to study whether these proteins bind Cu(2+), circular dichroism (CD), and nuclear magnetic resonance (NMR) were measured for four synthetic peptides corresponding to sections of the sequences of these proteins; 1 (HGGGY), 2 (HGGGYGH), 3 (YGHGGGY), and 4 (YGHGGGYGHGGGY), where all peptides were chemically blocked with an acetyl group at the N-terminus and an -NH(2) group at the C-terminus. Visible CD spectra of the four peptides show positive peaks near 580 and 340nm, which were observed at pH 7.4 but not pH 6.0, indicating clearly that the four peptides bind Cu(2+). The NMR spectra indicate that the addition of small amounts of CuSO(4) to 3 (Y1-G2-H3-G4-G5-G6-Y7) causes significant broadening of resonances of the side chain protons (C(beta)H, C(epsilon1)H, and C(delta2)H) of His3 and the side chain C(beta)H of Tyr1 at pH 7.4. In addition, the backbone C(alpha)H resonances of Gly2 and Gly4 were broadened more strongly than those of Gly5 and Gly6. CD titration experiment suggested that two repeats of YGHGGG comprise the fundamental Cu(2+) binding unit. Thus, the ozone-inducible proteins are capable of binding at least four or five copper ions per protein. These copper-binding proteins would function as active oxygen scavengers.     

Anti-crosslinking properties of carnosine: significance of histidine

Carnosine, a histidine-containing dipeptide, is a potential treatment for Alzheimer's disease. There is evidence that carnosine prevents oxidation and glycation, both of which contribute to the crosslinking of proteins; and protein crosslinking promotes beta-amyloid plaque formation. It was previously shown that carnosine has anti-crosslinking activity, but it is not known which of the chemical constituents are responsible. We tested the individual amino acids in carnosine (beta-alanine, histidine) as well as modified forms of histidine (alpha-acetyl-histidine, 1-methyl-histidine) and methylated carnosine (anserine) using glycation-induced crosslinking of cytosolic aspartate aminotransferase as our model. beta-Alanine showed anti-crosslinking activity but less than that of carnosine, suggesting that the beta-amino group is required in preventing protein crosslinking. Interestingly, histidine, which has both alpha-amino and imidazolium groups, was more effective than carnosine. Acetylation of histidine's alpha-amino group or methylation of its imidazolium group abolished anti-crosslinking activity. Furthermore, methylation of carnosine's imidazolium group decreased its anti-crosslinking activity. The results suggest that histidine is the representative structure for an anti-crosslinking agent, containing the necessary functional groups for optimal protection against crosslinking agents. We propose that the imidazolium group of histidine or carnosine may stabilize adducts formed at the primary amino group.     

Activation of carbonic anhydrase II by active-site incorporation of histidine analogs

The hydration of CO2 catalyzed by human carbonic anhydrase II (HCA II) is accompanied by proton transfer from the zinc-bound water of the enzyme to solution. We have replaced the proton shuttling residue His 64 with Ala and placed cysteine residues within the active-site cavity by mutating sites Trp 5, Asn 62, Ile 91, and Phe 131. These mutants were modified at the single inserted cysteine with imidazole analogs to introduce new potential shuttle groups. Catalysis by these modified mutants was determined by stopped-flow and 18O-exchange methods. Specificity in proton transfer was demonstrated; only modifications of the Cys 131-containing mutant showed enhancement in the proton transfer step of catalysis compared with unmodified Cys 131-containing mutant. Modifications at other sites resulted in up to 3-fold enhancement in rates of CO2 hydration, with apparent second-order rate constants near 350 microM(-1) s(-1). These are among the largest values of kcat/Km observed for a carbonic anhydrase.     

Lipopolysaccharide and Interleukin-1β Augmented Histidine Decarboxylase Activity in Cultured Cells of the Rat Embryonic Brain

Abstract: We investigated the effect of lipopolysaccharide (LPS) and various inflammatory cytokines on the histidine decarboxylase (HDC) activity in cultured cells of the rat embryonic brain. Histaminergic neuronal cell bodies were supposed to exist in cultured cells of the diencephalon but not in those of the cortex. The HDC activity was elevated by adding LPS and interleukin-1 β (IL-1β) but not by tumor necrosis factor-α (TNF-α) and IL-6 to the mixed primary cultures of diencephalon. In the adherent cell fraction of the cultured diencephalon cells, HDC activity was also enhanced by LPS and IL-1β. In a similar manner, LPS augmented HDC activity in the mixed primary culture of cerebral cortical cells and in its adherent cell fraction. The effects of IL-1β but not LPS in the mixed primary culture of diencephalon were canceled by a prior exposure to cytosine-β- d -arabinofuranoside. The changes in HDC activity after exposure to LPS for 12 h were not accompanied by increased mRNA levels. In these cell cultures, mast cells were not detected by Alcian Blue staining. These results indicated the presence of the third type of HDC-bearing cell besides neurons and mast cells in the brain. The increase of HDC activity by IL-1β might be due to cell proliferation.     

Hemoglobin crystals immersed in liquid oxygen reveal diffusion channels

Human hemoglobin (HbA) transports molecular oxygen (O₂) from the lung to tissues where the partial pressure of O₂ is lower. O₂ binds to HbA at the heme cofactor and is stabilized by a distal histidine (HisE7). HisE7 has been observed to occupy opened and closed conformations, and is postulated to act as a gate controlling the binding/release of O₂. However, it has been suggested that HbA also contains intraprotein oxygen channels for entrances/exits far from the heme. In this study, we developed a novel method of crystal immersion in liquid oxygen prior to X-ray data collection. In the crystals immersed in liquid oxygen, the heme center was oxidized to generate aquomethemoglobin. Increases of structural flexibility were also observed in regions that are synonymous with previously postulated oxygen channels. These regions also correspond to medically relevant mutations which affect O₂ affinity. The way HbA utilizes these O₂ channels could have a profound impact on understanding the relationship of HbA O₂ transport within these disease conditions. Finally, the liquid oxygen immersion technique can be utilized as a new tool to crystallographically examine proteins and protein complexes which utilize O₂ for enzyme catalysis or transport.     

Conservation of primary structure in the hisI gene of the archaebacterium, Methanococcus vannielii, the eubacterium Escherichia coli, and the eucaryote Saccharomyces cerevisiae

Summary A 2.7 kilobase pair (Kb) fragment of DNA, which complements mutations in the hisI locus of Escherichia coli, has been cloned and sequenced from the genome of the methanogenic archaebacterium Methanococcus vannielii. The cloned DNA directs the synthesis of three polypeptides, with molecular weights of 71,000, 29,000 and 15,600 in minicells of E. coli. Subcloning and mutagenesis demonstrates that hisI complementation results from the activity of the 15,600 molecular weight polypeptide. The primary structure of this archaebacterial gene and its gene product have been compared with the functionally equivalent gene and protein from the eubacterium E. coli (hisI) (Chiariotti et al. 1986) and from the eucaryote Saccharomyces cerevisiae (his4A) (Donahue et al. 1982). The DNA sequences of the archaebacterial and eubacterial genes are 40% homologous, the archaebacterial and eucaryotic DNA sequences are 47% homologous and, as previously reported (Bruni et al. 1986) the eubacterial and eucaryotic DNA sequences are 45% homologous. In E. coli the hisI locus is part of a bifunctional gene (hisI/E) within the single his operon. In S. cerevisiae the his4A locus is part of a multifunctional gene (his4) which encodes a protein with at least four enzymatic activities. The his genes of S. cerevisiae do not form an operon and are not physically linked. The M. vannielii hisI gene does not appear to be part of a multifunctional DNA sequence and, although it does appear to be within an operon, the open reading frames (ORFs) 5 and 3 to the M. vannielii hisI gene are not related to any published his sequences. The hisI and hisA genes (Cue et al. 1985) of M. vannielii are not closely linked in its genome.     

Properties and Ontogenic Development of Membrane-Bound Histidine Decarboxylase from Rat Brain

Abstract: Histidine decarboxylase (HD) activity was determined in high-speed fractions (100,000 g for 60 min) obtained from whole rat brain homogenates. Twenty-eight percent of the HD activity was associated with membranes, and the remaining was soluble. Several properties of the soluble and membrane-bound HD were compared. No significant differences in the values of K _m for histidine and pyridoxal 5'-phosphate were observed. The solubilization of membrane-bound HD with Triton X-100 resulted in an increase of 60% over the nonsolubilized activity with no changes in the K _m for substrate and cofactor. The proportion of free pyridoxal 5'-phosphate-independent activity was identical in both fractions. The soluble and membrane-bound forms of the enzyme differ slightly in their pH-activity profiles, although both enzymes showed an optimum pH near 6.5. The HD activities present in soluble and membrane fractions were determined at different postnatal ages. The soluble activity increased until day 90, whereas the membrane-bound activity became stabilized from day 20.     

Histidine decarboxylase from rat and rabbit brain: Partial purification and characterization

Histidine decarboxylase, the synthetic enzyme for histamine, was partially purified from regions of rat or rabbit brain rich in the enzyme. The enzyme was purified using ion exchange and hydrophobic column chromatography and chromatofocusing. Approximately 70-fold and 110-fold enrichments were attained from rat and rabbit brain, respectively. Rat and rabbit brain histidine decarboxylase had isoelectric points of pH 5.4 and 5.6, Km values of 80 M and 120 M histidine and Vmax values of 210 and 625 pmol histamine formed/hr-mg protein, respectively. The partially purified histidine decarboxylase from both sources was dependent on pyridoxal phosphate for maximal activity and was inhibited by -fluoromethylhistidine, nickel chloride and cobaltous chloride but was not inhibited by impromidine, -methyldopa, DTNB, zinc chloride or mercuric chloride. The enzyme had a broad pH optimum between pH 7.2 and 8.0. These studies provide further information on the characteristics of mammalian histidine decarboxylase from brain.     

Identification of histidyl and cysteinyl residues essential for catalysis by 5'-nucleotidase

Inactivation of both cytosolic 5'-nucleotidase and ecto-5'-nucleotidase by diethylpyrocarbonate indicated the presence of an essential histidyl residue which in the cytosolic enzyme was conclusively located at the active site. Inactivation by thiol reagents indicated the presence of an essential cysteinyl residue in both enzymes. The data suggest that both 5'-nucleotidases belong to a group of histidine phosphatases which also includes glucose-6-phosphatase and acid phosphatase. A working hypothesis for the catalytic mechanism of these enzymes is proposed.