Differential effect of total food withdrawal and dietary protein restriction on brain content of free histidine in the rat

Total withdrawal of food from young rats for 72–120 h produced an increase in brain content of free histidine which was less pronounced than the effect of prolonged dietary protein deficiency. The data suggested that the elevated brain content of histidine in both fasting and protein deficiency was due partly to increased plasma level of the amino acid but mainly to diminished plasma concentrations of the neutral amino acids known to share the same transport system across the blood-brain barrier. The results also support the idea that total starvation, and most likely, prolonged caloric restriction, like protein malnutrition, elicit increased formation of histamine in brain since the key regulatory enzyme,l-histidine carboxylyase (EC 4.1.1.22) functions at less than maximal efficiency under normal brain levels of histidine. These findings in the rat are probably relevant to the human in view of evidence that theK _m of blood-brain barrier neutral amino acid transport in the latter is low and therefore similar to the situation in the rat.     

The dimer-monomer conversion of Cerithidea myoglobin coupled with the heme iron oxidation

With regard to the distal (E7) residue, gastropod sea mollusc contains both types of myoglobin, one with and the other lacking the distal histidine. We have isolated a myoglobin from the radular muscle of Cerithidea rhizophorarum, a small whelk found on the Japanese coast. Unlike Aplysia myoglobin having a single histidine residue at position 95, Cerithidea myoglobin contains three histidines at positions 48, 66 and 98. Moreover, Cerithidea MbO₂ exists as homodimers and is oxidized, not to the usual form of metMb but to the hemichrome monomers. It was also found that the hemichrome monomers thus produced can easily be converted back to the dimerized oxy-form, if the ferric protein was reduced carefully with a slight excess of sodium hydrosulfite. This dimer-monomer conversion coupled with the heme-iron oxidation in Cerithidea myoglobin is very unique, and the distal histidine at position 66 is probably responsible for its reversible formation of hemichrome from the ferric met-form that occurred transiently in due course of the oxidation reaction of Cerithidea MbO₂     

Signal transduction systems in prokaryotes

The main components of chemosignaling systems of prokaryotes are multifunctional receptor molecules that include both sensor domains specifically recognizing external signals and effector domains converting these signals into an adequate cell response. This review summarizes and analyzes data on structural-functional organization, molecular mechanisms of action, and regulation of receptor forms of histidine kinases, adenylyl kinases, diguanylyl cyclases, and phosphodiesterases. These enzymes have been shown to be precursors of the receptor and effector components of the eukaryote hormonal signaling systems. This confirms the hypothesis developed by the authors about formation of the main archetypes of chemosignaling systems at the early evolution stages and about the evolutionary relationship of the signaling systems of prokaryotes and eukaryotes.     

Adenylylsulfate–ammonia adenylyltransferase activity is another inherent property of Fhit proteins

Fhits (fragile histidine triad proteins) occur in eukaryotes but their function is largely unknown, although human Fhit is believed to act as a tumour suppressor. Fhits also exhibit dinucleoside triphosphatase, adenylylsulfatase and nucleoside phosphoramidase activities that in each case yield nucleoside 5′-monophosphate as a product. Due to the dinucleoside triphosphatase activity, Fhits may also be involved in mRNA decapping. In the present study, we demonstrate Fhit-catalysed ammonolysis of adenosine 5′-phosphosulfate, which results in the formation of adenosine 5′-phosphoramidate. This reaction has previously been associated with adenylylsulfate–ammonia adenylyltransferase (EC 2.7.7.51). Our finding shows that the capacity to catalyse ammonolysis is another inherent property of Fhits. Basic kinetic parameters and substrate specificity of this reaction catalysed by human Fhit are presented.     

Screening of l‐histidine‐based ligands to modify monolithic supports and selectively purify the supercoiled plasmid DNA isoform

The growing demand of pharmaceutical‐grade plasmid DNA (pDNA) suitable for biotherapeutic applications fostered the development of new purification strategies. The surface plasmon resonance technique was employed for a fast binding screening of l ‐histidine and its derivatives, 1‐benzyl‐l ‐histidine and 1‐methyl‐l ‐histidine, as potential ligands for the biorecognition of three plasmids with different sizes (6.05, 8.70, and 14 kbp). The binding analysis was performed with different isoforms of each plasmid (supercoiled, open circular, and linear) separately. The results revealed that the overall affinity of plasmids to l ‐histidine and its derivatives was high (K_D > 10⁻⁸ M), and the highest affinity was found for human papillomavirus 16 E6/E7 (K_D = 1.1 × 10⁻¹⁰ M and K_D = 3.34 × 10⁻¹⁰ M for open circular and linear plasmid isoforms, respectively). l ‐Histidine and 1‐benzyl‐l ‐histidine were immobilized on monolithic matrices. Chromatographic studies of l ‐histidine and 1‐benzyl‐l ‐histidine monoliths were also performed with the aforementioned samples. In general, the supercoiled isoform had strong interactions with both supports. The separation of plasmid isoforms was achieved by decreasing the ammonium sulfate concentration in the eluent, in both supports, but a lower salt concentration was required in the 1‐benzyl‐l ‐histidine monolith because of stronger interactions promoted with pDNA. The efficiency of plasmid isoforms separation remained unchanged with flow rate variations. The binding capacity for pDNA achieved with the l ‐histidine monolith was 29‐fold higher than that obtained with conventional l ‐histidine agarose. Overall, the combination of either l ‐histidine or its derivatives with monolithic supports can be a promising strategy to purify the supercoiled isoform from different plasmids with suitable purity degree for pharmaceutical applications. Copyright © 2015 John Wiley & Sons, Ltd.     

Penta- and hexa-coordinate ferric hemoglobins display distinct pH titration profiles measured by Soret peak shifts

Hemoglobins with diverse characteristics have been identified in all kingdoms of life. Their ubiquitous presence indicates that these proteins play important roles in physiology, though function for all hemoglobins are not yet established with certainty. Their physiological role may depend on their ability to bind ligands, which in turn is dictated by their heme chemistry. However, we have an incomplete understanding of the mechanism of ligand binding for these newly discovered hemoglobins and the measurement of their kinetic parameters depend on their coordination at the heme iron. To gain insights into their functional role, it is important to categorize the new hemoglobins into either penta- or hexa-coordinated varieties. We demonstrate that simple pH titration and absorbance measurements can determine the coordination state of heme iron atom in ferric hemoglobins, thus providing unambiguous information about the classification of new globins. This method is rapid, sensitive and requires low concentration of protein. Penta- and hexa-coordinate hemoglobins displayed distinct pH titration profiles as observed in a variety of hemoglobins. The pentacoordinate distal histidine mutant proteins of hexacoordinate hemoglobins and ligand-bound hexacoordinate forms of pentacoordinate hemoglobins reverse the pH titration profiles, thus validating the sensitivity of this spectroscopic technique.     

Active Recombinant Rat Dipeptidyl Aminopeptidase I (Cathepsin C) Produced Using the Baculovirus Expression System

An active form of rat dipeptidyl aminopeptidase I (DPPI, cathepsin C) was obtained by heterologous expression in insect cells. Baculoviruses carrying a cDNA sequence encoding the entire rat DPPI precursor was used to infect High Five cells in a serum-free medium. Recombinant DPPI (rDPPI) was secreted into the medium from which it was purified by a combination of ammonium sulfate fractionation, hydrophobic interaction chromatography (HIC), and ion-exchange chromatography. A polyhistidine-tagged form of the enzyme (HT-rDPPI) was purified from the medium by immobilized metal affinity chromatography (IMAC).In vivoactivation of native rat DPPI involves at least three chain cleavages per subunit and the ability of the expression system to imitate this processing was investigated. Both rDPPI and HT-rDPPI were secreted into the medium as unprocessed and inactive proenzymes and gradually converted into their active forms in the medium. This process was not completed at the time of harvest but mature enzyme processed similarly to native rat and human DPPI could be obtained by incubating the eluates from the HIC and IMAC columns at pH 4.5 and 5°C for 18–40 h. The yield of purified and matured enzyme was approximately 50 mg/liter, and it was shown that rDPPI and HT-rDPPI were active against both a dipeptide–p-nitroanilide substrate and human growth hormone N-terminally extended with an Ala-Glu dipeptide.     

Expression,purification and activities of the entire family of intact membrane sensor kinases from Enterococcus faecalis

Two-component signal transduction systems are the main mechanism by which bacteria sense and respond to their environment, and their membrane-located histidine protein kinases generally constitute the sensory components of these systems. Relatively little is known about their fundamental mechanisms and precise nature of the molecular signals sensed, because of the technical challenges of producing sufficient quantities of these hydrophobic membrane proteins. This study evaluated the heterologous production, purification and activities of the 16 intact membrane sensor kinases of Enterococcus faecalis. Following the cloning of the genes into expression plasmid pTTQ18His, all but one kinase was expressed successfully in Escherichia coli inner membranes. Purification of the hexa-histidine ‘tagged’ recombinant proteins was achieved for 13, and all but one were verified as intact. Thirteen intact kinases possessed autophosphorylation activity with no added signal when assayed in membrane vesicles or as purified proteins. Signal testing of two functionally-characterized kinases, FsrC and VicK, was successful examplifying the potential use of in vitro activity assays of intact proteins for systematic signal identification. Intact FsrC exhibited an approximately 10-fold increase in activity in response to a two-fold molar excess of synthetic GBAP pheromone, whilst glutathione, and possibly redox potential, were identified for the first time as direct modulators of VicK activity in vitro. The impact of DTT on VicK phosphorylation resulted in increased levels of phosphorylated VicR, the downstream response regulator, thereby confirming the potential of this in vitro approach for investigations of modulator effects on the entire signal transduction process of two-component systems.     

Peptide histidine valine-42 stimulates gastric acid secretion in man

The effect of peptide histidine valine-42 (PHV-42) on gastric acid secretion was studied in man. PHV-42 was infused into 5 healthy volunteers at a dose of 10 pmol/kg/min. This dose caused a significant stimulation of basal gastric acid and potassium output. there were no significant changes in circulating gastrin throughout the infusion. In 2 subjects with a background of submaximal pentagastrin stimulation, PHV-42 infusion at the same dose did not alter acid secretion in either subject. The previous observation that PHV-42 is found particularly in the stomach and the new finding that it stimulates basal gastric secretion suggest the possibility that PHV-42 could have a role in local control of acid secretion.