Lena Hammar

Chromatographic purification of a mammalian histidine decarboxylase on charged and non-charged alkyl derivatives of agarose.

Histidine decarboxylase (EC 4.1.1.22) from a mouse mastocytoma has been purified by chromatography on charged and non-charged n-alkyl derivatives of agarose. The former was represented by the coupling product of CNBr-activated agarose and alkylmonoamines (alkylamino-agarose), the latter by the coupling of agarose and alkylglycidyl ehters (alkyl agarose). The choice of fractionation medium was restricted by the enzyme stability; excessively high ionic strength media could not be used. Under the conditions investigated, the best result was obtained with the non-charged ocytl agarose. The enzyme was adsorbed to this gel at a relatively high ionic strength, and on stepwise decrease in ionic stength of the eluting buffer it was desorbed with a total recovery of 80%. There was an approx. 10-fold increase in specific activity. The histidine decarboxylase, thus purified, retained 90-100% of its activity for 10 days or more at 6-8 degrees C. Some general comments on protein fractionation on charged and non-charged alkyl derivatives of agarose are given. The complexity of protein interaction with the charged alkyl derivatives is illustrated by experiments with a colored protein, phycoerythrin.

Purification and immunochemical analysis of histidine decarboxylase from murine mastocytoma

Application of a new scheme of purification for histidine decarboxylase (E.C. 4.1.1.22) leads to a highly purified enzyme (5000 nmol/mg·h, i.p. 5.0, M.W.: 110 kD) with reasonable stability (rest activity 80% after 3 weeks at 6–8°C). A major protein contaminant seen on electrofocusing (i.p. 4.6) shows immunological identity with the enzyme-containing protein (i.p. 5.0) and might be involved in the aggregation of the enzyme.

Purification of simian immunodeficiency virus, SIVMAC251, and of its external envelope glycoprotein, gp148☆

Two-phase extraction in a system composed of dextran and polyethylene glycol was used to purify simian immunodeficiency virus, SIVMAC251 (32H isolate) from 25 l of culture supernatant. The virus partitioned to the interphase with 80% recovery of gag peptide p27 and reverse transcriptase and an about 25% recovery of the external env glycoprotein, gp148. The virus was treated with octylglycoside and its subcomponents separated. Two gag-p27 containing fractions were obtained; gag-1, which also contained reverse transcriptase and nucleopeptides, and gag-2, which contained the major portion of the p27. The env gp148 was purified by chromatography through a series of lectin columns. The prepared materials are characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immuno- and lectin blotting.

Peptide inhibition of mammalian histidine decarboxylase

The hypothesis thatN-terminal histidine peptides might act as inhibitors to histidine decarboxylase was investigated. A murine mastocytoma was utilized as enzyme source. the crude extract of this tissue exhibits high rates of decarboxylation of both histidine and DOPA and was used to establish the specificity in the effect of the compounds tested. For kinetic analyses a highly purified histidine decarboxylase fraction was used. The effect of some representative peptides on both enzyme activities were recorded.Histidine decarboxylase exclusively was inhibited byN-terminal histidine peptides. None of the other peptides investigated interfered negatively with this enzyme. This inhibition was consistent in the purified preparation and appeared to be more pronounced with increasing hydrophobicity in the second amino acid. Histidyl-phenylalanine was found to be about 100-fold as potent as the commonly used specific histidine decarboxylase inhibitor α-methyl histidine.It is concluded that small peptides with histidine as theN-terminal amino acid might act as specific inhibitors for mammalian histidine decarboxylase. An analog effect of small tyrosyl or phenylalanyl peptides was not seen for the DOPA decarboxylase.

Mammalian histidine decarboxylase

SummaryHistidine decarboxylase (EC 4.1.1.22) prepared from a murine mastocytoma is activated up to six-fold when the concentration of phosphate in the assay medium is increased from 1 mM to 150 mM. Chloride and sulfate, on the other hand, are inhibitory and appear to interfere with the binding of pyridoxal phosphate to the enzyme. The inhibition by chloride is relatively less pronounced at high than at low concentrations of phosphate.The enzyme is inhibited by heavy metal ions and to some extent by alkylation and oxidation, but also by strong reduction.The histidine decarboxylase activity is stabilized by 150 mM potassium phosphate, 1 mM dithiothreitol and 10 μM pyridoxal phosphate when stored at 6–8 °C. This holds true for both crude extract enzyme and enzyme purified by molecular sieving and hydrophobic interaction chromatography.ZusammenfassungHistidin-Decarboxylase (EC 4.1.1.22), isoliert aus einem Mäuse-Mastocytom, zeigt eine bis zu sechsfach erhöhte Aktivität, wenn die Konzentration von Phosphat im Testmedium von 1 mM auf 150 mM gesteigert wird. Chlorid und Sulfat dagegen inhibieren und scheinen die Bindung von Pyridoxalphosphat an das Enzym zu verhindern. Die Inhibition von Chlorid ist bei hohen Phosphatkonzentrationen relativ geringer als bei niedrigen.Das Enzym wird außerdem durch Schwermetallionen sowie in geringem Maße durch Alkylierung und Oxidation, aber auch durch starke Reduktion, inhibiert.Die Histidin-Decarboxylase-Aktivität wird durch 150 mM Kaliumphosphat, 1 mM Dithiothreitol und 10 μM Pyridoxalphosphat bei 6–8°C stabilisiert. Dieses gilt sowohl für das ungereinigte als auch für das durch Gelfiltration und »Hydrophobic interaction«-Chromatographie gereinigte Enzym.

Characterization of glycoproteins from Chlamydia trachomatis using lectins

Glycoproteins in Chlamydia trachomatis, serotype L1, elementary bodies were studied by lectin blotting. A panel of 23 lectins representing a variety of sugar specificities was used. The pattern of lectin‐binding specificities at a peptide band was studied in order to determine the type and structure of its glycoconjugate. To establish chlamydial origin of the glycopeptide bands in the blot, control samples from non‐infected host cell membranes were run in parallel. Terminal mannosidic structures were demonstrated in a 72 kDa glycopeptide (gp72) by its selective binding of Galanthus nivalis lectin (GNA). Sialic acids were found in two chlamydial glycopeptides, gp40 and gp64, which appear to carry O‐linked glycoconjugates as they bound the peanut agglutinin (PNA, both gp40 and gp64) and jackfruit lectin (Jac, only gp40). Such structures were also present in other chlamydial glycopeptides. Lectins with specificities for fucose in different links, galactose and N‐acetyl glucosamine bound to several chlamydial peptides. On the basis of our results we suggest an alternative mechanism for uptake of chlamydial elementary bodies into host cells, namely phagocytosis mediated by eukaryotic cell surface lectins.

Mammalian histidine decarboxylase; Changes in molecular properties induced by oxidation and reduction

Histidine decarboxylase from a murine mastocytoma has been submitted to different separation methods. In these experiments the activity peaks were often very broad. This heterogeneity of the enzyme is traced back to the formation of aggregates, differing in apparent molecular weight by a multiple of about 55,000, as a result of oxidation.Under non-oxidative conditions the histidine decarboxylase activity is confined to one peak in both molecular sieve chromatography, hydrophobic interaction chromatography, chromatography on hydroxy apatite, pore gradient electrophoresis and electrofocusing.The molecular weight of the enzyme is estimated to be 110,000 by pore gradient electrophoresis (alkylated enzyme). The isoelectric point is pH 4.9–5.0, determined by electrofocusing under reducing conditions.

Mammalian histidine decarboxylase: Effect by protein kinase on mouse mastocytoma histidine decarboxylase

Mouse mastocytoma histidine decarboxylase is decreased in activity when, in crude preparations, incubated with a cAMP-dependent protein kinase. This effect was not seen with purified preparations of the histidine decarboxylase (specific activity 7–13 μmol·mg−1·h−1). Further, no incorporation of32P from AT32P during incubation of this enzyme with the protein kinase could be demonstrated. Therefore, if protein phosphorylation operates in the regulation of the histidine decarboxylase, factors removed during the purification must be involved.

Effect of His-Phe, a competitive inhibitor of histidine decarboxylase, on gastric acid secretion in chronic gastric fistula rats: Delay in acid secretion response to pentagastrin

The dipeptide His-Phe, earlier shown to inhibit mammalian histidine decarboxylase, was analysed concerning its effectin vivo on pentagastrin-induced gastric acid secretion. Chronic gastric fistula rats were used and the effectors in saline were given as continuous i.v. infusions while acid was collected from the fistula. Addition of petagastrin to the infusion solution resulted in an immediate increase in the acid output of the control runs. In the His-Phe experiments the dipeptide was introduced one hour before pentagastrin. A significant decrease in the acid output was obtained. This effect was optimal at a dose of about 6 mg/h and during the first few hours of the experiments. In spite of the continuous His-Phe infusion the acid secretion increased with time to the control values.These results are discussed in relation to preliminary observations on effects of α-fluoromethyl histidine on gastric acid secretion and the effect of this and His-Phe on gastric histamine content and histidine decarboxylase activity.

Binding of Galanthus nivalis lectin to Chlamydia trachomatis and inhibition of in vitro infection

A glycoprotein present in Chlamydia trachomatis, serotype L1, elementary bodies (EBs) was earlier found to bind the lectin from Galanthus nivalis (GNA). In the present paper we investigate the interaction of GNA with chlamydial EBs and its effect on in vitro infectivity. The binding affinity was studied with 125I‐GNA lectin. Within 15 min about 80% maximal binding was obtained. The chlamydia‐GNA interaction was inhibited by α‐methylmannoside, causing a decrease of about 50% at 1 mM. Curve fit analyses indicated two types of binding sites for GNA on the EBs. The affinity to these differed by a factor of 15. The influence of the lectin on the ability of C. trachomatis to infect McCoy cells was also investigated. There was a GNA‐dependent inhibition with a 50% reduction in the number of intracellular inclusions at 0.2 μuM of the lectin. The findings indicate the presence of terminal mannose structures on the chlamydial surface at or in the proximity of the cell‐binding domains. Mannose‐binding proteins of eukaryotic cells could be important for the initial uptake of EBs.