Purification and detection of multiple forms of histidine decarboxylase in rat gastric mucosa (author's transl)]

A specific histidine decarboxylase from rat gastric mucosa has been obtained at high purity and good yield (purification about 600-fold). The purification procedure included double (NH4)2SO4 fractionation, ion-exchange chromatography, preparative isoelectric focusing in a granulated gel and gel filtration. Only the specific histidine enzyme was obtained by that procedure; DOPA decarboxylase, a non-specific enzyme, was absent in our final preparation. Each step of the purification was visualized by polyacrylamide gel electrophoresis and analytical isoelectric focusing. The purified enzyme was apparently homogenous by criteria of electrophoresis and gel filtration and has a molecular weight of 94 000. Several protein bands appeared after isoelectric focusing and the enzyme activity was localized in 3 distinct peaks. The gastric enzyme consists of 3 active forms which could be distinguished by their isoelectric points: 5.4, 5.75 and 6. Moleculare weights estimated by SDS polyacrylamide gel electrophoresis were 97 000, 93 000 and 90 000, and no subunits were observed. Pyridoxal phosphate was required as a coenzyme and resolution of the holoenzyme agreed with a portion of the coenzyme tightly bound to the apoenzyme. The purified enzyme was stable at low ionic strength, near neutral pH; concentrated reducing agents inhibit the enzyme.     

Histidine decarboxylase from rat gastric mucosa: heterogeneity and enzyme forms modification

Rat gastric mucosal histidine decarboxylase is shown to exist in the crude extract as three active charged forms which are separable by isoelectric focusing. The distribution of enzyme activity in the three forms is independent of the homogenizing medium and of the isoelectric focusing procedure indicating that the heterogeneity does not arise during isolation. Multiple forms correspond to histidine decarboxylase and are related neither to 3,4-dihydroxyphenylalanine decarboxylase nor to the result of aggregation. This charge difference between the enzyme forms changes according to the time of storage and to the temperature, leading to the generation of less negatively charged species. The conversion cannot be attributed to proteolytic degradation nor to differences in stability between forms. The data indicate that these alternative charged states may really result from an in vivo post-translational modification of the enzyme.     

Relationship between the multiple forms of rat gastric histidine decarboxylase: effects of conditions favouring phosphorylation and dephosphorylation

Conditions favouring protein phosphorylation and dephosphorylation are examined for their effects on activity and charge heterogeneity of the rat gastric mucosal histidine decarboxylase. Incubation of gastric supernatant with various combinations of ATP, Mg2+, cyclic AMP and protein kinase under the blockade of endogenous phosphodiesterase and phosphatase fails to alter significantly enzyme activity as assayed with or without pyridoxal 5'-phosphate. Similar results are found with the purified enzyme. No change occurs in the distribution of activity between the charged forms. In contrast, treatment with alkaline phosphatase both inactivates the enzyme with preservation of heterogeneity, full reactivation being achieved by pyridoxal 5'-phosphate, and reduces the number of forms and converts forms II and III to form I with preservation of the catalytic potentialities. The data suggest that the enzyme heterogeneity may be related in part to the phosphorylation state; the possibility that the gastric enzyme is susceptible to several post-translational modifications is discussed.     

Inactivation of rat gastric mucosal histidine decarboxylase by phosphatase

Histidine decarboxylase of supernatants as well as of purified preparations from rat gastric mucosa is inactivated by a non-specific phosphatase in the absence of pyridoxal 5'-phosphate. The inactivation is a time and concentration-dependent process. Pyridoxal 5'-phosphate, but not histidine, protects the enzyme against phosphatase action. The inactivation is reversible, only pyridoxal 5'-phosphate reactivates the inactivated enzyme. Pyridoxamine 5'-phosphate is ineffective for histidine decarboxylase, but is converted into an active coenzyme only in gastric supernatant. Evidence for the occurrence of an active phosphatase in gastric tissue is also presented; its properties are those of an acid phosphatase and are similar to those of phosphatases hydrolyzing pyridoxal 5'-phosphate in other tissues. The data indicate that phosphatase promotes apoenzyme formation and may play a role in the regulation of histamine synthesis.     

Detection of multiple forms of rat liver ornithine decarboxylase.

Rat liver ornithine decarboxylase induced by injection of thioacetamide has been separated into at least two fractions by covalent chromatography on an activated thiol-Sepharose 4B column. The two major fractions could be distinguished by ion exchange chromatography and electrophoresis on acrylamide gels. In addition, the two forms displayed different Km values for ornithine. Although the two forms are separable, they display identical antigenic properties, pH optima, and they appear to be the same molecular size. The biological significance or the relationship between multiple forms of ornithine decarboxylase is not understood.     

Effects of dexamethasone on the activity of histidine decarboxylase, ornithine decarboxylase, and dopa decarboxylase in rat oxyntic mucosa

Since accelerated turnover of histamine in oxyntic mucosa may be an important factor in the pathogenesis of peptic ulcers, the effect of dexamethasone and other glucocorticoids on the activity of gastric histidine decarboxylase (HDC) was studied in the rat. The activity of HDC in rat oxyntic mucosa increased significantly after dexamethasone was injected s.c. to rats at doses larger than 0.4 mg/kg body weight. The maximum response of the HDC activity to dexamethasone (4 mg/kg) was observed 8 h after the treatment. The activity of ornithine decarboxylase (ODC) increased at 4 h, while that of DOPA decarboxylase showed no significant change throughout the 16-h period following a single injection of dexamethasone. The mucosal levels of histamine, putrescine, and spermidine rose significantly after the steroid treatment, while the spermine levels remained nearly constant. There was no sex difference in these responses to dexamethasone. Betamethasone showed nearly the same effects as dexamethasone on the decarboxylase activities and the mucosal levels of diamines. Serum gastrin levels showed no significant change for the first 4 h and then rose significantly 8 and 16 h after dexamethasone treatment. Pentagastrin (0.5 mg/kg) increased the HDC activity, while it showed no significant effect on either the mucosal ODC activity or levels of polyamines and histamine. These data suggest that dexamethasone influences the metabolism of histamine and polyamines in rat oxyntic mucosa both directly and via stimulation of gastrin release.     

Isolation and characterization of multiple forms of rat liver UDP-glucuronate glucuronosyltransferase.

UDP-glucuronosyltransferase (EC 2.4.1.17) activity was solubilized from male Wistar rat liver microsomal fraction in Emulgen 911, and six fractions with the transferase activity were separated by chromatofocusing on PBE 94 (pH 9.4 to 6.0). Fraction I was further separated into Isoforms Ia, Ib and Ic by affinity chromatography on UDP-hexanolamine-Sepharose 4B. UDP-glucuronosyltransferase in Fraction III was further purified by rechromatofocusing (pH 8.7 to 7.5). UDP-glucuronosyltransferases in Fractions IV and V were purified by UDP-hexanolamine-Sepharose chromatography. The transferase isoforms in Fractions II, III, IV and V were finally purified by h.p.l.c. on a TSK G 3000 SW column. Purified UDP-glucuronosyltransferase Isoforms Ia (Mr 51,000), Ib (Mr 52,000), Ic (Mr 56,000), II (Mr 52,000), IV (Mr 53,000) and V (Mr 53,000) revealed single Coomassie Blue-stained bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoform III enzyme showed two bands of Mr 52,000 and 53,000. Comparison of the amino acid compositions by the method of Cornish-Bowden [(1980) Anal. Biochem. 105, 233-238] suggested that all UDP-glucuronosyltransferase isoforms are structurally related. Reverse-phase h.p.l.c. of tryptic peptides of individual isoforms revealed distinct 'maps', indicating differences in primary protein structure. The two bands of Isoform III revealed distinct electrophoretic peptide maps after limited enzymic proteolysis. After reconstitution with phosphatidylcholine liposomes, the purified isoforms exhibited distinct but overlapping substrate specificities. Isoform V was specific for bilirubin glucuronidation, which was not inhibited by other aglycone substrates. Each isoform, except Ia, was identified as a glycoprotein by periodic acid/Schiff staining.     

Spectroscopic analysis of recombinant rat histidine decarboxylase

Mammalian histidine decarboxylases have not been characterized well owing to their low amounts in tissues and instability. We describe here the first spectroscopic characterization of a mammalian histidine decarboxylase, i.e. a recombinant version of the rat enzyme purified from transformed Escherichia coli cultures, with similar kinetic constants to those reported for mammalian histidine decarboxylases purified from native sources. We analyzed the absorption, fluorescence and circular dichroism spectra of the enzyme and its complexes with the substrate and substrate analogues. The pyridoxal-5'-phosphate-enzyme internal Schiff base is mainly in an enolimine tautomeric form, suggesting an apolar environment around the coenzyme. Michaelis complex formation leads to a polarized, ketoenamine form of the Schiff base. After transaldimination, the coenzyme-substrate Schiff base exists mainly as an unprotonated aldimine, like that observed for dopa decarboxylase. However, the coenzyme-substrate Schiff base suffers greater torsion than that observed in other L-amino acid decarboxylases, which may explain the relatively low catalytic efficiency of this enzyme. The active center is more resistant to the formation of substituted aldamines than the prokaryotic homologous enzyme and other L-amino acid decarboxylases. Characterization of the similarities and differences of mammalian histidine decarboxylase with respect to other homologous enzymes would open new perspectives for the development of new and more specific inhibitors with pharmacological potential.     

Polymorphism of rat gastric mucosal histidine decarboxylase: effects of the coenzyme and sulfhydryl groups

Several factors are examined for their implication in the charge heterogeneity and form conversion of rat gastric mucosal histidine decarboxylase. The apoenzyme and the holoenzyme are undistinguishable with respect to their pI and to the distribution of enzyme activity in the three forms. The latter are not produced by differential coenzyme binding. Studies for glycoprotein characterization provide evidence that the heterogeneity does not arise from enzyme-bound carbohydrate. Oxidative or reductive environments change the distribution between forms without modifying the molecular weight. Conversion of form III to forms I and II can be effected by treatment with dithiothreitol. A similar loss of negatively charged form occurs upon ageing and is not prevented by an alkylating agent. All three forms show equal sensitivity to N-ethylmaleimide and dithiothreitol inhibitions. The oxidation-reduction state of exposed sulfhydryl groups may be responsible at least in part for the charge heterogeneity.     

Isolation of pepsinogen granules from rabbit gastric mucosa

Pepsinogen granules were isolated from rabbit stomachs by a combination of differential and isopycnic gradient centrifugation. The isolation procedure utilized 1 M sucrose and alkaline pH to stabilize the granules. The isolated granules were shown to be 8.4-fold enriched in pepsinogen and free of mitochondria and microsome enzyme markers. In addition to pepsinogen, a cation-insensitive but anion-sensitive Mg2+-ATPase co-purified with the zymogen. The enzyme was unaffected by aurovertin, oligomycin, and ouabain, but inhibited by high concentrations of vanadate, N,N'-dicyclohexylcarbodiimide, and azide. The enzyme activity was stimulated by tetrachlorosalicylanilide and the combination of valinomycin and nigericin in K+-containing media. The similarities between this enzyme and other secretory granule ATPases are discussed.     

Purification and characterization of histidine decarboxylase from mouse kidney.

Histidine decarboxylase was purified 800-fold from the kidneys of thyroxine-treated mice. The purification procedure included precipitation of protein from a crude supernatant after heating it to 55 degrees C at pH 5.5, fractionation with (NH4)2SO4, phosphocellulose column chromatography, chromatofocusing, DEAE-Sepharose column chromatography, gel filtration on Sephacryl S-300 and preparative polyacrylamide-gel electrophoresis. The native enzyme had an estimated Mr of 113 000. The protein was analysed in SDS/10%-polyacrylamide gels and formed a single band corresponding to a subunit Mr of 55 000, indicating that it is a dimer. Three forms of the enzyme were resolved on isoelectrofocusing gels, with pI 5.3, 5.5 and 5.7.     

In vitro study of proteolytic degradation of rat histidine decarboxylase.

Mammalian ornithine decarboxylase (ODC) is a very unstable protein which is degraded in an ATP-dependent manner by proteasome 26S, after making contact with the regulatory protein antizyme. PEST regions are sequences described as signals for protein degradation. The C-terminal PEST region of mammalian ODC is essential for its degradation by proteasome 26S. Mammalian histidine decarboxylase (HDC) is also a short-lived protein. The full primary sequence of mammalian HDC contains PEST-regions at both the N- and C-termini. Rat ODC and different truncated and full versions of rat HDC were expressed in vitro. In vitro degradation of rat ODC and rat 1-512 HDC were compared. Like ODC, rat 1-512 HDC is degraded mainly by an ATP-dependent mechanism. However, antizyme has no effect on the degradation of 1-512 HDC. The use of the inhibitors MG-132 and lactacystine significantly inhibited the degradation of 1-512 HDC, suggesting that a ubiquitin-dependent, proteasome 26S proteolytic pathway is involved. Results obtained with the different modifications of rat HDC containing all three PEST regions (full version, 1-656 HDC), only the N-terminal PEST region (1-512 HDC), or no PEST region (69-512 HDC), indicate that the N-terminal (1-69) fragment, but not the C-terminal fragment, determines that the HDC protein is a proteasome substrate in vitro.     

Isolation of a branched-chain alpha-keto acid decarboxylase from rat liver.

An enzyme which catalyses oxidative decarboxylation of branched-chain alpha-keto acids was extracted from rat liver mitochondria with the aid of NaClO4. Purification yielded a product which appeared homogenous upon electrophoresis. Some kinetic data are reported; however, the enzyme is inactive with alpha-ketoisovalerate. The tenacity of binding to mitochondria, specificity, and other features, suggest that the decarboxylase may be a component of an enzyme complex named alpha-ketoisocaproate: alpha-keto-beta-methylvalerate dehydrogenase.     

Isolation of 5,8-oxyretinoic acid from rat intestinal mucosa.

Polar metabolites of retinoic acid accumulate in the intestine of vitamin A-deficient rats 3 h after administration of 450 μg of [11,12-³H]retinoic acid. Using new Chromatographic procedures developed for the purification of vitamin A metabolites, a major polar derivative of retinoic acid was isolated from intestine in pure form as its methyl ester and positively identified as 5,8-oxyretinoic acid.     

Marked increase in gastric histidine decarboxylase activity in patients with hypergastrinemia.

Histidine decarboxylase (HDC) activity and histamine content were measured in endoscopic gastric biopsy specimens of 19 control subjects with normogastrinemia and 6 patients with hypergastrinemia. In controls, the HDC activity was 3 fold higher in fundic mucosa (120 +/- 13 fmol/min/mg protein, mean +/- S.E.) than in antral mucosa (39 +/- 5 fmol/min/mg protein). In patients with hypergastrinemia, an extremely high HDC activity (713 +/- 181 fmol/min/mg protein) was observed in fundic mucosa, although the HDC activity in antral mucosa was not significantly different from that of controls. The histamine content in fundic mucosa was also significantly higher in patients with hypergastrinemia than in controls but no significant difference was seen in histamine content in antral mucosa between the two groups. These results are compatible with the hypothesis that in man, as well as in rat, histamine synthesis in fundic mucosa is enhanced by gastrin.     

Isolation and functional properties of an arginine-selective endoprotease from rat intestinal mucosa. A putative prosomatostatin convertase.

The endoproteolytic activity previously detected in rat intestinal mucosal extracts (Beinfeld M., Bourdais, J., Kuks, P., Morel, A., and Cohen, P. (1989) J. Biol. Chem. 264, 4460-4465), was purified to homogeneity as a 65-kDa molecular species. This putative proprotein-processing enzyme cleaves the peptide bond on the carboxyl side of a single arginine residue in hepta-[Leu62-Gln-Arg-Ser-Ala-Asn-Ser68] or trideca-[Asp56-Glu-Met-Arg-Leu-Glu-Leu-Gln-Arg-Ser-Ala-Asn-+ ++Ser68] peptides, reproducing the prosomatostatin sequence around Arg64, the locus for endoproteolytic release of either somatostatin-28 or its NH2-terminal fragment, somatostatin-28-(1-12), from their common precursor. This enzyme exhibits a strict selectivity for arginyl residues, as demonstrated with related substrates, and did not cleave at lysyl residues. Moreover, only arginyl residues belonging to peptides of the prosomatostatin family were cleaved, since no hydrolysis of peptides from other prohormones was detected. In addition, the arginine residue situated at position -5 on the NH2-terminal side of Arg64 not only did not function as a cleavage locus, but had no effect on the overall cleavage kinetics of the prosomatostatin-(56-68) peptide substrate. This enzyme also cleaved, but with much less efficiency, the peptide bond on the carboxyl side of an arginine in peptides containing either an Arg-Lys or a Lys-Arg doublet corresponding to prohormone cleavage sites. This enzyme was insensitive to divalent cation chelators, was completely inhibited by aprotinin and leupeptin, and was somewhat inhibited by other serine-protease inhibitors. It is concluded that this endoprotease is a serine protease and could be involved in prohormone or proprotein post-translational processing at single arginine cleavage sites.     

Hyperosmotic oxidation of glucose and decarboxylation of histidine in the gastric mucosa.

Paired slices of rat gastric mucosa were incubated with labeled glucose or histidine in isosomotic solution of 3-fold hyperosmotic solutions concentrated in NaCl, KCl, or ethanol. The rate of (1-14C)glucose oxidation to 14CO2 in isosmotic solution was reduced by 74% in hyperosmotic NaCl and by 28% in hyperosmotic KCl. The rate of (6-14C)glucose oxidation to 14CO2 in isosmotic solution was reduced by 64% in hyperosmotic NaCl and by 53% in hyperosmotic KCl. Reductions of glucose oxidation in hyperosmotic ethanol were not significant. The ratio of 14CO2 formed from (1-14C)glucose to that formed from (6-14C)glucose was not significantly changes by hyperosmotic NaCl or ethanol, but was significantly raised by hyperosmotic KCl. The rate of (carboxyl-14C)histidine decarboxylation in isosomotic solution was reduced significantly by 48% in hyperosmotic NaCl, by 30% in hyperosmotic KCl, and by 27% in hyperosmotic ethanol. We conclude that hyperosmotic solutions reduce glucose oxidation and histidine decarboxylation by rat gastric mucosa in the order of potency: NaCl greater than Kcl greater than or equal to ethanol. Thus hyperosmotic solutions inhibit the source of metabolic energy for stimulated acid secretion, the citric acid cycle, and the formation of the secretagogue histamine.