Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides.

Rhodopseudomonas spheroides can grow in a defined medium with either light or oxygen as an energy source. Cells grown anaerobically or at very low oxygen tensions are rich in the photosynthetic pigment bacteriochlorophyll, whereas this pigment is virtually absent in cells grown under high oxygen tensions. Aminolaevulinate synthetase, the first enzyme on the pathway to bacteriochlorophyll, appears to play an important role in the control of bacteriochlorophyll synthesis. Thus, the enzyme has a high activity in extracts of pigmented cells and a low activity in extracts of non-pigmented cells. Further, oxygenation of a pigmented culture causes immediate cessation of pigment synthesis and produces a rapid fall in the activity of aminolaevulinate synthetase. This loss of activity appears to be due to the loss of an endogenous activator of the enzyme. Thus, pigmented cells contain cystine trisulphide, which at muM concentrations is an activator of aminolaevulinate synthetase, while oxygenation causes a rapid fall in the cellular content of this trisulphide. Cystathionase (EC 4.2.1.15) extracted from pigmented cells can catalyse the formation of cystine trisulphide from cystine, while rhodanese (EC 2.8.1.1) extracted from the same cells can catalyse the degradation of cystine trisulphide in the presence of sulphite to form cystine and thiosulphate. It is proposed that the cellular content of cystine trisulphide is controlled by changes in the levels of substrates for cystathionase and possibly rhodanese rather than changes in the amounts of these enzymes. Cystine trisulphide controls the activity of aminolaevulinate synthetase by converting a low-activity form of the enzyme (b-form) into a high-activity form (a-form). The fall in aminolaevulinate synthetase activity on oxygenation appears to be the result of cessation of conversion of b-form into a-form, along with a conversion of a-form into b-form. Factors affecting the equilibrium between the forms and the possible mechanisms for their interconversion are discussed.     

Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. The involvement of sulphur metabolism

1. The ;initial' 5-aminolaevulinate synthetase activity, that is the activity observed immediately after cell disruption, in extracts prepared from unharvested semianaerobically grown Rhodopseudomonas spheroides, was twice that observed under the same assay conditions in extracts prepared from harvested cells. 2. The effect of oxygenation of a culture on the ;maximum' aminolaevulinate synthetase activity, that is the activity observed 1h after disruption of harvested cells, is markedly influenced by the contents of the growth medium. Oxygenation of organisms for 1h in the medium in which they have grown produces an 80-90% decrease in maximum activity, whereas similar treatment of organisms resuspended in fresh medium produces less than a 40% decrease. 3. This protective effect of fresh medium is absolutely dependent on the presence of sulphate. When cells are suspended in sulphate-deficient fresh medium, the maximum activity falls by 65-75% even without oxygenation. A high maximum activity is regenerated when sulphate is resupplied. 4. When organisms are oxygenated in the medium in which they have grown, the cellular contents of GSH+GSSG and cysteine+cystine fall very markedly and homolanthionine is formed. Both the fall in aminolaevulinate synthetase activity and the changes in sulphur metabolism are largely prevented by the addition of compounds which stimulate synthesis of cysteine de novo or inhibit the conversion of cysteine S into homocysteine S. 5. The maximum aminolaevulinate synthetase activity was directly proportional to the GSH+GSSG content of all cell preparations. In glutathione-depleted extracts the ;low'-activity enzyme could be re-activated in vitro by the addition of GSH, GSSG, cysteine or cystine, whereas in extracts with a high glutathione content the ;high'-activity enzyme was unaffected by these sulphur compounds. 6. The activation of low-activity enzyme with exogenous sulphur compounds was prevented by excluding air or by adding NADH. Studies with purified enzyme indicate that sulphur compounds do not interact directly with the enzyme, but that their effect is mediated by a number of other endogenous factors.     

Glycogenolysis during recovery from muscular work. The time course of phosphorylase activity is dependent on Pi concentration in the abdominal muscle of the shrimp Crangon crangon

1) Glycogen is degraded in the abdominal muscle of the shrimp Crangon crangon (Decapoda, Crustacea) during the recovery period following work. The regulation of post-exercise glycogen breakdown and the properties of glycogen phosphorylase (EC 2.4.1.1) have been studied: 2) Glycogen phosphorylase exists as unphosphorylated b-form and phosphorylated a-form, the latter contains 1 molecule phosphate/subunit. Both forms of phosphorylase are dimers, isoenzymes have not been detected. 3) The purified b-form is inactive in absence of AMP and has very low affinities for AMP and Pi. For half-maximum activation 0.33 +/- 0.04 mM AMP is necessary, and the Km-value for Pi at 1 mM AMP is 48 +/- 5 mM. IMP does not affect the activity of the b-form. 4) The a-form is active without effectors, its Km-value for Pi is 5.3 +/- 1.5 mM. The proportion of phosphorylase a increases in vivo, from about 25% at rest, to approximately 90% upon work and remains at this high level during the first minutes of recovery. 5) It is concluded that the glycogenolytic flux in the abdominal muscle of the shrimp even during post-exercise periods depends on the level of the a-form the activity of which is restricted in time and extent by the cytoplasmic Pi concentration (Kamp, G. & Juretschke, H. P. (1987) Biochim. Biophys. Acta 929, 121-127).     

Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. Purification and properties of the high-activity form of the enzyme.

1. The high-activity form of aminolaevulinate synthetase has been prepared from extracts of semi-anaerobically grown cells of Rhodopseudomonas spheroides, which were allowed to become activated in air. Specific activity was 130 000--170 000 nmol of aminolaevulinate/h per mg of protein at 37 degree C. 2. Enzyme fraction Ia prepared on DEAE-Sephadex was a mixture of four active enzymes, pI5.55, 5.45, 5.35 and 5.2, when prepared in either Tris or phosphate buffers and when extracts were activated by air or by cystine trisulphide. 3. The enzyme was further purified by preparative polyacrylamide-gel electrophoresis in imidazole/veronal buffer, pH 7.6, followed by gel filtration on Sephadex G-100 and concentration with DEAE-Sephadex. 4. The most active enzyme, pI 5.55, ran as a single protein band, mol.wt. 49 000, in sodium dodecyl sulphate and 2-mercaptoethanol. The apparent molecular weight under non-denaturing conditions was 62 000--68 000 on Sephadex G-100 or G-200, pH 7.5, and on polyacrylamide-gel electrophoresis, pH 8.5, at enzyme concentrations below 10 000 units/ml, i.e. less than 60 microgram of protein/ml, and the enzyme was mainly monomeric. 5. The enzyme was homogeneous by gel disc electrophoresis at pH 8.9 and 7.6, but a slightly more diffuse band of protein was obtained during electrophoresis in glycine buffer, pH 7.4. 6. Enzyme samples possessed an intrinsic yellow fluorescence when viewed under u.v. light and this fluorescence coincided exactly with enzymic activity on gel electrophoresis. Fluorescence maxima were 420 nm (excitation) and 495 nm (emission). 7. Radioactive 35S-labelled enzyme had 14 atoms of sulphur/mol of protein (or/40 leucine residues) of which 5--6 residues were cyst(e)ine and 8--9 residues were methionine. 8. Mo carbohydrate was detected apart from glucose, which prevented accurate determination of tryptophan with methanesulphonic acid and tryptamine.     

Enzymatic synthesis of novel glutathione analogs

A strain of Escherichia coli enriched in its content of gamma-glutamylcysteine synthetase and glutathione synthetase by recombinant DNA techniques has been immobilized in a carrageenan matrix and used for the synthesis of various types of isotopically labeled glutathione (L-gamma-glutamyl-L-cysteinyl-glycine) (K. Murata, W. A. Abbott, R. J. Bridges, and A. Meister (1985) Anal. Biochem. 150, 235-237). In the present work, this E. coli matrix was used as the basis of a method for the synthesis of glutathione analogs. Thus, amino acid analogs were used in place of the corresponding amino acid constituents of glutathione (e.g., 4-fluoroglutamate was substituted for glutamate) in the reaction mixtures. Using this method we have synthesized several analogs of glutathione including L-gamma-glutamyl-(beta-chloro)-L-alanyl-glycine, (R,S)-4-fluoro-DL-gamma-glutamyl-L-cysteinyl-glycine, D-gamma-glutamyl-L-cysteinyl-glycine, and L-gamma-glutamyl-L-homocysteinyl-glycine. This method may also be used for the synthesis of a number of L- and D-gamma-glutamyl amino acids. The analogs are purified by gel-filtration and ion-exchange chromatography. The analogs are used to examine the substrate specificity and mechanisms of action of glutathione-utilizing enzymes and for studies on glutathione metabolism and function. Fluorine-containing analogs may be used for NMR studies. The enzymatically prepared compounds may also be used as intermediates in the chemical synthesis of other analogs of glutathione and glutathione disulfide.     

Aminolaevulinate synthetase of Micrococcus denitrificans. Purification and properties of the enzyme, and the effects of growth conditions on the enzyme activity in cells

1. 5-Aminolaevulinate synthetase was detected in extracts of the non-photosynthetic bacterium Micrococcus denitrificans. 2. Activity is high in cells grown anaerobically in a defined nitrate medium, but is low in cells grown in an iron-deficient medium, and in cells grown aerobically. 3. Aminolaevulinate synthetase was purified extensively; it has a molecular weight of about 68000; apparent K(m) values for glycine, succinyl-CoA and pyridoxal phosphate are 12mm, 10mum and 11mum respectively; 2mum-haemin and 14mum-protoporphyrin inhibit by 50%. 4. The low activity of aminolaevulinate synthetase in iron-deficient cells increases on adding iron salts to cells only under conditions where protein synthesis can occur. 5. In defined nitrate medium with a high Ca(2+) concentration anaerobic growth yield is higher, but aminolaevulinate synthetase activity is lower than in cells grown in the medium with a low Ca(2+) concentration. In medium made from AnalaR constituents, growth yield is low and aminolaevulinate synthetase activity is high even in the presence of high concentrations of Ca(2+); on adding Cu(2+) (0.1mum) to the medium growth yield and aminolaevulinate synthetase activity become the same as in non-AnalaR medium. 6. Cells incubated under conditions where protein synthesis does not occur but where electron transport does, lose their aminolaevulinate synthetase activity rapidly. 7. The activities of aminolaevulinate dehydratase and succinic thiokinase do not change under any of the conditions of growth examined. 8. The possible mechanisms controlling aminolaevulinate synthetase activity and the role of this enzyme in controlling the synthesis of haem in this organism are discussed.     

Glutathione metabolism in normal and cystinotic fibroblasts

Intracellular concentrations of glutathione and activities of the enzymes gamma-glutamylcysteine synthetase, glutathione synthetase, and gamma-glutamyl transpeptidase were measured in confluent cultured human fibroblasts cell lines from 14 normal cell lines and four cystinotic cell lines. gamma-Glutamyl transpeptidase had a wide range of variability while the glutathione synthetic enzymes, gamma-glutamylcysteine synthetase and glutathione synthetase, had narrower variations and also exhibited no apparent relationship to glutathione content. No differences in the activities of these enzymes were found between normal and cystinotic cells in confluent cell cultures. The activities of the above enzymes and the cell number and content of glutathione, cystine, DNA, and total protein in two normal and two cystinotic fibroblast cell lines were measured during growth. The following growth-dependency patterns were observed: (1) gamma-glutamylcysteine synthetase activity increased markedly in lag and early log phases in both normal and cystinotic cells and decreased rapidly to low confluent levels thereafter. (2) gamma-Glutamyl transpeptidase showed the same wide range of activity noted at confluency but activities decreased in the log phase of growth, a pattern also seen in cystinotic cells. (3) Glutathione synthetase activity remained relatively constant during growth of normal cells but exhibited a peak of activity during lag and early growth of cystinotic cells. (4) Comparative glutathione levels of normal and cystinotic cells were not significantly different and exhibited similar fluctuations with time. (5) The cystine content of normal and cystinotic cells unexpectedly rose to high levels in the lag phase, then decreased to 0.1 nmol 1/2 cystine/mg protein in normal cells and to 0.3 to 1.2 nmol 1/2 cystine/mg protein in cystinotic cells during the log phase. As confluency was approached, normal cell cystine remained at low levels while cystinotic cell cystine rose to characteristically high levels of 50- to 100-fold greater than normal cells at late confluency. These studies extend our understanding of the regulation of glutathione and cystine content in cultured fibroblasts and suggest that glutathione content is closely controlled throughout the cell cycle in the face of varying activities of its anabolic and catabolic enzymes.     

Identification of rabbit uterine cervical procollagenase activator as rabbit matrix metalloproteinase 3 (stromelysin).

1. We previously reported an endogenous activator of procollagenase from the culture medium of rabbit uterine cervical fibroblasts (Ishibashi et al. (1987) Biochem. J. 241, 527-534). 2. Similar activator was also purified and characterized from rabbit synovial fibroblasts (Vater et al. (1983) J. biol. Chem. 258, 9374-9382), but its mode of activation of procollagenase was reported to be different from that of purified activator from uterine cervical fibroblasts. 3. Here we report the comparative studies of the two activators of procollagenase and demonstrate that they are identified as matrix metalloproteinase 3 (stromelysin) by their immunological and functional criteria. The specific role of the activator in procollagenase activation is also described.     

Enzymatic activities of P-450(11 beta)s expressed by two cDNAs in COS-7 cells

Expression plasmids were constructed using two cDNA clones of P-450(11 beta), pcP-450-(11 beta)-2, and pcP-450(11 beta)-3 (Morohashi et al. (1987) J. Biochem. 102, 559-568 and Kirita et al. (1988) J. Biochem. 104, 683-686), and introduced into COS-7 cells by electroporation. The expression of P-450(11 beta) proteins and their localization in the mitochondria were demonstrated by immunoblotting, immunofluorescence microscopy, and immunoelectron microscopy. The enzymatic activities of the expressed P-450(11 beta)s were determined using deoxycorticosterone (DOC), deoxycortisol, and corticosterone as substrates. Though the activities of the two P-450(11 beta)s for 11-, 18-, and 19-hydroxylation of DOC were almost equal, the production of 18-hydroxycorticosterone and aldosterone from corticosterone by P-450(11 beta)-3 was greater than that by P-450(11 beta)-2.     

Evidence for a new activator of rat liver phosphofructokinase

A low molecular weight compound that activates purified rat liver phosphofructokinase has been isolated and partially purified from rat hepatocyte extracts. It can be separated from both fructose bisphosphate and AMP on DEAE-Sephadex. Incubation of rat hepatocytes with glucagon lowers the level of this activator, and this accounts for the inhibition of phosphofructokinase that was observed in hepatocyte extracts (S. Pilkis, et al. (1979) Biochem. Biophys. Res. Commun. $\text{88}$, 960–967). Other characteristics of this activator are described which suggest that it is not any of the known effectors of rat liver phosphofructokinase.     

Paraquat toxicity and pyridine nucleotide coenzyme synthesis: a data correction

The decrease in pyridine nucleotide coenzymes which occurs during poisoning of Escherichia coli by hyperbaric oxygen or paraquat is not due to impairment of nicotinatemononucleotide pyrophosphorylase (carboxylating) [EC 2.4.2.19] as was previously proposed (Brown, O.R. et al. Biochem. Biophys. Res. Commun. 91:982-990; 1979). This was shown directly using extracts of E. coli, prepared after exposure to 1 mM paraquat or 4.2 atmospheres of oxygen. The enzyme also was not impaired in Neurospora crassa by 1 mM paraquat. A naturally-occurring, non-dialyzable inhibitor of the enzyme was found in E. coli extracts. The inhibitor caused the erroneous, low nicotinatemononucleotide pyrophosphorylase (carboxylating) activities previously reported in extracts of E. coli poisoned by paraquat.     

Interplay between lipoic acid and glutathione in the protection against microsomal lipid peroxidation

Reduced glutathione (GSH) delays microsomal lipid peroxidation via the reduction of vitamin E radicals, which is catalyzed by a free radical reductase (Haenen, G.R.M.M. et al. (1987) Arch. Biochem. Biophys. 259, 449-456). Lipoic acid exerts its therapeutic effect in pathologies in which free radicals are involved. We investigated the interplay between lipoic acid and glutathione in microsomal Fe2+ (10 microM)/ascorbate (0.2 mM)-induced lipid peroxidation. Neither reduced nor oxidized lipoic acid (0.5 mM) displayed protection against microsomal lipid peroxidation, measured as thiobarbituric acid-reactive material. Reduced lipoic acid even had a pro-oxidant activity, which is probably due to reduction of Fe3+. Notably, protection against lipid peroxidation was afforded by the combination of oxidized glutathione (GSSG) and reduced lipoic acid. It is shown that this effect can be ascribed completely to reduction of GSSG to GSH by reduced lipoic acid. This may provide a rationale for the therapeutic effectiveness of lipoic acid.     

Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. Binding of pyridoxal phosphate to 5-aminolaevulinate synthetase.

1. Pyridoxal 5'-phosphate is a cofactor essential for the enzymic activity of aminolaevulinate synthetase from Rhodopseudomonas spheroides. It also aids activation of the low-activity enzyme by trisulphides such as cystine trisulphide, whereas inactivation of enzyme is facilitated by its absence. 2. The fluorescence spectrum of purified high-activity enzyme is that expected for a pyridoxal phosphate--Schiff base, but the firmly bound cofactor does not appear to be at the active centre. In dilute solutions of enzyme this grouping is inaccessible to nucleophiles such as glycine, hydroxylamine, borohydride and cyanide, at pH 7.4. 3. An active-centre Schiff base is formed between enzyne and added pyridoxal phosphate, which is accessible to nucleophiles. Concentrated solutions of this enzyme--Schiff base on treatment with glycine yield apo- and semi-apoenzyme, which can re-bind pyridoxal phosphate. 4. Two types of binding of pyridoxal phosphate are distinguishable in dilute solution of enzyme, but these become indistinguishable when concentrated solutions are treated with cofactor. A change occurs in the susceptibility towards borohydride of the fluorescence of the "structural" pyridoxal phosphate. 5. One or two molecules of cofactor are bound per subunit of mol. wt. 50 000 in semiapo- or holo-enzyme. The fluorescence of pyridoxamine phosphate covalently bound to enzyme also indicates one to two nmol of reducible Schiff base per 7000 units of activity in purified and partially purified samples of enzyme. 6. Cyanide does not convert high-activity into low-activity enzyme, but with the enzyme-pyridoxal phosphate complex it forms a yellow fluorescent derivative that is enzymically active.     

Thiyl radicals react with nitric oxide to form S-nitrosothiols with rate constants near the diffusion-controlled limit

A possible route to S-nitrosothiols in biology is the reaction between thiyl radicals and nitric oxide. D. Hofstetter et al. (Biochem. Biophys. Res. Commun.360:146-148; 2007) claimed an upper limit of (2.8+/-0.6)x10(7) M(-1)s(-1) for the rate constant between thiyl radicals derived from glutathione and nitric oxide, and it was suggested that under physiological conditions S-nitrosation via this route is negligible. In the present study, thiyl radicals were generated by pulse radiolysis, and the rate constants of their reactions with nitric oxide were determined by kinetic competition with the oxidizable dyes 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) and a phenothiazine. The rate constants for the reaction of nitric oxide with thiyl radicals derived from glutathione, cysteine, and penicillamine were all in the range (2-3) x10(9) M(-1)s(-1), two orders of magnitude higher than the previously reported estimate in the case of glutathione. Absorbance changes on reaction of thiyl radicals with nitric oxide were consistent with such high reactivity and showed the formation of S-nitrosothiols, which was also confirmed in the case of glutathione by HPLC/MS. These rate constants imply that formation of S-nitrosothiols in biological systems from the combination of thiyl radicals with nitric oxide is much more likely than claimed by Hofstetter et al.     

Heme biosynthesis pathway regulation in a model of hepatocarcinogenesis pre-initiation.

1. Heme regulation before the appearance of hyperplastic nodules was investigated in mice models of hepatocarcinogenesis. 2. With this aim 5-aminolaevulinate synthetase (ALA-S), microsomal heme-oxygenase (MHO), mitochondrial and cytoplasmic rhodanese activities were examined throughout a period of 35 days in animals exposed to dietary p-dimethylaminoazobenzene (DAB). 3. ALA-S activity was significantly diminished (50%) on day 14, then showing a sharply rising profile from day 28 onwards, and reaching 350% on day 35. 4. A similar profile was observed for mitochondrial rhodanese activity. 5. Changes in MHO and cytoplasmic rhodanese activities were almost the opposite to those observed for ALA-S. 6. The distinctive alteration in mitochondrial and cytoplasmic rhodanese would suggest that it plays a subtle role in ALA-S regulation during carcinogenesis initiation through a mechanism that appears to involve subcellular localization controls perhaps by means of the breakage of cystine trisulphide postulated to act as an ALA-S activator. 7. Taking into account the present results, we suggest a probable mechanism for the onset of hepatocarcinogenesis that includes a primary activating liver status, provoking biochemical aberration leading to the stage of initiation of hepatocarcinogenesis involving the whole organ.     

Purification of major basic glutathione transferase isoenzymes from rat liver by use of affinity chromatography and fast protein liquid chromatofocusing

Seven major isoenzymes of glutathione transferase with isoelectric points ranging from pH 6.9 to 10 were isolated from rat liver cytosol. The purification procedure included affinity chromatography on immobilized S-hexylglutathione followed by high-performance liquid chromatofocusing. Characteristics, such as physical properties, reactions with antibodies, specific activities with various substrates, kinetic constants, and sensitivities to a set of inhibitors, are given for discrimination and identification of the different isoenzymes. The multiple forms of the enzyme correspond to glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4, and 4-4 in the recently introduced nomenclature [W.B. Jakoby et al. (1984) Biochem. Pharmacol. 33, 2539-2540]. A seventh form appears to be a heterodimeric protein composed of subunit 3 and an as yet unidentified subunit.     

Sample processing alters glutathione and cysteine values in blood

The accurate assessment of glutathione status of blood is essential for its use as an index of health and aging. A major variable in glutathione analysis is sample processing, and identification of optimal standard conditions is needed. Thus our objective was to evaluate several methods to determine which one yields maximal levels of free and bound glutathione and cyst(e)ine in blood. Reduced glutathione (GSH), glutathione disulfide (GSSG), cysteine (Cys), and cystine were analyzed specifically by an HPLC-dual electrochemical method. The highest GSH levels were found in ultrafiltrates of hemolysates, which were 58% greater than those in acid extracts of whole blood, and accounted for 96% of the free and bound GSH in borohydride-reduced samples; GSSG was undetected. The next highest values were in acid extracts of hemolysates which were 13% greater than in extracts of whole blood; both extracts contained GSH and GSSG. Their GSSG contents expressed in GSH equivalents comprised 7-9% of GSH + GSSG. Cys levels were highest in ultrafiltrates which were 11-fold greater than in acid extracts of whole blood, accounting for 62% of the total cyst(e)ine pool. In summary, the results indicate that ultrafiltration of hemolysates is the blood processing method of choice to obtain maximal values of free and bound GSH and cyst(e)ine.     

D-Xylose metabolism in Rhodosporidium toruloides

Höfer et al. (Biochem. Biophys. Acta 1971. 252:1-12) presented circumstantial evidence that suggested that Rhodosporidium toruloides produced a xylose isomerase. We were unable to detect this activity in cell-free extracts of this yeast, however, xylose reductase and xylitol dehydrogenase activities were detected.     

Cobinding of bilirubin and laurate to human serum albumin: spectroscopic characterization of stoichiometric complexes

Light absorption and CD spectra of bound bilirubin and albumin fluorescence spectra have been recorded from mixtures containing albumin, A, bilirubin, B, and laurate, L, in Tris-NaCl buffer at pH 8.2, 25 degrees C. Concentrations of the corresponding stoichiometric complexes, ABiLj, for i = 0/3 and j = 0/3, have been calculated from previously determined stoichiometric cobinding constants (H. Sato et al. (1988) Arch. Biochem. Biophys. 260, 811-821). Spectral data of the complexes have finally been found by iterative computer fitting using the principle of several acceptable solutions (R. Brodersen et al. (1987) Eur. J. Biochem. 169, 487-495). The results were utilized at the microscopic level to investigate ligand-induced conformational changes. When laurate was bound to AB, a decrease of the distance between Trp-214 and the bound bilirubin occurred, as measured according to F?rster's principle. The distances were 21.9 +/- 0.3 A in AB, 19.7 +/- 0.3 A in ABL, and 17.9 +/- 0.2 A in ABL2.     

Localization of an endogenous ADP-ribose acceptor, p33, in polymorphonuclear cell granules in chicken liver interlobular connective tissue

We investigated immunohistochemically the localization of p33, an endogenous substrate protein for an arginine-specific ADP-ribosyltransferase in chicken liver. Polymorphonuclear-pseudo-eosinophilic granulocytes (heterophils) in interlobular connective tissues of the liver were exclusively and strongly stained with the antibody against p33. Strong reactivity was associated with granules in cytoplasm of the heterophils. When the chicken liver nuclear fraction was washed, the transferase activity was released into the 600 x g supernatant fraction while a nuclear enzyme poly(ADP-ribose) synthetase was retained in the pellet fraction. These results indicate that p33 and probably also ADP-ribosyltransferase, found in the liver nuclear fraction [Tanigawa et al. (1984) J. Biol. Chem. 259, 2022-2029, Mishima et al. (1988) Eur. J. Biochem. 179, 267-273], originate from interlobular heterophils of the chicken liver.