Preliminary characterization of phosphotyrosine phosphatase activities in human peripheral blood lymphocytes: Identification of CD45 as a phosphotyrosine phosphatase

A preliminary characterization of the protein phosphotyrosine phosphatase (PT-Pase) activity in human peripheral blood lymphocytes (PBL) has been made using two tyrosine-containing peptides and the epidermal growth factor receptor from A-431 cells as substrates. High PTPase activity with a pH optimum near 7.4 was observed in both the membrane and the cytosolic fractions. At least three distinct fractions with PTPase activity were separated on DEAE cellulose columns, indicating that the enzyme is heterogeneous. Vanadate, molybdate, and salts of zinc, copper, and mercury were all effective enzyme inhibitors, although the inhibition was generally incomplete and showed some variation with the enzyme preparation. The difficulty in completely inhibiting PTPase activity in lymphocytes may help explain the variation between laboratories in studies of tyrosine phosphorylation in these cells. Studies with highly purified T lymphocytes obtained by filtration of PBL through nylon wool columns indicated that the activity is present in T cells. Absorption with agarose containing anti-HLe-I, a mouse monoclonal lgGi antibody specific for the leukocyte-specific surface protein T-200 (CD45), removed up to 40% of the PTPase activity. Enzyme activity was recovered on the immunoadsorbent after extensive washing, confirming that the enzyme was being bound to the beads. Immunoabsorbents containing other mouse lgGi antibodies failed to bind PTPase activity, indicating that the binding to beads with anti-HLe-I antibody is specific. Further characterization of the CD45 and PTPase activities in lymphocytes may provide a better under standing of the role of protein tyrosine phosphorylation in the regulation of proliferation and differentiation in these cells.     

Immunomodulating activities of water-soluble exopolysaccharides obtained from submerged culture of Lentinus lepideus

Immunomodulating activities of water-soluble exopolysaccharides (LL-EX) obtained from submerged mycelial culture of Lentinus lepideus were studied and their effectiveness was compared with lipopolysaccharide (LPS). The influence of the LL-EX on macrophage cellular lysosomal enzyme activity was to stimulate up to 267%, 392%, and 464% at the level of 10, 50, and 100 mug/ml, respectively. When the LL-EX was further fractionated into LL-Fr.I and Fr.II by Sepharose CL-6B gel chromatography, the cellular lysosomal enzyme activity of LL-Fr.II (2.1- fold) was higher than Fr.I (1.2-fold). Moreover, both LL-Fr.I and Fr.II stimulated the cytokines IL-1beta, TNF-alpha, and IL-6 in macrophages. In mixed lymphocyte reaction, LL-Fr.I and Fr.II enhanced the splenocyte proliferation up to 1.2-fold and 1.4-fold (50 mg/ml), respectively, stimulating only T lymphocytes. The fractions of LL-EX not show any direct toxicity against human gastric adenocarcinoma cell (AGS). The molecular masses of LL-Fr.I and Fr.II were estimated to be about 1,986 kDa and 21 kDa, respectively. The total sugar and protein contents of the two fractions were 84.97% and 69.88% and 15.03% and 30.12%, respectively. The sugar and amino acid compositions of the LL-Fr.I and Fr.II were also analyzed in detail.     

Semiquantitative cytochemical determination of acid phosphatase, beta-glucuronidase, and beta-glucosaminidase activity in peripheral blood lymphocytes.

Activity of acid phosphatase, beta-glucuronidase, and beta-glucosaminidase in peripheral blood lymphocytes was determined cytochemically in 20 normal subjects, 10 male and 10 female, by the use of BARKA and ANDERSON's (1962), HAYASHI et al. (1964) and HAYASHI's (1965) methods, respectively. Results obtained were semiquantitatively according to subdivision of lymphocytes into enzyme-negative and enzyme-positive cells. Enzyme-positive lymphocytes were divided into cells with granular, mixed granular and diffuse enzymatic reaction type. In the first two types of cytochemical reaction a number of enzyme-positive lysosomal granules were counted and expressed in terms of both absolute count and percentage of circulating lymphocytes. Enzyme-positive lymphocytes represented 80.3%, 40.5% and 41.5% of the total lymphocyte count in regard to the presence of acid phosphatase, beta-glucuronidase, and beta-glucosaminidase, respectively.     

STUDIES ON LYSOSOMES : XII. Redistribution of Acid Hydrolases in Human Lymphocytes Stimulated by Phytohemagglutinin

Subcellular fractions were isolated by differential centrifugation from pure suspensions of human blood lymphocytes incubated with and without phytohemagglutinin (PHA). Between 30 and 120 min after addition of PHA to intact cells, redistribution of acid hydrolases (beta glucuronidase, acid phosphatase), from a 20,000 g x 20 min granular fraction into the corresponding supernatant, was observed. No increase in total acid hydrolase activity was found at these times. The mitochondrial marker enzyme, malate dehydrogenase, did not undergo redistribution. Granules derived from PHA-treated cells became more fragile upon subsequent incubation with membrane-disruptive agents in vitro (streptolysin S, filipin). These changes were associated with an increase in the over-all permeability of the stimulated cell to substances in the surrounding medium, such as neutral red. Augmentation of dye entry into lymphocytes required intact metabolism as judged by response to temperature and inhibitors (cyanide, antimycin A, 2,4-dinitrophenol). PHA, however, did not release enzyme activity from hydrolase-rich granules in vitro or render them more susceptible to subsequent challenge with membrane-disruptive agents. These studies suggest that PHA induces early changes in the surface of lymphocytes. The consequent redistribution of acid hydrolases may play a role in remodeling processes of the stimulated cells.     

Different baseline sister chromatid exchange levels in density fractionated human lymphocytes

Summary Different activation states of B and T lymphocytes, as manifested by differences in cell density, were obtained by Percoll density centrifugation of unstimulated human lymphocytes. Four different density fractions were defined: B cells with low (1.043 g/ml) and high (1.056) density, and T cells with low (1.067) and high (1.077) density, respectively. Sister chromatid exchange (SCE) conditions and proliferation rates were determined. Total B cells, stimulated by the bacterial mitogen Branhamella, had 4.6 SCE per cell, the lowest mean baseline SCE level recorded among lymphocytes. The growth rate was intermediate between that of low and high density T cells. The two T cell fractions stimulated by phytohemagglutinin (PHA) had different baseline SCE frequencies and different growth characteristics: the low density cells had 5.7 SCEs per cell and a short cell cycle, whereas high density cells had 12.5 SCEs per cell and a longer cell cycle. The differences in baseline SCE frequency and growth characteristics between the two T cell fractions seem to be correlated with the differences in the activation state as reflected by the cell density. Both high and low density T cell are G₀ populations which supposedly differ with respect to previous history in vivo such as age and contact with antigens. The reason why these cells react differently to bromodeoxyuridine (BrdU) is unknown, but differences in intracellular DNA precursor pools and enzyme activities might play a role.     

Studies on the leukotriene D4-metabolizing enzyme of rat leukocytes, which catalyzes the conversion of leukotriene D4 to leukotriene E4

Leukotriene D4-metabolizing enzyme was studied using rat neutrophils, lymphocytes and macrophages. These leukocyte sonicates converted leukotriene D4 to leukotriene E4. However, the leukotriene D4-metabolizing activity varied with cell type, and macrophages showed the highest activity among these leukocytes. The subcellular localization of the leukotriene D4-metabolizing enzyme of macrophages was examined, and the leukotriene D4-metabolizing activity was found to be present in the membrane fraction, but not in the nuclear, granular and cytosol fractions. When macrophages were modified chemically with diazotized sulfanilic acid, a poorly permeant reagent which inactivates cell-surface enzymes selectively, the leukotriene D4-metabolizing activity of macrophages decreased significantly (about 95%) without any inhibition of marker enzymes of microsome, cytosol, lysosome and mitochondria. When neutrophils and lymphocytes were modified with diazotized sulfanilic acid, the leukotriene D4-metabolizing activity was also inhibited about 90% by the modification. Among various enzyme inhibitors used, o-phenanthroline, a metal chelator, remarkably inhibited the leukotriene D4-metabolizing activity of leukocytes, and the o-phenanthroline-inactivated enzyme activity was fully reactivated by Co2+ and Zn2+. These findings seem to indicate that rat neutrophils, lymphocytes and macrophages possess the leukotriene D4-metabolizing metalloenzyme which converts leukotriene D4 to leukotriene E4, on the cell surface, although macrophages have a higher enzyme activity than the other two.     

Functional properties of T and B cells isolated by affinity chromatography from rat thoracic duct lymph.

Rat thoracic duct lymphocytes (TDL) were separated into two fractions by passing the cells through a column of rabbit anti-rat F (ab′)₂ antibody coupled to Sephadex G-200. Cells with readily detectable surface immunoglobulin (Ig) were retained on the gel, whereas those without surface Ig were recovered in the effluent. Adherent cells were retrieved by eluting the column with rat Ig. Both dividing and nondividing lymphocytes were separated by this procedure. The adherent and non-adherent fractions contained functionally active lymphocytes as judged by a thymidine incorporation technique and the immunological performance of the cells after transfer to normal recipients. Antibody forming cells and B memory cells were concentrated in the adherent fraction. The non-adherent fraction contained antigen-sensitive T cells which initiate graft versus host reaction and specifically sensitized lymphocytes of the kind which transfer resistance to L. monocytogenes.     

Separation of human lymphocyte subpopulations by density gradient electrophoresis. I. Different mobilities of T (T mu, T alpha) and B lymphocytes from human tonsils

T and B lymphocytes from human tonsils were separated by density gradient electrophoresis on the basis of their surface charge. The high-mobility cell fractions were found to be highly enriched in T lymphocytes with only very small proportions of B cells. In contrast, the low-mobility fractions were predominantly B lymphocytes, and had only 10 to 30% contamination of T cells. The intermediate-mobility fractions contained both T and B lymphocytes in approximately equal proportions. IgM-bearing lymphocytes, as well as cells with receptors for mouse erythrocytes, the Fc portion of IgG, and complement were found in the intermediate- and low-mobility fractions. T lymphocytes, prepared by E rosetting, were also electrophoresed by this method and found to be of higher mobility as compared with peripheral blood T lymphocytes. T cells with Fc receptors for IgM (Tμ) or IgA (Tα) were found to be considerably heterodisperse with regard to surface charge and were present in all fractions. The separated cell fractions were treated in vitro with various concentrations of concanavalin A and thereafter examined for Tμ, Tγ, and Tα phenotypes. Low concentrations of Con A (2.5 μg/ml) had no effect on cell surface phenotypes. However, higher concentrations of Con A (20μg/ml) significantly reduced the numbers of T cells having IgM receptors (Tμ), but failed to alter the expression of the Tγ phenotype. The latter finding contrasts to that observed with T cells from the peripheral blood where high concentrations of Con A increase the proportions of the Tγ cells. This study demonstrates that density gradient electrophoresis can be used for the separation and study of lymphocyte subpopulations from human tonsils.     

On Extraction and Quantitation of Plant Peroxidase Isoenzymes

Peroxidase in tobacco callus tissue differed in extract-ability depending on the subcellular distribution of the enzyme. Based on extractability it consisted of four fractions: freely soluble and less freely soluble in phosphate buffer, KCl-soluble, and insoluble. The latter two fractions were un-extractable by a phosphate buffer alone. The different fractions contained varied proportions of peroxidase isoenzymes. The extractability of indoleacetic acid oxidase was similar. A medium of high ionic strength is essential for quantitative extraction of peroxidase and indoleacetic acid oxidase isoenzymes. For quantitation of isoperoxidase activity on polyacryl-amide gel following electrophoretic separation, benzidine and o-dianisidine were better hydrogen donors than guaiacol and pyrogallol. The optimum pH was 4.5, but a citrate buffer was inhibitory. The optimum conditions included an acetate buffer at pH 4.5, a substrate concentration of 0.03 %, benzidine as the hydrogen donor, and a 3-minute treatment with 7 % acetic acid after staining. The color intensity of the bands remained unchanged for at least three days. With appropriate sample size and reaction time there was a linear relationship between enzyme concentration and activity.     

Ferritin in normal human peripheral blood T-lymphocyte subpopulations

Six peripheral blood lymphoid fractions (total lymphocytes, non-T, T, Tar (autologous rosette-forming T cells/precursor), T mu (helper), and T gamma (suppressor) lymphocytes) isolated through rosetting procedures were examined for the presence of ferritin by a direct immunofluorescence technique. Although ferritin was present in all lymphoid fractions studied, a significantly higher proportion of ferritin-containing cells were detected in the T-cell fraction than in the non-T-cell fraction, (mean +/- SD = 7.9 +/- 1.6% and 5.0 +/- 1.2%, respectively). T mu- and T gamma-cell fractions showed a twofold increase in the number of ferritin-positive cells (14.1 +/- 1.4% and 15.4 +/- 2.6%, respectively), as compared with Tar (7.0 +/- 0.9%)-and total lymphocyte (6.9 +/- 1.3%)-cell fractions. These results indicate that ferritin is preferentially distributed in T mu and T gamma lymphocytes and may constitute the basis for explaining some of the roles exercised by these cells in the control of other biological systems.     

Leukotriene A synthase activity of purified mouse skin arachidonate 8-lipoxygenase expressed in Escherichia coli

Mouse skin 8-lipoxygenase was expressed in COS-7 cells by transient transfection of its cDNA in pEF-BOS carrying an elongation factor-1alpha promoter. When crude extract of the transfected COS-7 cells was incubated with arachidonic acid, 8-hydroxy-5,9,11, 14-eicosatetraenoic acid was produced as assessed by reverse- and straight-phase high performance liquid chromatographies. The recombinant enzyme also reacted on alpha-linolenic and docosahexaenoic acids at almost the same rate as that with arachidonic acid. Eicosapentaenoic and gamma-linolenic acids were also oxygenated at 43% and 56% reaction rates of arachidonic acid, respectively. In contrast, linoleic acid was a poor substrate for this enzyme. The 8-lipoxygenase reaction with these fatty acids proceeded almost linearly for 40 min. The 8-lipoxygenase was also expressed in an Escherichia coli system using pQE-32 carrying six histidine residues at N-terminal of the enzyme. The expressed enzyme was purified over 380-fold giving a specific activity of approximately 0.2 micromol/45 min per mg protein by nickel-nitrilotriacetate affinity chromatography. The enzymatic properties of the purified 8-lipoxygenase were essentially the same as those of the enzyme expressed in COS-7 cells. When the purified 8-lipoxygenase was incubated with 5-hydroperoxy-6,8,11, 14-eicosatetraenoic acid, two epimers of 6-trans-leukotriene B4, degradation products of unstable leukotriene A4, were observed upon high performance liquid chromatography. Thus, the 8-lipoxygenase catalyzed synthesis of leukotriene A4 from 5-hydroperoxy fatty acid. Reaction rate of the leukotriene A synthase was approximately 7% of arachidonate 8-lipoxygenation. In contrast to the linear time course of 8-lipoxygenase reaction with arachidonic acid, leukotriene A synthase activity leveled off within 10 min, indicating suicide inactivation.     

Electron microscopic cytochemical localization of alpha-hydroxyacid oxidase in rat kidney cortex. Heterogeneous staining of peroxisomes

The substrate specificity of alpha-hydroxyacid oxidase in the rat kidney has been investigated cytochemically by the cerium technique and biochemically with a luminometric assay applied to isolated renal peroxisomes. Rat kidneys were fixed by perfusion via the abdominal aorta with a low concentration (0.25%) of glutaraldehyde. Vibratome sections were incubated for 60 min at 37 degrees C in a medium containing 3 mM CeCl3, 100 mM NaN3 and 5 mM of an alpha-hydroxyacid in 0.1 M Pipes or 0.1 M Tris-maleate buffer both adjusted to pH 7.8. Ten aliphatic alpha-hydroxyacids with chain lengths between 2 and 8 carbon atoms and two aromatic substrates were tested. The alpha-hydroxyacid oxidase in the kidney exhibited a markedly different substrate specificity than the corresponding enzyme in the liver. Thus glycolate gave a negative reaction while two aromatic substrates, mandelic acid and phenyllactic acid, stained prominently. With aliphatic substrates a stronger reaction was obtained in Pipes than in the Tris-maleate buffered incubation media. The best reaction in the kidney was obtained with hydroxybutyric acid. These cytochemical findings were confirmed by the luminometric determination of the oxidase activity in isolated purified peroxisome fractions. By electron microscopy the electron dense reaction product of cerium perhydroxide was found in the matrix of peroxisomes in the proximal tubules. The intensity of reaction varied markedly in neighbouring epithelial cells but also in different peroxisomes within the same cell. Thus heavily stained particles were seen next to lightly reacted ones. These observations establish the substrate specificity of alpha-hydroxyacid oxidase in the rat kidney and demonstrate the marked heterogeneity in the staining of renal peroxisomes for this enzyme.     

Analysis of enzymatic activities of subcellular and chromatographic fractions by an automated colorimetric microassay system

A simple, automated colorimetric microassay system has been designed to quantitate enzyme activities commonly used as markers for subcellular compartments. This system relies on the spectrophotometric reading of microtiter wells containing the chromophore products. The microassay allows rapid, economical, and quantitative analysis of enzyme activities associated with sucrose or Percoll gradient fractions used for subcellular fractionation studies as well as the screening of a large number of fractions derived from HPLC and other separation columns used for enzyme purification. We describe its use for the quantitation of activities associated with acid and alkaline phosphatases, alkaline phosphodiesterase, beta-glucuronidase, alpha-N-acetylglucosaminidase, alpha-mannosidase, alpha-L-fucosidase, glycosidases, serine esterases, and succinate dehydrogenase, and give the range of their sensitivities. This microassay system has been applied to the isolation of granules of cytolytic lymphocytes and to the identification and purification of a serine esterase from the isolated granules of these cells.     

Biochemical characterization of the reverse activity of rat brain ceramidase. A CoA-independent and fumonisin B1-insensitive ceramide synthase

We have previously purified a membrane-bound ceramidase from rat brain and recently cloned the human homologue. We also observed that the same enzyme is able to catalyze the reverse reaction of ceramide synthesis. To obtain insight into the biochemistry of this enzyme, we characterized in this study this reverse activity. Using sphingosine and palmitic acid as substrates, the enzyme exhibited Michaelis-Menten kinetics; however, the enzyme did not utilize palmitoyl-CoA as substrate. Also, the activity was not inhibited in vitro and in cells by fumonisin B1, an inhibitor of the CoA-dependent ceramide synthase. The enzyme showed a narrow pH optimum in the neutral range, and there was very low activity in the alkaline range. Substrate specificity studies were performed, and the enzyme showed the highest activity with d-erythro-sphingosine (Km of 0.16 mol %, and Vmax of 0.3 micromol/min/mg), but d-erythro-dihydrosphingosine and the three unnatural stereoisomers of sphingosine were poor substrates. The specificity for the fatty acid was also studied, and the highest activity was observed for myristic acid with a Km of 1.7 mol % and a Vmax of 0.63 micromol/min/mg. Kinetic studies were performed to investigate the mechanism of the reaction, and Lineweaver-Burk plots indicated a sequential mechanism. Two competitive inhibitors of the two substrates were identified, l-erythro-sphingosine and myristaldehyde, and inhibition studies indicated that the reaction followed a random sequential mechanism. The effect of lipids were also tested. Most of these lipids showed moderate inhibition, whereas the effects of phosphatidic acid and cardiolipin were more potent with total inhibition at around 2.5-5 mol %. Paradoxically, cardiolipin stimulated ceramidase activity. These results define the biochemical characteristics of this reverse activity. The results are discussed in view of a possible regulation of this enzyme by the intracellular pH or by an interaction with cardiolipin and/or phosphatidic acid.     

Localization of arachidonate 12-lipoxygenase in parenchymal cells of porcine anterior pituitary

12-Lipoxygenases oxygenate arachidonic acid producing its 12S-hydroperoxy derivative and are well known as platelet and leukocyte enzymes. When a peroxidase-linked immunoassay of the enzyme according to the avidin-biotin method was applied to the cytosol fractions from various parts of porcine brain, a considerable amount of the enzyme was found in the anterior pituitary. The enzyme level (about 200 ng/mg cytosol protein) corresponded to about 6% of the enzyme content in porcine peripheral leukocytes. Posterior and intermediate lobes showed about one-tenth of the enzyme level of anterior pituitary. Other parts of porcine brain contained the 12-lipoxygenase in amounts below 7 ng/mg cytosol protein. The cytosol fraction (0.7 mg of protein) of anterior pituitary produced 12S-hydroxy-5,8,10,14-eicosatetraenoic acid from 25 microM arachidonic acid in about 34% conversion at 24 degrees C for 5 min, giving a specific enzyme activity about 3 nmol/min/mg protein. Furthermore, various octadecapolyenoic acids were oxygenated almost as fast as the arachidonate 12-oxygenation. When anterior pituitary was investigated immunohistochemically with anti-12-lipoxygenase antibody, most of the immunostained cells were certain parenchymal cells with granules, which were not blood cells. These biochemical and immunohistochemical results provide a good reason for considering that 12-lipoxygenase does play an important role in pituitary function.     

Tryptophan degradation in transplanted tumor cells undergoing rejection

The depletion of an essential amino acid, tryptophan, caused by indoleamine 2,3-dioxygenase induction in vitro, has been shown to be due to a mechanism that is used in self-defense against inhaled microorganisms and tumor growth. In this communication, we report the results of measuring dioxygenase activity in the peritoneal exudate cells and tumor cytotoxicity at the transplantation loci after in vivo transplantation of tumor cells into the peritoneal cavity of syngeneic or allogeneic strains of mice. The enzyme was induced only when the tumor cells were being rejected from allogeneic animals and no change was observed when the cells continued to grow in syngeneic animals. Furthermore, when the syngeneic tumor cells in a diffusion chamber were i.p. transplanted simultaneously with i.p. injection of allogeneic tumor cells, the enzyme was induced not only in allografted tumor cells but also in the syngeneic tumor cells. Under these conditions, the tumor cells in the diffusion chamber ceased to grow and 50% of the cells were rejected. To determine the type of cells containing the induced enzyme, the peritoneal exudate cells (tumor cells and host cells--mostly small lymphocytes) were separated into six fractions by sedimentation under gravity and by differential centrifugation. Approximately 80% of total enzyme activity was localized in a tumor-rich fraction (98.9% purity), whereas only 0.2% of the activity was found in a lymphocyte-rich fraction (99.5% purity). The localization of indoleamine 2,3-dioxygenase in the tumor cells was confirmed by complement-dependent lysis with specific antibodies against tumor and host cells.     

Cytochemical reactivity of T mu lymphocytes in human lymphatic tissue for dipeptidylaminopeptidase IV

Summary The enzyme dipeptidylaminopeptidase IV (DAP IV; EC 3.4.14.-) was recently shown cytochemically to be confined, in blood and bone marrow, to human T cells bearing, Fc receptors for IgM (T lymphocytes). This observation, confirmed by direct biochemical tests, stimulated us to study the histochemical distribution of DAP IV in normal human lymphatic tissue. In cryostat sections of lymph node, tonsil and thymus, DAP IV was detectable only in lymphocytes, Hassal's corpuscles and the endothelia of vessels. The distribution pattern of DAP IV-positive lymphocytes accorded well with results obtained with human T cell antisera. Compared to cytochemical reactions for other enzymes, such as acid esterase, DAP IV has the advantage that it does not stain monocytes, B lymphocytes or other mononuclear cells. Further, it does not depend on a particular type of staining pattern like, for example, the dot-like reaction product of acid esterase in T lymphocytes. Since the reaction for DAP IV remains more or less unchanged in month-old sections, it is easily adaptable to routine work and has the potentiality of being applied to the diagnosis of T cell lymphomas.     

Linoleic acid isomerase in Lactobacillus plantarum AKU1009a proved to be a multi-component enzyme system requiring oxidoreduction cofactors

Linoleic acid isomerase in Lactobacillus plantarum was found to be a novel multi-component enzyme system widespread in membrane and soluble fractions. The isomerization reaction involved a hydration step, 10-hydroxy-12-octadecenoic acid production from linoleic acid, as part of the reaction, and the hydration reaction was catalyzed by the membrane fraction. Both membrane and soluble fractions were required for the whole isomerization reaction, i.e., conjugated linoleic acid (CLA) production from linoleic acid, and for CLA production from 10-hydroxy-12-octadecenoic acid, a reaction intermediate. The multi-component enzyme system was inhibited by o-phenanthroline, and divalent metal ions such as Ni(2+) and Co(2+) restored activity. Metal oxides such as VO(4)(3+), MoO(4)(2+), and MnO(4)(2+) enhanced activity. The multi-component enzyme systems required oxidoreduction cofactors such as NADH together with FAD or NADPH for total activity.     

Chemical modification of Salmonella typhimurium phosphoribosylpyrophosphate synthetase with 5'-(p-fluorosulfonylbenzoyl)adenosine. Identification of an active site histidine

Liquid chromatographic procedures have been developed for rapidly locating the site of reaction of chemical modification reagents with Salmonella typhimurium 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) synthetase. The enzyme was reacted with the active site-directed reagent 5'-(p-fluorosulfonylbenzoyl)adenosine (FSBA). FSBA bound to the enzyme with an apparent KD of 1.7 +/- 0.4 mM. The enzyme was inactivated during the reaction, and a limiting stoichiometry of 1.2 mol of FSBA/mol of enzyme subunit corresponded to complete inactivation. Inclusion of ATP in the reaction protected the enzyme from inactivation and incorporation of the reagent. Inclusion of ribose 5-phosphate increased the rate of reaction of PRPP synthetase with FSBA. Amino acid analyses of acid hydrolysates of modified enzyme failed to detect any known FSBA-amino acid adducts. Tryptic digestion of 5'-(p-fluorosulfonylbenzoyl)-[3H]adenosine-modified enzyme at pH 7.0 yielded a single radioactive peptide. The peptide, TR-1, was subjected to combined V8 and Asp-N protease digestion, and a single radioactive peptide was isolated. This radioactive peptide yielded the sequence Asp-Leu-His-Ala-Glu, which corresponded to amino acid residues 128-132 in S. typhimurium PRPP synthetase. No radioactivity was associated with any of the phenylthiohydantoin-amino acid fractions, all of which were recovered in good yield. A majority of the radioactivity was found in the waste effluent (64%) and on the glass fiber filter loaded into the sequenator (23%). The lability of the modification and the sequence of this peptide indicate His130 as the site of reaction with FSBA.     

Interaction of bilirubin with human lymphocytes and granulocytes — the effect on bilirubin metabolism

An in vitro degradation of bilirubin by isolated human granulocytes and lymphocytes is described. Changes in the bilirubin concentration during interaction with these cells were determined spectrophotometrically. The dependence of the reaction velocity on the original bilirubin concentration followed an adsorption isotherm, typical of enzyme processes with a Km of 79 microM at a cell concentration of 500/microL. The primary event is the adsorption of bilirubin to the cell surface and, in addition, its detoxication. Cyanide and sonic treatment of cells inhibit the reaction, salicylic acid enhances the cell activity. The bilirubin-detoxicating effect is discussed with respect to the therapy of hyperbilirubinaemia in neonates.