Analysis of the importance of arginine 102 in neutral endopeptidase (enkephalinase) catalysis.

Neutral endopeptidase 24.11 contains an active site arginine believed to function in substrate binding. This arginine is thought to form an ionic interaction with the COOH-terminal carboxylate of NEP substrates. The functionality of arginine 102 has been investigated by using site-directed mutagenesis to produce mutants in which this residue was converted to a lysine, glycine, glutamine, or glutamate. All of the mutants exhibited essentially full activity as determined with a synthetic peptide amide, glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide. In contrast, activity was detected only with the wild-type enzyme and the lysine mutant using a synthetic substrate containing a free COOH-terminal carboxylate, dansyl-Gly-Trp-Gly. Inhibition studies with the physiologically active peptide substrates substance P, endothelin, and angiotensin I, as well as substance P free acid, [D-Ala2,Leu5]enkephalin, and [D-Ala2,Leu5]enkephalinamide indicated a lack of importance of arginine 102 in substrate binding. With [D-Ala2,Met5]enkephalin and the chemotactic peptide, N-formyl-Met-Leu-Phe, a significant decrease in affinity is observed with the arginine 102 mutants. These results suggest that the contribution of arginine 102 to substrate binding is dependent upon the strength of other subsite interactions. Examination of dipeptides as inhibitors indicates that the nature and orientation of the P'2 residue is important in determining the strength of the interaction of arginine 102 with its substrates.     

Anti-tumor antibody produced by human tumor-infiltrating and peripheral blood B lymphocytes

Cell suspensions from 69 human tumor biopsies and malignant effusions depleted of infiltrating T cells were incubated for 10–14 days with mitomycin-C-treated cells of the transformed T cell line MOT as feeder cells. B lymphocytes proliferated and differentiated as indicated by immunoglobulin (Ig) seerction in the culture supernatants (B cell expansion). Ig was present in culture supernatants of tumor cell suspensions incubated without MOT feeder cells (non-expanded cells), but the addition of MOT feeder cells to these cultures invariably resulted in a significant increase in Ig concentration. While IgG, IgA. and IgM isotypes were all detected in supernatants of both expanded- and nonexpanded tumor cell suspensions, the increase in total Ig induced by MOT feeder cells was mainly due to an increase in IgG. Peripheral blood B lymphocytes (PBBL) from 15 cancer patients and 4 healthy individuals were also successfully expanded by the same method. In these it was shown that IgA was the predominant Ig isotype. Using a modified enzyme-linked immunosorbent assay, IgG of 25/36 expansions from tumor cell suspensions showed reactivity with autologous tumor targets, and that from 10/13 expansions reacted with allogeneic tumor targets of the same histological diagnosis. No reactivity was found against tumor targets of different histology. IgG of 4/10 expansions of PBBL from cancer patients showed reactivity with allogeneic tumor targets of the same histology, while no reactivity was demonstrated against tumor targets of different histology. IgG of expanded PBBL from healthy individuals showed no reactivity against tumor targets. This method allows detailed study of the specific humoral antitumor immune response of intratumoral and peripheral blood B lymphocytes in cancer.Work supported by grants from the Share and Concern Foundations and grant CA MOPP from the National Institutes of Health, C.J.A.P. is a visiting scientist from the University of Nijmegen, Department of Medical Oncology, Nijmegen, The Netherlands, and is supported by a Fulbright Senior Research Grant and grants from the Dutch Cancer Society and the Regional Cancer Center of the East Netherlands (IKO). J.A.M.B. is a visiting scientist from the University of Sao Paolo, Department of Immunology, Brazil, and is supported by grant 90/1844-4 from the FAPESP     

The hydrolysis of endothelins by neutral endopeptidase 24.11 (enkephalinase)

Endothelins 1-3 are a family of 21-amino acid peptides whose structure consists of two rings formed by intra-chain disulfide bonds and a linear "COOH-terminal tail." These peptides were originally described on the basis of their potent vasoconstrictor activity. The hydrolytic inactivation of endothelin action has recently been implicated to be attributed, at least in part, to the enzyme neutral endopeptidase 24.11 (Scicli, A. G., Vijayaraghavan, J., Hersh, L., and Carretero, O. (1989) Hypertension 14, 353). The kinetic properties and mode of hydrolysis of the endothelins by this enzyme are reported in this study. The Km for endothelins 1 and 3 hydrolysis is approximately 2 microM while endothelin2 exhibits a 5-fold higher Km. Endothelins 1 and 2 exhibit similar Vmax values while endothelin3 is hydrolyzed considerably more slowly. The initial cleavage site in endothelin1 is at the Ser5-Leu6 bond located within one of the cyclic structures. Thermolysin, a bacterial neutral endopeptidase with a similar substrate specificity to neutral endopeptidase 24.11 initially cleaves endothelin1 between His16-Leu17 which lies within the COOH-terminal linear "tail" portion of the molecule. The cleavage of endothelins 2 and 3 by neutral endopeptidase 24.11 differs from that observed with endothelin1 in that cleavage of these endothelins occurs at Asp18-Ile19 within the linear COOH-terminal tail structure. These results demonstrate that the endothelins are good substrates for neutral endopeptidase 24.11 and suggest that their mode of cleavage is dependent upon both amino acid sequence as well as peptide conformation.     

Use of 5-deazaFAD to study hydrogen transfer in the D-amino acid oxidase reaction.

The apoprotein of hog kidney D-amino acid oxidase was reconstituted with 5-deazaflavin adenine dinucleotide (5-deazaFAD) to yield a protein which contains 1.5 mol of 5-deazaFAD/mol of enzyme. The deazaFAD-containing enzyme forms complexes with benzoate, 2-amino benzoate, and 4-aminobenzoate which are both qualitatively and quantitatively similar to those observed with native enzyme. The complex with 2-aminobenzoate exhibits a new long wavelength absorption band characteristic of a flavin charge-transfer complex. The reconstituted enzyme exhibits no activity when assayed by D-alanine oxidation. However, the bound chromophore can be reduced by alanine, phenylalanine, proline, methionine, and valine, but not by glutamate or aspartate, indicating the deazaFAD enzyme retains the substrate specificity of the native enzyme. Reduction of the enzyme by D-alanine exhibits a 1.6-fold deuterium isotope effect. Reoxidation of the reduced enzyme occurred in the presence of pyruvate plus ammonia, but not with pyruvate alone or ammonia alone. beta-Phenylpyruvate and alpha-ketobutyrate, but not alpha-ketoglutarate could replace pyruvate. Reduced enzyme isolated following reaction with [alpha-3H]alanine was found to contain 0.5 mol of tritium/mol of deazaFADH2. After denaturation of the tritium-labeled enzyme, the radioactivity was identified as deazaFADH2. Reaction of the reduced tritium-labeled enzyme with pyruvate plus ammonia prior to denaturation yields [alpha-3H]alanine and unlabeled deazaFAD. These results suggest that reduction and reoxidation of enzyme-bound deazaFAD involves the stereo-specific transfer of alpha-hydrogen from substrate to deazaFAD.     

Studies on the Kinetic Mechanism and Salt Activation of Bovine Brain Choline Acetyltransferase

Abstract: :The kinetic mechanism of bovine brain choline acetyltransferase has been studied using acetylaminocholine as a dead-end inhibitor and di-methylaminoethanol as an alternate substrate. Acetylaminocholine inhibition is competitive with respect to acetylcholine and noncompetitive with respect to choline. Dimethylaminoethanol exhibits one-sixth the V_max obtained with choline. These results suggest that the reaction obeys a sequential random kinetic mechanism. Salt activation of the enzyme is nonspecific with respect to monovalent anions, and results in a parallel increase in the K^m for choline and the Kⁱ for acetylcholine. These results support the conclusion that salt activation of choline acetyltransferase is a nonspecific effect and that no specific chloride ion regulation of this enzyme occurs in vivo .     

Cellular immunity in man: correlation of leukocyte migration inhibition factor formation and delayed hypersensitivity

The formation of leukocyte migration inhibition factor (MIF) by the lymphocytes of 13 normal persons immune to the protein antigen keyhole limpet hemocyanin (KLH) has been investigated. KLH-induced MIF formation expressed as percent migration was compared with delayed hypersensitivity, antibody, and in vitro lymphocyte blastogenic responses to this antigen. Individuals were studied 404–840 days (median 540 days) after their last exposure to KLH. Nine persons had delayed hypersensitivity to KLH and 10 had circulating KLH antibody. The lymphocytes of 11 showed an in vitro blastogenic response to KLH stimulation, while the lymphocytes of nine produced MIF after KLH stimulation. The mean percent migration for the subjects with KLH delayed hypersensitivity was 48.2 (range 20.4–70.4) compared with 133 (range 120–161) for the four persons who did not have KLH delayed hypersensitivity (P < 0.05). The correlation coefficient between the precent migration and delayed hypersensitivity was ?0.78 (P < 0.01). No correlation was demonstrated between migration inhibition and the other parameters of immunity.     

The phosphorylation of choline acetyltransferase

Human placental Choline Acetyltransferase (ChAT) has been shown to be phosphorylated in vitro by kinases present in rat brain. Phosphorylation occurs at a single site with the exclusive phosphoamino acid being serine. ChAT phosphorylation was shown to be calcium, and not cyclic nucleotide, dependent and was inhibited by inhibitors of calcium/calmodulin protein kinases including anti-calmodulin anti-sera. ChAT phosphorylation was stimulated by calmodulin (9 fold) and, to a lesser extent, by phosphatidylserine (4 fold). These results indicate the involvement of a calcium/calmodulin and possibly also a calcium/phosopholipid kinase. This finding was confirmed by demonstrating ChAT phosphorylation using both purified multifunctional calcium/calmodulin protein kinase (CaMK) and calcium/phospholipid protein kinase C (PKC) from rat brain. A stoichiometric incorporation of 0.9 mol phosphate/mol ChAT was achieved by CaMK. Phosphorylated ChAT could be isolated from freshly prepared rat brain synaptosomes. The results obtained with this model system support the hypothesis that in vivo a fraction of ChAT exists phosphorylated.     

Identification of the active-site arginine in rat neutral endopeptidase 24.11 (enkephalinase) as arginine 102 and analysis of a glutamine 102 mutant

Neutral endopeptidase 24.11 contains an active-site arginine residue involved in binding the free carboxylate of substrate peptides and inhibitors. This arginine reacts rapidly with [14C]phenylglyoxal, and its reaction is selectively blocked by the presence of either the substrate Met5-enkephalin, the competitive inhibitor phenylalanylalanine, or the transition state analog phosphoramidon. The phenylglyoxal-modified peptide was isolated by a procedure involving limited digestion by trypsin, separation of the tryptic peptides by high pressure liquid chromatography (HPLC), further digestion of the modified peptide by pepsin, and a final purification by HPLC. By this procedure arginine 102 was identified as the active-site arginine. Verification of this finding came from the use of site-directed mutagenesis in which this arginine was replaced by glutamine. Both the mutant and wild-type enzyme reacted equally well with an amide containing substrate, glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide. However, reaction of the mutant enzyme with a substrate containing a free COOH-terminal carboxylate, 5-dimethylaminonaphthalene-1-sulfonyl-D-Ala-Gly-(NO2)Phe-Gly, was barely detectable with the mutant enzyme. Similarly the mutant enzyme showed a loss of selectivity in inhibition by D-Ala2-Met5-enkephalin compared to the corresponding amide but exhibited no difference in the maximal velocity for hydrolysis of D-Ala2-Met5-enkephalin and its amide.     

Depression of murine natural killer cell cytotoxicity by isobutyl nitrite

Summary We have investigated the effect of isobutyl nitrite on murine NK-cell antitumor-directed cytotoxicity. This agent has been suggested as one of the factors underlying immunodeficiency syndrome (AIDS) in man. We demonstrated that two injections, each of 0.25 ml isobutyl nitrite, resulted in significant depression of endogenous splenic and peripheral blood natural killer (NK) cell cytotoxicity against T-cell lymphoma, YAC-1. In addition to endogenous NK cells, activity of pyrimidinol-activated NK cells was also substantially depressed by this agent. The latter observation is of the utmost importance, since it suggests that the attempt to augment NK-cell activity (to promote resistance to infections and malignancies) could fail in patients with AIDS who are isobutyl nitrite users. Isobutyl nitrite was NK-cell-suppressive not only after in vivo administration but, most importantly, also after inhalation. This indicates that isobutyl nitrite, via its NK-cell suppressive effect, could contribute to immunodeficiency in AIDS. Studies on the mechanism of NK-cell depression by isobutyl nitrite demonstrated that the NK-cell tumor-binding properties as well as NK-cell cytotoxic potential were substantially depressed. Mixing experiments failed to reveal any regulation by suppressor cell activities. The results of these studies clearly indicate that isobutyl nitrite is an immunosuppressive agent and that its use should be avoided. Abbreviations used: NK, natural killer; S-RPMI 1640, supplemented tissue culture medium 1640; HEPES, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; TBC, tumor-binding cells; C-TBC, cytotoxic tumor-binding cells; AIPP, 2-amino-5-iodo-6-phenyl-4 pyrimidinol; LU, lytic units; T:E, target-to-effector; AIDS, acquired immunodeficiency syndrome     

Fate of the nuclear lamina during Caenorhabditis elegans apoptosis

Invertebrates and in Drosophila, lamins and lamin-associated proteins are primary targets for cleavage by caspases. Eliminating mammalian lamins causes apoptosis, whereas expressing mutant lamins that cannot be cleaved by caspase-6 delay apoptosis. Caenorhabditis elegans has a single lamin protein, Ce-lamin, and a caspase, CED-3, that is responsible for most if not all somatic apoptosis. In this study we show that in C. elegans embryos induced to undergo apoptosis Ce-lamin is degraded surprisingly late. In such embryos CED-4 translocated to the nuclear envelope but the cytological localization of Ce-lamin remained similar to that in wild-type embryos. TUNEL labeling indicated that Ce-lamin was degraded only after DNA is fragmented. Ce-lamin, Ce-emerin, or Ce-MAN1 were not cleaved by recombinant CED-3, showing that these lamina proteins are not substrates for CED-3 cleavage. These results suggest that lamin cleavage probably is not essential for apoptosis in C. elegans.