Aminopeptidase B from the rat testis is a bifunctional enzyme structurally related to leukotriene-A4 hydrolase

An aminopeptidase B (Ap-B) was previously purified to homogeneity from rat testis extracts and characterized. In the present work, by using oligonucleotides selected on the basis of partial amino acid microsequences of pure Ap-B and PCR techniques, the nucleotide sequence of a 2.2-kb cDNA was obtained. The deduced amino acid sequence corresponds to a 648-residue protein (72.3 kDa) containing the canonical "HEXXHX18E" signature, which allowed its classification as a member of the M1 family of metallopeptidases. It exhibits 33% identity and 48% similarity with leukotriene-A4 hydrolase, a relation further supported by the capacity of Ap-B to hydrolyze leukotriene A4. Both enzymes also were closely related to a partially sequenced protein from Dictyostelium discoideum, which might constitute the putative common ancestor of either aminopeptidase or epoxide hydrolase, or both. Ap-B and its mRNA were detected in the germ line and in the Sertoli and peritubular cells of the seminiferous tubules. Because the enzyme was found in the medium conditioned by spermatocytes and spermatids and in the acrosome during spermatozoa formation, together these observations suggested an involvement of this exometallopeptidase in the secretory pathway. It is concluded that this ubiquitous enzyme may be involved in multiple processing mechanisms.     

Amino acid residue 104 in an alpha-class glutathione S-transferase is essential for the high selectivity and specificity of the enzyme for 4-hydroxynonenal

By using resonance-enhanced two-photon ionization in a supersonic beam, photoionization spectra of the 0(0)0 band of the first singlet-singlet electronic transition of 35Cl-, 37Cl-, 79Br-, and 81Br-C6H5 have been obtained at a rotational temperature of 1 K. The ions have been analyzed according to their masses in a time-of-flight mass spectrometer. From the spectra of 35Cl- and 37Cl-C6H5 obtained simultaneously, an isotope shift of 0.14 +/- 0.02 cm-1 has been determined. Similarly, from the spectra of 79Br- and 81Br-C6H5 the isotope shift resulted to be smaller than 0.02 cm-1. The spectra obtained have been interpreted by considering the geometrical structure and symmetry of each molecule, and values for the rotational constants and geometrical parameters in the upper and lower states have been obtained in agreement with the experimental results.     

The rat dermorphin-like immunoreactivity is supported by an aminopeptidase resistant peptide

Site-directed antibodies against synthetic related dermorphin peptides were previously produced and characterized. One of them, which specifically recognizes the crucial 'opioid message' (the N-terminal part of the dermorphin molecule (i.e. Tyr-D-Ala-Phe-Gly) was selected in order to detect and locate endogenous dermorphin-like molecules in rat, mouse and guinea pig tissues. Dermorphin-like peptides were found to be present in tissues known to contain peptides such as neurons in the central nervous system, nerve fibers in the gut and B and T immune cells. With all the tissues assayed, the HPLC profile obtained on the immunoreactive material showed the same main peak eluted at a retention time of 32 +/- 1 min. The results of biochemical experiments in which enzymatic treatments were performed on the dermorphin-like immunoreactivity indicate the immunoreactivity is a peptide resistant to aminopeptidase hydrolysis. This finding suggests the presence of a residue conferring resistance to proteolytic processes of this kind, which is likely to be a D-amino acid residue.     

Crossreactivity, efficiency and catalytic specificity of an esterase-like antibody

The antibody D2.3 catalyzes the hydrolysis of several p-nitrobenzyl and p-nitrophenyl esters with significant rate enhancement; product inhibition is observed with the former compounds but not with the latter. Whereas enzyme specificity has been extensively studied by X-ray crystallography, structural data on catalytic antibodies have thus far related only to one of the reactions they catalyze. To investigate the substrate specificity and the substrate relative to product selectivity of D2.3, we have determined the structures of its complexes with two p-nitrophenyl phosphonate transition state analogs (TSAs) and with the reaction product, p-nitrophenol. The complexes with these TSAs, determined at 1.9 A resolution, and that with p-nitrobenzyl phosphonate determined previously, differ mainly by the locations and conformations of the ligands. Taken together with kinetic data, the structures suggest that a hydrogen bond to an atom of the substrate distant by eight covalent bonds from the carbonyl group of the hydrolyzed ester bond contributes to catalytic efficiency and substrate specificity. The structure of Fab D2.3 complexed with p-nitrophenol was determined at 2.1 A resolution. Release of p-nitrophenol is facilitated due to the unfavourable interaction of the partial charge of the nitro group of p-nitrophenolate with the hydrophobic cavity where it is located, and to the absence of a direct hydrogen bond between the product and the Fab. Catalytic specificity and the manner of product release are both affected by interactions with substrate atoms remote from the reaction center that were not programmed in the design of the TSA used to elicit this antibody. Selection of a catalytic antibody that makes use of TSA unprogrammed features has been made practical because of the screening for catalytic efficiency incorporated in the procedure used to obtain it.     

Characterization of the aminopeptidase activity of epidermal leukotriene A4 hydrolase against the opioid dynorphin fragment 1-7

Leukotriene A4 hydrolase is a bifunctional cytosolic enzyme, which both hydrolyses leukotriene A4 (LTA4) into leukotriene B4 (LTB4) and exerts aminopeptidase activity against opioid peptides. In the present study we have investigated whether the peptides angiotensin I and II, bradykinin, kallidine, histamine, dynorphin fragment 1-7 and substance P can act as substrates for epidermal and neutrophil LTA4 hydrolase. Among the tested substrates, dynorphin fragment 1-7 was found to be the best substrate for the enzyme. The aminopeptidase activity of epidermal and neutrophil LTA4 hydrolase against dynorphin fragment 1-7 was further characterized. The enzyme was purified from human epidermis and human neutrophils by anion exchange chromatography (Q-Sepharose) and affinity chromatography on a column with the LTA4 hydrolase inhibitor bestatin coupled to AH-Sepharose. The incubation of the dynorphin fragment 1-7 with LTA4 hydrolase resulted in the formation of tyrosine. The presence of the N-terminal amino acid tyrosine is essential for the interaction of opioids with their receptors, and this finding indicates that the LTA4 hydrolase can inactivate dynorphin fragment 1-7. After the two purification steps no other aminopeptidases acting at the N-terminal tyrosine of dynorphin fragment 1-7 was present in the preparation. This was demonstrated by the abolishment of the degradation at the N-terminal end of dynorphin fragment 1-7 when preincubating the enzyme preparation with LTA4 before the incubation with the dynorphin fragment 1-7. The abolishment of the aminopeptidase activity shows that activation of the hydrolase part of the enzyme, with conversion of LTA4 into the potent proinflammatory compound LTB4, results in an inhibition of the aminopeptidase activity of the enzyme. As a result, the catabolism of dynorphin fragment 1-7 and probably of other opioid peptides is inhibited, resulting in sustained biological effects of these opioids. This phenomenon may be important for the maintenance of inflammation in skin conditions, such as psoriasis and atopic dermatitis, in which LTB4 is formed.     

Prolyl aminopeptidase is not a sulfhydryl enzyme: identification of the active serine residue by site-directed mutagenesis

Prolyl aminopeptidase (PAP) has been classified as a sulfhydryl enzyme on the basis of its high sensitivity to p-chloromercuribenzoate and heavy metals. Recently, however, the possibility of PAP being instead a serine enzyme has been raised as a result of two observations--the conservation of some residues among the PAPs hitherto sequenced, and a similarity to some serine hydrolases. This is the first report describing an attempt to identify the active residue by site-directed mutagenesis. The pap genes from Bacillus coagulans and Aeromonas sobria, were used for the study. The changes made were Cys62Ser and Ser101Ala for the first enzyme, and Thr92Ala and Ser146Ala for the second. For both enzymes, only the changes made on the serine residues resulted in their complete inactivation, indicating that PAP is a serine peptidase.     

The specificity requirements of bacteriophage T4 lysozyme

The role of the sinoatrial ring bundle (SARB) in internodal conduction was examined by the microelectrode technique in excised rabbit hearts. The spread of the sinus impluse to the surrounding tissues was shown to proceed anteriorly toward the right branch of the crista terminalis significantly faster than toward the other direction. Thus the right SARB and the right branch of the crista terminalis close to the sinus node were the earliest areas excited by the sinus impulse in the areas surrounding the sinus node. It was further shown that the activation sequence does not initiate from the right SARB to the right branch of the crista terminalis via the junction of these two structures. Cutting the SARB did not produce any delay in conduction from the sinus node to the atrioventricular (AV) node. The conduction velocity measured at the endocardial surface by two microelectrodes has proved that conduction in the crista terminalis was significantly faster than in the SARB. The upstroke of the action potential from the crista terminalis was also steeper than that from the SARB. These results suggest that the SARB is not the main route for impulse propagation from the sinus node to the AV node; the fastest internodal conduction therefore takes place with wide wave fronts, along the crista terminalis.     

The amino acid following an asn-X-Ser/Thr sequon is an important determinant of N-linked core glycosylation efficiency

Many eukaryotic proteins are modified by Asn-linked (N-linked) glycosylation. The number and position of oligosaccharides added to a protein by the enzyme oligosaccharyltransferase can influence its expression and function. N-Linked glycosylation usually occurs at Asn residues in Asn-X-Ser/Thr sequons where X not equal Pro. However, many Asn-X-Ser/Thr sequons are not glycosylated or are glycosylated inefficiently. Inefficient glycosylation at one or more Asn-X-Ser/Thr sequons in a protein results in the production of heterogeneous glycoprotein products. These glycoforms may differ from one another in their level of expression, stability, antigenicity, or function. The signals which control the efficiency of N-linked glycosylation at individual Asn residues have not been fully defined. In this report, we use a site-directed mutagenesis approach to investigate the influence of the amino acid at the position following a sequon (the Y position, Asn-X-Ser/Thr-Y). Variants of rabies virus glycoprotein containing a single Asn-X-Ser/Thr sequon at Asn37 were generated. Variants were designed with each of the twenty common amino acids at the Y position, with either Ser or Thr at the hydroxy (Ser/Thr) position. The core glycosylation efficiency of each variant was quantified using a cell-free translation/glycosylation system. These studies reveal that the amino acid at the Y position is an important determinant of core glycosylation efficiency.     

The amino acid at the X position of an Asn-X-Ser sequon is an important determinant of N-linked core-glycosylation efficiency

N-Linked glycosylation is a common form of protein processing that can profoundly affect protein expression, structure, and function. N-Linked glycosylation generally occurs at the sequon Asn-X-Ser/Thr, where X is any amino acid except Pro. To assess the impact of the X amino acid on core glycosylation, rabies virus glycoprotein variants were generated by site-directed mutagenesis with each of the 20 common amino acids substituted at the X position of an Asn-X-Ser sequon. The efficiency of core glycosylation at the sequon in each variant was quantified in a rabbit reticulocyte lysate cell-free translation system supplemented with canine pancreas microsomes. The presence of Pro at the X position completely blocked core glycosylation, whereas Trp, Asp, Chi, and Leu were associated with inefficient core glycosylation. The other variants were more efficiently glycosylated, and several were fully glycosylated. These findings demonstrate that the X amino acid is an important determinant of N-linked core-glycosylation efficiency.     

Redesigning the substrate specificity of an enzyme by cumulative effects of the mutations of non-active site residues

Directed evolution was used to change the substrate specificity of aspartate aminotransferase. A mutant enzyme with 17 amino acid substitutions was generated that shows a 2.1 x 10(6)-fold increase in the catalytic efficiency (kcat/Km) for a non-native substrate, valine. The absorption spectrum of the bound coenzyme, pyridoxal 5'-phosphate, is also changed significantly by the mutations. Interestingly, only one of the 17 residues appears to be able to contact the substrate, and none of them interact with the coenzyme. The three-dimensional structure of the mutant enzyme complexed with a valine analog, isovalerate (determined to 2.4-A resolution by x-ray crystallography), provides insights into how the mutations affect substrate binding. The active site is remodeled; the subunit interface is altered, and the enzyme domain that encloses the substrate is shifted by the mutations. The present results demonstrate clearly the importance of the cumulative effects of residues remote from the active site and represent a new line of approach to the redesign of enzyme activity.     

Probing the substrate specificity of an enzyme catalyzing inactivation of streptogramin B antibiotics using LC-MS and LC-MS/MS

LC-MS and LC-MS/MS analyses indicated that an enzyme responsible for inactivating the antibiotic etamycin is specific for streptogramins and acts on both type B-I and B-II streptogramin subgroups. No enzymatic activity was detected for other cyclodepsipeptides such as surfactins and viscosin. It was demonstrated using analogs of etamycin that the picolinyl moiety is essential to obtain enzyme-generated ring-opened compounds. Because the picolinyl moiety is also essential for the biological activity of streptogramins, it is proposed that this residue is a distinctive topographic feature in the binding of this group of antibiotics to enzyme active sites.     

Cleavage of rhesus rotavirus VP4 after arginine 247 is essential for rotavirus-like particle-induced fusion from without

We recently described our finding that recombinant baculovirus-produced virus-like particles (VLPs) can induce cell-cell fusion similar to that induced by intact rotavirus in our assay for viral entry into tissue culture cells (J. M. Gilbert and H. B. Greenberg, J. Virol. 71:4555-4563, 1997). The conditions required for syncytium formation are similar to those for viral penetration of the plasma membrane during the course of viral infection. This VLP-mediated fusion activity was dependent on the presence of the outer-layer proteins, viral protein 4 (VP4) and VP7, and on the trypsinization of VP4. Fusion activity occurred only with cells that are permissive for rotavirus infection. Here we begin to dissect the role of VP4 in rotavirus entry by examining the importance of the precise trypsin cleavage of VP4 and the activation of VP4 function related to viral entry. We present evidence that the elimination of the three trypsin-susceptible arginine residues of VP4 by specific site-directed mutagenesis prevents syncytium formation. Two of the three arginine residues in VP4 are dispensable for syncytium formation, and only the arginine residue at site 247 appears to be required for activation of VP4 functions and cell-cell fusion. Using the recombinant VLPs in our syncytium assay will aid in understanding the conformational changes that occur in VP4 involved in rotavirus penetration into host cells.     

Membrane localisation of a UDP-sugar hydrolase, in Salmonella, is by an uncleaved N-terminal signal peptide

Many immunologic aspects of atopic dermatitis have been studied, but basic pathobiologic mechanisms of this disease remain unknown. In this study, we measured the production of interleukin-6 (IL-6) by peripheral blood T cells and monocytes from patients with atopic dermatitis in comparison to normal control subjects and patients with chronic psoriasis. We found that peripheral blood T cells isolated from patients with atopic dermatitis produced significantly higher levels of IL-6 (36.1 +/- 5.1 units/ml, n = 22) than T cells derived from either normal subjects (12.6 +/- 1.9 units/ml, n = 22) or patients with chronic psoriasis (26.7 +/- 4.1 units/ml, n = 7). T-cell activation was also measured in the patients with atopic dermatitis by soluble serum IL-2 receptor levels and were found to be significantly higher (623.7 +/- 8.1 units/ml, n = 8) than normal subjects (357.2 +/- 26.0 units/ml, n = 8). In contrast to the increased production of IL-6 by T cells in atopic dermatitis, there was no significant difference in the IL-6 production by peripheral blood monocytes derived from patients with atopic dermatitis compared to normal subjects. Thus, peripheral blood T cells derived from patients with AD spontaneously produce increased amounts of IL-6 compared to T cells from normal subjects, which may reflect the increased activation state of T cells in atopic dermatitis. These data support the concept that activated T cells or subsets of T cells may be important effector cells in mediating inflammatory activity in atopic disease.     

The differential specificity of chymotrypsin A and B is determined by amino acid 226

The A and B isoforms of the pancreatic serine proteinase, chymotrypsin are known to cleave substrates selectively at peptide bonds formed by some hydrophobic residues, like tryptophan, phenylalanine and tyrosine. We found, however, that the B forms of native bovine and recombinant rat chymotrypsins are two orders of magnitude less active on the tryptophanyl than on the phenylalanyl or tyrosyl substrates, while bovine chymotrypsin A cleaves all these substrates with comparable catalytic efficiency. Analysing the structure of substrate binding pocket of chymotrypsin A prompted us to perform an Ala226Gly substitution in rat chymotrypsin B. The specificity profile of the Ala226Gly rat chymotrypsin B became similar to that of bovine chymotrypsin A suggesting that only the amino acid at sequence position 226 is responsible for the differential specificities of chymotrypsin A and B isoenzymes.     

Effects of ions on adenosine binding and enzyme activity of purified S-adenosylhomocysteine hydrolase from bovine kidney

The present investigation was undertaken to determine the effect of various ions on the characteristics of S-adenosylhomocysteine (SAH) hydrolase from bovine kidney. The binding sites of [3H]-adenosine to purified SAH hydrolase were not influenced by phosphate, magnesium, potassium, sodium, chloride or calcium ions at physiological cytosolic concentrations. To test whether NAD+ in the SAH hydrolase is essential for adenosine binding, we prepared the apoenzyme by removing NAD+ with ammonium sulfate. The resulting apoenzyme did not exhibit any [3H]-adenosine binding. Since the apoenzyme was enzymatically inactive, it is suggested that adenosine binds to the active site and not to an allosteric site of the intact enzyme. The kinetics of the hydrolysis and the synthesis of SAH catalyzed by the enzyme SAH hydrolase were measured in the presence and absence of phosphate and magnesium. Phosphate increased the Vmax for both synthesis and hydrolysis. However, only the affinity of adenosine for SAH synthesis was significantly enhanced from 10.1+/-1.3 microM to 5.4+/-0.5 microM by phosphate. This effect was already maximal at a phosphate concentration of 1 mM. All other tested ions were without effect on the enzyme activity. Our results show that phosphate at physiological concentrations shifts the thermodynamic equilibrium of SAH hydrolase in the direction of SAH synthesis. These findings imply that SAH-sensitive transmethylation reactions are inhibited during renal hypoxia when intracellular levels of phosphate, adenosine, and SAH are elevated.