The role of the quaternary structure in the activation of human L-asparaginase.

Human L-asparaginase-like protein 1 (ASRGL1) has hydrolytic activity against L-asparagine and isoaspartyl dipeptides. As an N-terminal nucleophile hydrolase family member, its activation depends on an intramolecular autoprocessing step between G167 and T168. In vitro, autoprocessing reaches only 50% completion, which restrains the activity and hampers the full understanding of the activation process. The ASRGL1 dimer interface plays a critical role in intramolecular processing, and the interactions within oligomers can offer relevant information about autoprocessing. In this work, a fully processed trimeric conformation of ASRGL1 was observed for the first time, and we combined biophysical and structural proteomics assays to characterize trimeric ASRGL1. Our analyses show that oligomerization is critical for autoprocessing, hydrolytic activity and thermal stability. The newest trimeric ASRGL1 conformation enhances protein activity and presents a melting temperature deviation of 4.33 °C in comparison to the monomeric conformation. The interaction of the third monomer in the trimeric conformation is driven by an α-helix comprising residues KVNLARLTLF (227-236).

Parameters of the reproductive tract, spermatogenesis, daily sperm production and major seminal plasma proteins of tropically adapted morada nova rams.

This study describes the reproductive parameters of Morada Nova rams, a breed of hair sheep from Brazil and with unique adaption to tropical environments. At 42 weeks of age, 15 rams were subjected to semen collection and, 1 week later, animals were slaughtered for collection of testes, epididymis and accessory sex glands. We conducted 2-D electrophoresis of seminal plasma proteins and major spots of stained gels were identified by LC-MS/MS. Total RNA was isolated from testis, epididymis and vesicular glands and subjected to qPCR. At slaughter, scrotal circumference and testicular weight were 27.5 ± 0.5 cm and 109.5 ± 6.0 g, respectively. Seminiferous tubule (ST) diameter was 188.3 ± 4.0 µm and each testis contained 1.9 ± 0.1 Sertoli cells (×10(9) ). Each Sertoli cell supported 0.1 ± 0.01 A spermatogonia, 3.0 ± 0.2 pachytene spermatocytes and 7.7 ± 0.5 round spermatids/tubule cross section. Daily sperm production reached 5.6 × 10(6)  cells/g of testis parenchyma. Testis size appeared as indicative of ST diameter and associated with epididymal measurements, as well as with the population of round spermatids and Sertoli cells/testis. Rams with heavier testes had greater daily sperm production and more Sertoli cells/testis. We detected 90.9 ± 9.6 spots per 2-D gel of seminal plasma. Major seminal proteins were identified as ram seminal vesicle proteins at 14 and 22 kDa, representing 16.2% and 12.8% of the total intensity of valid spots in the gels, respectively. Expression of both genes was greater in the vesicular glands as compared to testis and epididymis. Pixel intensity for those proteins in the 2-D gels was significantly correlated with seminal vesicle weight. This is the first description of the basic reproductive aspects of Morada Nova rams, including protein profiles of their seminal plasma. These findings will allow a better understanding of their reproductive physiology.

Chemical composition of lipids present in cat and dog oocyte by matrix-assisted desorption ionization mass spectrometry (MALDI- MS).

The aim of the present study was to investigate the level of information on the chemical structures and relative abundances of lipids present in cat and dog oocytes by matrix-assisted laser desorption mass spectrometry (MALDI-MS). The MALDI-MS approach requires a simple analysis workflow (no lipid extraction) and few samples (two or three oocytes per analysis in this work) providing concomitant profiles of both intact phospholipids such as sphingomyelins (SM) and phosphatidylcholines (PC) as well as triacylglycerols (TAG). The lipids were detected in oocytes by MALDI using dihydroxybenzoic acid (DHB) as the matrix. The most abundant lipid present in the MS profiles of bitch and queen oocytes was a PC containing 34 carbons and one unsaturation [PC (34:1)]. Oocytes of these two species are characterized by differences in PC and TAG profiles detected qualitatively as well as by means of principal component analysis (PCA). Cat oocytes were mainly discriminated by more intense C52 and C54 TAG species and a higher number of unsaturations, indicating predominantly linoleic and oleic fatty acyl residues. Comparison of the lipid profile of bitch and queen oocytes with that of bovine oocytes revealed some similarities and also some species specificity: TAG species present in bovine oocytes were also present in bitches and queens; however, a more pronounced contribution of palmitic, stearic and oleic fatty acid residues was noticed in the lipid profile of bovine oocytes. MALDI-MS provides novel information on chemical lipid composition in canine and feline oocytes, offering a suitable tool to concomitantly monitor, in a nearly direct and simple fashion the composition of phospholipids and TAG. This detailed information is highly needed to the development of improved protocols for in vitro culture and cryopreservation of cat and dog oocytes.

Clonostachys rosea BAFC3874 as a Sclerotinia sclerotiorum antagonist: mechanisms involved and potential as a biocontrol agent.

AIMS: To establish the modes of action of the antagonistic fungal strain Clonostachys rosea BAFC3874 isolated from suppressive soils against Sclerotinia sclerotiorum and to determine its potential as a biocontrol agent. METHODS AND RESULTS: The antagonistic activity of C. rosea BAFC3874 was determined in vitro by dual cultures. The strain effectively antagonized S. sclerotiorum in pot-grown lettuce and soybean plants. Antifungal activity assays of C. rosea BAFC3874 grown in culture established that the strain produced antifungal compounds against S. sclerotiorum associated with secondary metabolism. High mycelial growth inhibition coincided with sclerotia production inhibition. The C. rosea strain produced a microheterogeneous mixture of peptides belonging to the peptaibiotic family. Moreover, mycoparasitism activity was observed in the dual culture. CONCLUSIONS: Clonostachys rosea strain BAFC3874 was proved to be an effective antagonist against the aggressive soil-borne pathogen S. sclerotiorum in greenhouse experiments. The main mechanisms involve peptaibiotic metabolite production and mycoparasitism activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Clonostachys rosea BAFC3874 may be a good fungal biological control agent against S. sclerotiorum. In addition, we were also able to isolate and identify peptaibols, an unusual family of compounds in this genus of fungi.

ACE gene titration in mice uncovers a new mechanism for ACE on the control of body weight.

Mice harboring 1, 2, or 3 copies of the angiotensin-converting enzyme (ACE) gene were used to evaluate the quantitative role of the ACE locus on obesity. Three-copy mice fed with a high-fat diet had lower body weight and peri-epididymal adipose tissue than did 1- and 2-copy mice (P < 0.05). On regular diet, 3-copy mice had to eat more to maintain the same body weight; on a high-fat diet, they ate the same but weighed less than 1- and 2-copy mice (P < 0.05), indicating a higher metabolic rate in 3-copy mice that was not affected by ANG II AT(1) blocker treatment. A catalytically inactive form of thimet oligopeptidase (EC 3.4.24.15; EP24.15) was used to isolate ACE substrates from adipose tissue. Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) identified 162 peptide peaks; 16 peptides were present in both groups (1- and 3-copy mice fed with a high-fat diet), whereas 58 of the 72 unique peptides were found only in the 3-copy mice. Peptide size distribution was shifted to lower molecular weight in 3-copy mice. Two of the identified peptides, LVVYPWTQRY and VVYPWTQRY, which are ACE substrates, inhibited in vitro protein kinase C phosphorylation in a concentration-dependent manner. In addition, neurolysin (EC 3.4.24.16; EP24.16) activity was lower in fat tissue from 3- vs. 1-copy mice (P < 0.05). Taken together, these results provide evidence that ACE is associated with body weight and peri-epididymal fat accumulation. This response may involve the generation of oligopeptides that inhibit the activity of EP24.16 and other oligopeptidases within the adipose tissue.

Primary and secondary kinetic isotope effects in proton (H(+)/D(+)) and chloronium ion ((35)Cl(+)/(37)Cl(+)) affinities.

The Cooks' kinetic method and tandem-in-space pentaquadrupole QqQqQ mass spectrometry were used to measure primary and secondary kinetic isotope effects (KIEs) in H(+) and Cl(+) (X(+)) affinity for a series of A/A(') isotopomeric pairs. Gaseous, isotopomeric, and loosely bound dimers [A...X(+)...A(')] were formed in combinations in which X = H(+), D(+), (35)Cl(+) or (37)Cl(+) and A/A(') = acetonitrile/acetonitrile - d(3), acetonitrile/acetonitrile-(15)N, acetonitrile-d(3)/acetonitrile-(15)N, acetone/acetone-d(6), acetone/acetone-(18)O, acetone-d(6)/acetone-(18)O, pyridine/pyridine-d(5), pyridine/pyridine-(15)N, pyridine-d(5)/pyridine-(15)N, or 3-((35)Cl)chloropyridine/3-((37)Cl)chloropyridine. Under nearly the same experimental conditions, the dimers were mass-selected and then dissociated by low-energy collisions with argon, yielding AX(+) and A(')X(+) as the fragment ions. KIEs were measured from the changes in ion affinities of the neutrals (DeltaX(+)) as estimated by the AX(+)/A(')X(+) abundance ratios. Using [A...H(+)(D(+))...A(')] and [A...(35)Cl(+)((37)Cl(+))...A(')] dimers and by comparing their extent of dissociation under nearly identical collision-induced dissociation conditions, the kinetic method was also applied, for the first time, to measure primary KIEs of the central ion as well as their influence on secondary KIEs. Becke3LYP/6-311++G(2df,2p) calculations were found to provide Delta(DeltaZPE)s for the competitive dissociation reactions that accurately predict the nature (normal or inverse) of the measured KIEs.

Chiroselective self-directed octamerization of serine: implications for homochirogenesis.

Serine undergoes chiroselective self-directed oligomerization to form a singly protonated octamer under positive ion electrospray conditions, as identified by ion trap tandem mass spectrometry. The experiments also show a series of higher-order clusters (metaclusters) corresponding to [(Ser8H)n]n+, n = 1, 2, 3. There is a magic number effect favoring formation of the protonated octamer over its homologues and also a strong preference for octamer formation from homochiral serine molecules. Collision-induced dissociation suggests that the protonated octamer is composed of four hydrogen-bonded dimers, stabilized by further extensive hydrogen bonding. Density functional calculations support this model and show that the protonated homochiral octamer is energetically stabilized relative to its possible fragments (dimer plus protonated hexamer, etc). The calculations also show that heterochiral octamers are less stable than homochiral octamers (e.g., the protonated 7:1 cluster is 2.1 kcal/mol less stable than the 8:0 analogue). The implications of these results for the origin of homochirality are discussed.

Mass spectrometric quantitation of chiral drugs by the kinetic method.

A novel mass spectrometric method for rapid, accurate (2-4% ee) quantitation of chiral drugs is described. Copper(II)-bound complexes of seven model drugs (atenolol, DOPA, ephedrine, pseudoephedrine, isoproterenol, norepinephrine, propranolol) with chiral reference compounds (L-amino acids) are generated by electrospray ionization mass spectrometry. The trimeric complex ions (three chiral ligands--one of the analyte and two of the reference compound) are collisionally activated, and they undergo dissociation by competitive loss of either the neutral reference or the neutral drug molecule. The ratio of the two competitive dissociation rates, viz. the product ion branching ratio, is related via the kinetic method to the enantiomeric composition of the drug mixture. A two-point calibration curve, derived from the kinetic method, allows rapid quantitation of enantiomeric excess of drug mixtures. The chiral sensitivity of the method is such as to allow determination of mixtures with a few percent enantiomeric contamination.

Serine octamer metaclusters: formation, structure elucidation and implications for homochiral polymerization.

Multiply charged serine metaclusters (composed of two or more homochiral octameric units) are generated by electrospray ionization, and their unique fused structures (hydrogen-bonded through the sticky ends of the drum-shaped octameric units) have been elucidated using tandem mass spectrometry experiments and molecular mechanics calculations.

Multiple stage pentaquadrupole mass spectrometry for generation and characterization of gas-phase ionic species. The case of the PyC2H 5 (+·) isomers.

Eleven isomers with the PyC2H 5 (+·) composition, which include three conventional (1-3) and eight distonic radical cations (4-11), have been generated and in most cases successfully characterized in the gas phase via tandem-in-space multiple-stage pentaquadrupole MS(2) and MS(3) experiments. The three conventional radical cations, that is, the ionized ethylpyridines C2H5-C5H4N(+·) (1-3), were generated via direct 70-eV electron ionization of the neutrals, whereas sequences of chemical ionization and collision-induced dissociation (CID) or mass-selected ion-molecule reactions were used to generate the distonic ions H2C(·)-C5H4N(+)-CH3 (4-6), CH3-C5H4N(+)-CH 2 (·) (7-9), C5H5N(+)-CH2CH 2 (·) (10), and C5H5N(+)-CH(·)-CH3 (11). Unique features of the low-energy (15-eV) CID and ion-molecule reaction chemistry with the diradical oxygen molecule of the isomers were used for their structural characterization. All the ion-molecule reaction products of a mass-selected ion, each associated with its corresponding CID fragments, were collected in a single three-dimensional mass spectrum. Ab initio calculations at the ROMP2/6-31G(d, p)//6-31G(d, p)+ZPE level of theory were performed to estimate the energetics involved in interconversions within the PyC2H5 (+·) system, which provided theoretical support for facile 4⇌7 interconversion evidenced in both CID and ion-molecule reaction experiments. The ab initio spin densities for the a-distonic ions 4-9 and 11 were found to be largely on the methylene or methyne formal radical sites, which thus ruled out substantial odd-spin derealization throughout the neighboring pyridine ring. However, only 8 and 9 (and 10) react extensively with oxygen by radical coupling, hence high spin densities on the radical site of the distonic ions do not necessarily lead to radical coupling reaction with oxygen. The very typical "spatially separated" ab initio charge and spin densities of 4-11 were used to classify them as distonic ions, whereas 1-3 show, as expected, "localized" electronic structures characteristic of conventional radical ions.

The ionized methylene transfer from the distonic radical cation (+)CH 2-O-CH 2 to heterocyclic compounds. A pentaquadrupole mass spectrometric study.

Ion-molecule reactions of the mass-selected distonic radical cation (+)CH2-O-CH 2 (·) (1) with several heterocyclic compounds have been investigated by multiple stage mass spectro- metric experiments performed in a pentaquadrupole mass spectrometer. Reactions with pyridine, 2-, 3-, and 4-ethyl, 2-methoxy, and 2-n-propyl pyridine occur mainly by transfer of CH 2 (+·) to the nitrogen, which yields distonic N-methylene-pyridinium radical cations. The MS(3) spectra of these products display very characteristic collision-induced dissociation chemistry, which is greatly affected by the position of the substituent in the pyridine ring. Ortho isomers undergo a δ-cleavage cyclization process induced by the free-radical character of the N-methylene group that yields bicyclic pyridinium cations. On the other hand, extensive CH 2 (+·) transfer followed by rapid hydrogen atom loss, that is, a net CH(+) transfer, occurs not to the heteroatoms, but to the aromatic ring of furan, thiophene, pyrrole, and N-methyl pyrrole. The reaction proceeds through five- to six-membered ring expansion, which yields the pyrilium, thiapyrilium, N-protonated, and N-methylated pyridine cations, respectively, as indicated by MS(3) scans. Ion 1 fails to transfer CH 2 (+·) to tetrahydrofuran, whereas a new α-distonic sulfur ion is formed in reactions with tetrahydrothiophene. Unstable N-methylene distonic ions, likely formed by transfer of CH 2 (+·) to the nitrogen of piperidine and pyrrolidine, undergo rapid fragmentation by loss of the α-NH hydrogen to yield closed-shell immonium cations. The most thermodynamically favorable products are formed in these reactions, as estimated by ab initio calculations at the MP2/6-31G(d,p)//6-31G(d,p) + ZPE level of theory.