Stress susceptibility in pigs supplemented with ractopamine.

Ractopamine is a ß-adrenergic agonist used as an energy repartitioning agent in the diets of finishing pigs. Most ractopamine studies are limited to evaluations of growth performance and meat quality, and there is little information on the effects of this additive on the behavior and welfare of pigs. Therefore, the objective of this study was to evaluate various indicators of stress caused by feeding diets containing ractopamine. One hundred seventy barrows and 170 gilts weighing 107.3 kg were allocated to 30 pens with 10 to 12 barrows or gilts per pen. Pigs were offered 1 of the 3 dietary treatments (0, 5, or 10 mg ractopamine/kg) for 28 d with 5 barrow pens and 5 gilt pens per treatment. Pigs were evaluated for behavior 3 d per week 1 wk before the initiation of the experiment and throughout the experiment. Each pig was classified into 1 of the 13 activities (drinking water, lying alone, lying in clusters, standing, nosing pig, sitting, feeding, biting pig, walking, exploring, running away, playing, and mounting pen mates) and also grouped into 1 of the 3 categories (calm, moving, and feeding themselves) based on those activities. At the end of the experiment, 3 pigs from each pen were slaughtered, and blood samples were collected during exsanguination to determine physiological indicators of stress (cortisol, lactate, and creatine-kinase enzymes). The incidence of skin and carcass lesions was determined at shoulder, loin, and ham. Ractopamine had no effect (P > 0.05) on pig behavior, total number of skin and carcass lesions, or blood concentrations of cortisol or lactate. However, there was an increase (P < 0.05) of creatine kinase concentrations in pigs receiving ractopamine-supplemented feed. This finding is consistent with the concept that ractopamine may cause muscular disorders, and this warrants further investigation.

Meat quality of swine supplemented with ractopamine under commercial conditions in Brazil.

Commercial crossbred barrows and gilts (n = 340) were used to study the effects of different dietary inclusions of ractopamine hydrochloride (RAC) on quality of LM and semimembranosus muscle (SM). Pigs were blocked by BW (107.3 ± 0.76 kg) and allotted to gender-specific pens (10 to 12 pigs/pen), and within blocks, pens of barrows or gilts (10 pens/treatment) were randomly assigned to 1 of 3 dietary RAC inclusions (0, 5, or 10 mg/kg) fed during the last 28 d before slaughter. Initial (45-min) and ultimate (24-h) pH and temperature were measured in LM and SM. Visual and instrumental [lightness (L*), redness (a*), and yellowness (b*) values] color as well as drip loss percentages were measured in both muscles after the 24-h chilling period at 1 to 4 °C. The LM was also evaluated for marbling, and samples of the LM were used to measure intramuscular fat (IMF) content, cooking losses, and Warner-Bratzler shear force (WBSF). Pork quality characteristics of the LM (P ≥ 0.227) and SM (P ≥ 0.082) did not differ between barrows and gilts. Furthermore, neither pH nor temperature of the LM (P ≥ 0.164) or SM (P ≥ 0.284) was affected by feeding pigs RAC. The LM from pigs fed 10 mg/kg of RAC received lesser (P = 0.032) subjective color scores than LM from pigs fed 0 and 5 mg/kg of RAC, and LM from pigs fed 10 mg/kg of RAC was less (P = 0.037) red than LM from pigs fed 0 mg/kg of RAC. In addition, SM from pigs fed 10 mg/kg of RAC had lesser (P ≤ 0.015) a* and b* values than pork from control-fed pigs; however, L* values for LM and SM were not (P ≥ 0.081) affected by dietary RAC. Drip loss percentages of the LM were similar (P = 0.815) among RAC treatments, but the SM from RAC-fed pigs had smaller (P = 0.020) drip loss percentages than SM from pigs fed 0 mg/kg of RAC. Marbling scores and IMF content of the LM did not (P ≥ 0.133) differ among RAC treatments; however, WBSF values were greater (P = 0.005) for LM chops from pigs fed 10 mg/kg than chops from pigs fed 0 and 5 mg/kg of RAC. Even though feeding barrows and gilts 10 mg/kg of dietary RAC reduced (P = 0.050) cooking losses of LM chops compared with feeding 5 mg/kg of RAC, including 10 mg/kg of RAC in the diet of finishing pigs reduced pork tenderness. Therefore, results from this study support the recommendation that including 5 mg/kg of RAC in finishing diets should improve live pig performance without detrimental effects on fresh pork quality and cooked pork palatability.

ACE inhibitor-based treatment and antihypertensive effects in patients with type 2 diabetes.

AIM: The presence of hypertension significantly increases cardiovascular risk in diabetic patients. Different classes of antihypertensive drugs, by targeting different pathophysiological mechanisms and therapeutic targets, might provide different antihypertensive effects. The authors speculated that drugs specifically targeting the renin-angiotensin-aldosterone system provide better antihypertensive control than other therapeutic agents. METHODS: Fifty consecutive type 2 diabetic patients with hypertension (M:F 29:21) were followed for 3-9 yrs. Antihypertensive treatment was stable for the last 12 months and included angiotensin convertying enzyme (ACE) inhibitors (ACEI) alone in 8 patients (group IA), ACEI combined with other drugs in 11 patients (group IB) and non-ACEI treatment in 31 patients (group II), 23 of whom were treated with Ca-channel blockers and 8 were treated with beta-blockers alone or with diuretics. During the last month of the study a 3-7 days antihypertensive drugs wash-out was performed. Measurements were performed in sitting position in the same ambulatory conditions, in supine position after 20 min of absolute rest, and in motionless standing station after quickly rising up from sitting rest. RESULTS: Groups IA, IB, and II had similar blood pressure values during antihypertensive therapy within the last year. However, blood pressure values after antihypertensive drug wash-out were significantly higher in groups IA and IB vs. group II (SBP and DBP resting sitting position, P=0.039 and P=0.014 respectively; SBP and DBP in standing position, P=0.001 and P=0.016, respectively). CONCLUSION: These data show that the underlying condition in terms of pathophysiologic mechanisms is more severe in groups IA and IB, including a greater increase of peripheral resistance. Thus we may conclude that the antihypertensive effect of ACEI is greater than other classes of antihypertensive drugs.

Modelling neurotransmitter functions: a laser spectroscopic study of (1S,2S)-N-methyl pseudoephedrine and its complexes with achiral and chiral molecules.

Wavelength and mass resolved resonance-enhanced two photon ionization (R2PI) excitation spectra of (1S,2S)-N-methyl pseudoephedrine (MPE) and its complexes with several achiral and chiral solvent molecules, including water (W), methyl (R)-lactate (L(R)), methyl (S)-lactate (L(S)), (R)-2-butanol (B(R)), and (S)-2-butanol (B(S)), have been recorded after a supersonic molecular beam expansion and examined in the light of ab initio calculations. The spectral patterns of the selected complexes have been interpreted in terms of the specific hydrogen-bond interactions operating in the diastereomeric complexes, whose nature in turn depends on the structure and the configuration of the solvent molecule. The obtained results confirm the view that a representative neurotransmitter molecule, like MPE, "communicates" with the enantiomers of a chiral substrate through different, specific interactions. These findings can be regarded as a further contribution to modelling neurotransmitter functions in biological systems.

Chiral clusters in a supersonic beam: R2PI-TOF spectroscopy of diastereomeric carboxylic esters/(R)-(+)-1-phenyl-1-propanol complexes.

Wavelength and mass resolved resonance-enhanced two photon ionization (R2PI) excitation spectra of (R)-(+)-1-phenyl-1-propanol (P(R)) and its complexes with some chiral esters, i.e. methyl lactates (L(R) and L(S)), methyl 3-hydroxybutyrates (H(R) and H(S)), and methyl 2-chloropropionates (C(R) and C(S)), have been recorded after a supersonic molecular beam expansion and interpreted in the light of DFT calculations. The spectral features of the selected complexes were found to depend on the nature of hydrogen-bond interactions within the diasteromeric complexes, whose intensity in turn depends upon the structure and the configuration of the estereal moiety. The study further confirms resonant two-photon ionization spectroscopy, coupled with time-of-flight mass resolution (R2PI-TOF), as an excellent tool for gathering valuable information on the interactive forces in molecular clusters and for the enantiodiscrimination of chiral molecules in the gas phase.

Enantiodiscrimination of chiral alpha-aminophosphonic acids by mass spectrometry.

Diastereomeric clusters between first-group metal ions (M(+)) and chiral alpha-aminophosphonic acids (A and B) have been readily generated in the gas phase by electrospray ionization (ESI) and their fragmentation investigated by mass spectrometry. The complexes studied had the general formula [MA(S)B(2)](+) and [MA(R)B(2)](+), where M = H, Li, Na, or K, A(S) and A(R) are the two enantiomers of a given acid A, and B is a reference alpha-aminophosphonic acid of defined configuration. Collision-induced decomposition (CID) of [MA(S)B(2)](+) and [MA(R)B(2)](+) leads to fragmentation patterns characterized by [MAB](+)/[MB(2)] abundance ratios which depend on the configuration of ligand A. These different spectral features were correlated to the different stability of the diastereomeric [MA(S)B(2)](+) and [MA(R)B(2)](+) complexes in the gas phase. The results have been discussed in the light of MM2 Molecular Mechanics Force Field calculations.

R2PI Study of intermolecular hydrogen bond in solvent-free chiral complexes.

One- and two-color, mass selected R2PI spectra of the S(1)<--S(0) transitions in the bare (+)-(R)-1-phenyl-1-ethanol (E(R)) and its complexes with different solvent molecules (solv) (-)-(R)-2-butanol (B(R)) or (+)-(S)-2-butanol (B(S)) and (-)-(R)-2-butylamine (A(R)) or (+)-(S)-2-butylamine (A(S)), have been recorded after a supersonic molecular beam expansion. The one-color R2PI excitation spectra of the diastereomeric complexes are characterized by significant shifts of their band origin relative to that of bare E(R). The extent and the direction of these spectral shifts are found to depend on the structure and the configuration of solv and are attributed to different short-range interactions in the ground and excited states of the complexes. In analogy with other diastereomeric complexes, the phenomenological binding energy of the homochiral cluster is found to be greater than that of the heterochiral one.

Avaliaçäo de testes de ELISA para diagnóstico sorológico de infecçöes pelos sorotipos 3, 5 e 7 de Actinobacillus pleuropneumoniae em suínos / Evaluation of ELISAs for the diagnosis of Actinobacillus pleuropneumoniae infections serotypes 3, 5 e 7 in pigs

Compararam-se dois antígenos no teste de ELISA para o diagnóstico sorológico dos sorotipos 3, 5 e 7 de A. pleuropneumoniae prevalentes no Brasil. Um compunha-se de da fase aquosa da extraçäo fenólica de suspensäo bacteriana (FAF) e o outro de lipopolissacarídeos de cadeia longa (LPS-CL). Com esses antígenos foram padronizados ELISAs monovalente e polivalente para os sorotipos prevalentes no Brasil. Com os resultados dos testes de um conjunto de amostras de soro de suínos livres de infecçäo para A. pleuropneumoniae e um conjunto de amostras de soro obtidas de leitöes inoculados com os sorotipos citados e que apresentaram soroconversäo, determinaram-se as equaçöes discriminantes para os conjuntos de soros e compararam-se os testes quanto à distância generalizada de Mahalanobis, ao coeficiente de determinaçäo, ao teste F, ao coeficiente global, à sensibilidade e à especifidade. Na análise desses parâmetros observou-se que o antígeno FAF foi superior. Com o ELISA PFAF reaçöes positivas näo esperadas foram observadas com animais inoculados com os sorotipos heterólogos 2 e 9, näo observadas com o PLPS-CL. A sensibilidade dos testes polivalentes ficou entre 91,5 e 95,7 por cento com especifidade similar, indicando que ambos os testes säo adequados para triagem sorológica uma vez que detectam os sorotipos 3, 4, 5, 6, 7 e 8

Coccidiose e desempenho em frangos com anticoccidianos na raçäo a partir de diferentes idades / Coccidiosis and perfomance in broilers with anticoccidial medicated feed starting at different ages

Estudou-se o efeito da idade das aves, da reutilizaçäo da cama vegetal e de programas anticoccidianos na coccidiose e desempenho de frangos. Utilizaram-se quatro lotes de 24 pintos cada (um dia de idade) submetidos a quatro tratamentos (T): T1 - aves näo tratadas com anticoccidiano, T2 - aves medicadas com anticoccidiano a partir do 14º dia de vida, T3 - aves medicadas com anticoccidiano a partir do sétimo dia de vida, T4 - aves medicadas com anticoccidiano a partir do primeiro dia de vida. As contagens de oocistos aumentaram com a reutilizaçäo da cama, no terceiro lote. T1 (sem anticoccidiano) e T4 (anticoccidiano desde o primeiro dia) apresentaram as maiores contagens. Os escores por Eimeria acervulina aumentaram no terceiro lote e baixaram no quarto lote. Os escores por E. maxima aumentaram no segundo lote, e deste para o terceiro e quarto lotes. Os escores por E. tenella aumentaram no terceiro e quarto lotes. Com desafio alto, como ocorrido no terceiro lote, as medicaçöes anticoccidianas a partir do primeiro, sétimo e 14º dias resultaram em desempenhos semelhantes. Com desafios baixos ou moderados, como ocorridos nos lotes 1, 2 e 4, a medicaçäo a partir do 14º dia resultou em melhores desempenhos. Isso sugere que, em condiçöes de baixo desafio, como em frangos criados em cama de primeiro uso e em granjas com aves de uma única idade, a medicaçäo anticoccidiana a partir de 14º pode ser vantajosa em relaçäo aos programas de medicaçäo a partir do primeiro dia

Variability of resistance in goats infected with Haemonchus contortus in Brazil.

The variability between and within breeds with respect to nematode egg counts (EPG), packed cell volume (PCV) and hemoglobin (Hb) was studied in 36 yearling female goats of the Caninde (15), Bhuj (6) and Anglo-Nubian (15) breeds, exposed to Haemonchus contortus. Nematode-free goats were turned to a contaminated paddock in late February. From then on, fecal egg per gram counts (EPG), packed cell volume (PCV) and hemoglobin (Hb) were determined at 2-week intervals up to Week 18. The EPG, transformed as [log(EPG + 75)], varied (P < 0.01) between goats within breeds and between weeks of exposure, but not between goat breeds (P > 0.05). PCV and Hb were affected by goat breeds (P < 0.05), by goats within breeds (P < 0.01) and by weeks of exposure (P < 0.01). Anglo-Nubians had higher (P < 0.01) PCV and Hb than Caninde; Bhuj had intermediate values. There were two EPG rises; one between Weeks 6 and 10 and the other between Weeks 14 and 16. The within breed variability was marked during the EPG rise on Week 6, when individual egg counts ranged from 130 to 2500. The EPG rises coincided with drops in Hb. PCV presented a similar trend, though not as marked. Haemonchus was responsible for more than 95% of nematode eggs counted. Considering the goat as experimental unit, the correlation coefficients (r) were: -0.45, P = 0.0064, between log(EPG + 75) and PCV; and -0.53, P = 0.0009, between log(EPG + 75) and Hb. The negative correlation between egg counts and blood values suggested breed differences in PCV and Hb were related to resistance to H. contortus infection and/or to its effects.

Comparaçäo de métodos de isolamento de bactérias NAD-dependentes do trato respiratório superior de suínos sadios / Methods to isolate NAD-dependent bacteria from the upper respiratory tract of health pigs

Secreçöes nasais, tonsilares e tecido tonsilar foram coletados de 67 leitöes de 9 a 15 semanas de idade, provenientes de três rebanhos positivos para Actinobacillus pleuropneumoniae (App), e de 50 leitöes provenientes de dois rebanhos, negativos. Foram classificados como positivos aqueles rebanhos com isolamento prévio de sorotipos 3, 5 e 7 e rebanhos negativos aqueles submetidos a controle veterinário, sem notificaçäo de sintomas clínicos, lesöes de pleuropneumonia suína e sem isolamento do agente. O material coletado foi submetido a três diferentes métodos de cultivo: 1- semeadura direta em meio de cultivo sólido seletivo; 2- diluiçäo em caldo seletivo seguido de subsemeadura em meio de cultivo sólido seletivo; 3- diluiçäo em caldo seletivo seguido de subsemeadura em ágar sangue. Entre as amostras NAD-dependentes recuperadas 86 foram classificadas como App, 13 como grupo minor e 21 como grupo taxon (C, D, E e F). Dos rebanhos positivos foram recuperadas quatro amostras de App (sorotipos 3, 7 e 12) e 51 näo sorotipificáveis. Dos rebanhos negativos foram recuperadas 31 amostras de App näo sorotipificáveis, indicando que o App faz parte da flora normal do trato respiratório superior dos suínos. O melhor método de isolamento de amostras NAD-dependentes de leitöes portadores foi da biópsia de tecido tonsilar semeado diretamente em meio sólido seletivo (PPLO ágar adicionado de 2 mica grama de cristal violeta, 10 mica grama NAD, 1 mica grama de lincomicina, 1,4 mica grama de bacitracina por ml)

Utilizaçäo de um teste de ELISA polivalente para detecçäo de anticorpos contra Actinobacillus pleuropneumoniae (App) / Utilization of a polyvalent ELISA test for detection antibodies against of Actinobacillus pleuropneumoniae

Um teste de ELISA polivalente baseado em lipopolissacarídeos de cadeia longa (LPS - CL) de Actinobacillus pleuropneumoniae (App) sorotipos 3, 5 e 7 foi avaliado testando-se amostras do soro de leitöes e matrizes provenientes de 10 rebanhos positivos e de 10 rebanhos negativos. Foram classificados como positivos aqueles rebanhos com isolamento prévio do App sorotipos 3, 5 ou 7 e rebanhos negativos aqueles submetidos ao controle veterinário, sem notificaçäo de sintomas clínicos, sem lesöes de pleuropneumonia suína e sem isolamento do agente. Todos os rebanhos positivos apresentaram sorologia positiva e as matrizes apresentaram maior número de soroconversores (P<0,05) do que os leitöes. Entre os rebanhos negativos quatro apresentaram sorologia negativa, cinco sorologia positiva com valores preditivos altos (96 a 99 por cento) e um com valor preditivo considerado baixo (56 por cento). O teste apresentou 100 por cento de sensibilidade e aparentemente baixa especificidade, porém, como detectou os sorotipos prevalentes no Brasil e sorotipos que possuem LPS - CL homólogos (3, 4, 5, 6, 7 e 8), ele é aplicável somente como teste de triagem

Efeito de pré-infusöes uterinas sobre a fertilidade de Marräs (Sus scrofa domestica, Linnaeus, 1758) / The effect of intrauterine sensitilization on gilt (Sus scrofa domestica, Linnaeus, 1758) fertility

O objetivo deste estudo foi investigar o efeito da pré-sensibilizaçäo uterina com antígenos espermáticos ou seminais sobre a fertilidade da marrä de reposiçäo. Cento e quarenta e uma marräs cruzadas pré-púberes com 160-165 dias de idade foram alojadas. O estro foi induzido pela aplicaçäo de PMSG-hCG no alojamento. A detecçäo de estro foi realizada duas vezes ao dia com o auxílio do macho. As pré-infusöes uterinas foram realizadas 24 horas após o início do estro no primeiro e segundo estro. As marräs foram agrupadas em 5 diferentes tratamentos: espermatozóides mortos (EM, n = 29), plasma seminal (PS, n = 29), plasma seminal e espermatozóides mortos (PS + EM, n = 28), soluçäo salina fisiológica (SS, n = 28) e controle (n = 27). Marräs que sempre apresentaram reflexo de tolerância ao macho (RTM) positivo no momento da infusäo foram analisadas separadamente (Grupo 1) daquelas que tiveram no mínimo uma vez um RTM negativo no momento da infusäo (Grupo 2). Somente uma inseminaçäo artificial (IA) foi realizada 24 horas após a manifestaçäo do RTM no terceiro cio. Aos 28-32 dias após a IA, as marräs foram abatidas e os fetos e corpos lúteos foram contados. No Grupo 1, a taxa de prenhez do grupo PS (100 por cento) foi superior (p<0,05) ao controle (90 por cento), PS + EM (90,9 por cento) e SS (80 por cento), entretanto foi similar à do EM (92,9 por cento). No grupo 2, nao ocorreram diferenças entre os grupos (p<0,05). Näo houve diferenças entre os tratamentos com relaçäo à taxa ovulatória, taxa de perda embrionária e número de embriöes viáveis. Os resultados sugerem que melhora significativa causada pela pré-sensibilizaçäo uterina possa ser observada somente se, por alguma outra razäo, os índices reprodutivos do rebanho deixassem a desejar

The histidines reacting with ethoxyformic anhydride in porcine pancreatic lipase: their relationships with enzyme activity.

The activities of porcine pancreatic lipase (449 amino acid residues) toward two different substrates, p-nitrophenylacetate and tributyrylglycerol, and their dependence on histidine ethoxyformylation were studied. In parallel, the ethoxyformylation of the lipase fragment constituting the C-terminal sequence of lipase (residues 336 to 449) was also investigated. This fragment was found to have retained the ability of lipase to catalyse p-nitrophenylacetate hydrolysis. The first histidine to react either in lipase or in the lipase fragment was His-354. The activities of the two compounds toward p-nitrophenyl-acetate were lost but that of the enzyme toward tributyrylglycerol was almost entirely retained. When a larger excess of ethoxyformic anhydride was used for the lipase reaction, 2.8 histidine residues were ethoxyformylated and characterised as His-354, His-156 and His-75, which resulted in an 85% inhibition of the tributyrylglycerol hydrolysis by the enzyme. Hydroxylamine treatment reactivated most of the lipase and lipase fragment. This is the first demonstration that the two lipase activities are not associated with the same active site. The loss of activity toward triacylglycerol hydrolysis suggests that His-156 and/or His-75 belong(s) to the active site or that a conformational change resulting from the ethoxyformylation renders the lipase inactive.

Characterization in rat pancreatic juice of a protein homologous to the human pancreatic stone protein.

1. The pancreatic stone protein (PSP, Mr 15,000) which has been discovered in human calculi derives from the native glycosylated forms of the protein (Mrs 17,500-22,000) which are present in human pancreatic juice through tryptic cleavage of the Arg 11-Ile 12 bond. 2. In the present study, a homologous native form of the protein (Mr 17,000) was purified from rat pancreatic juice. 3. Its N-terminal amino acid sequence was found to display a high degree of homology with that of the human native protein forms, apart from the fact that it was not glycosylated. 4. In rat as in human, tryptic cleavage of the Arg 11-Ile 12 bond transforms a soluble protein into one which is practically insoluble at neutral pH.

Porcine pancreatic lipase. The disulfide bridges and the sulfhydryl groups.

Following complete sequence analysis of the 449 amino acids in porcine pancreatic lipase [J. De Caro et al. (1981) Biochim. Biophys. Acta, 671, 129-138], the position of the six disulfide bridges and of the two free thiols of the protein was investigated using a variety of techniques. Three bridges (Cys-4--Cys-10, Cys-237--Cys-261 and Cys-433--Cys-449) were easily identified in the peptic digest of lipase at pH 2.0. In the latter digest, two other bridges (Cys-285--Cys-296 and Cys-299--Cys-304) were also identified by means of the cystine peptide constituted by two peptide segments: Ala281-Gly-Phe-Pro-Cys-Asp-Ser287 and Thr292-Ala-Asn-Lys-Cys-Phe-Pro-Cys-Pro-Ser-Glu-Gly-Cys-Pro-Gln-Met307. A disulfide bridge, formed by Cys-285 and one of the three half-cystines of the other segment, connected the two peptide moieties. A second disulfide bridge linked the two remaining half-cystines. It was not possible to split any peptide bond between Cys-296 and Cys-304 with proteolytic enzymes. The determination of the pairing of the four half-cystines was resolved as follows. Bond Lys-295--Cys-296 was cleaved with trypsin. A single cycle of Edman degradation was then performed on the peptide compound, thus freeing, in particular, Cys-296 of the peptide bond (296-297). Cys-296 only retained its S-S connection with the half-cystine partner. At the completion of the above operations, the two peptide segments (282-287) and (297-307) could be separated. Consequently it was concluded that Cys-285 is linked to Cys-296. Therefore Cys-299 and Cys-304 are paired. The most reactive SHI group of the enzyme was localized on Cys-181 by condensation of the native protein with radioactive N-ethylmaleimide. The alkylation of the SHII group required previous denaturation of the molecule at alkaline pH. The results obtained for the characterization of the SHII group suggested the existence of two isomeric forms, the SHII being either on Cys-101 or Cys-103 and the bridge alternately between Cys-90--Cys-103 or Cys-90--Cys-101. It is not yet known whether these two forms pre-exist in native lipase or result from an exchange reaction. The bridge Cys-90--Cys-101 was characterized in a thermolytic digest of a cyanogen bromide fragment (CN II) of the protein. However, another bridge involving Cys-181 was also found in the digest. This bridge is considered as being an artefact. It is possible that considering the treatments undergone by the large peptide (CN II), the SH groups of lipase were oxidized and transformed in an S-S bridge. The disulfide bridges of lipase form relatively small loops along the main chain. This arrangement is consistent with a high flexibility of the molecule. As reported earlier [R. Verger et. al. (1971) Biochim. Biophys. Acta. 242, 580-592], the SHI group is not essential for lipase activity. The role of the SHII group should be more precisely investigated.

Porcine pancreatic lipase. Completion of the primary structure.

The complete primary structure of a lipase (triacylglycerol hydrolase; EC 3.1.1.3) is presented for the first time. The porcine pancreatic enzyme which was investigated is composed of a single chain of 449 amino acids. Upon fragmentation by CNBr, five peptides were obtained. The sequence of four of them (CN I-CN IV) has already been published. The present report deals with the arrangement of the 142 amino acids of the C-terminal peptide CN V, thus completing the analysis of the whole molecule. Special problems resulting from incomplete cleavage of some peptide bonds in CN V and aggregation of large peptides were overcome using Sephadex filtration of succinylated derivatives in 50% acetic acid, automated sequence analysis of peptide mixtures and subdigestion of material which could not be directly resolved. No obvious homology was found when the sequence of porcine lipase was compared with other protein, including pancreatic phospholipase A2 and colipase from the same species. However, a few similarities which might be significant were detected between the environment and relative position of certain half cystines in lipase and colipase, as well as between two tyrosine-rich regions existing in both proteins.

Characterization of the serine reacting with diethyl p-nitrophenyl phosphate in porcine pancreatic lipase.

The position in porcine pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) of the serine reacting specifically with emulsified or micellar diethyl p-nitrophenyl phosphate has been investigated. This serine which appears to be involved in lipase adsorption to insoluble triglyceride interfaces, is at position 152 in the enzyme chain. The sequence around this amino acid is: His-Val-Ile-Gly-His-Ser-Leu-Gly.

Porcine pancreatic lipase. Sequence of the first 234 amino acids of the peptide chain.

The single polypeptide chain of about 460 amino acids of porcine pancreatic lipase (EC 3.1.1.3) has been fragmented into five peptides by cyanogen bromide cleavage [Rovery, M., Bianchetta, J. & Guidoni, A. (1973) Biochim. Biophys. Acta, 328, 391--395]. The sequence of the first three cyanogen bromide peptides (CNI, CNII, CNIII), including a total of 234 amino acids, was fully elucidated. Automatic or manual Edman degradation was performed on the different peptides. Fragmentations of the CN peptides were accomplished by digestions with trypsin (after citraconylation or 1,2-cyclohexanedione treatment), chymotrypsin and Staphylococcus aureus external protease. Hydrolysis of unreduced material by pepsin and thermolysin, performed in order to determine the S-S bridge positions, provided useful overlapping peptides. The glycan moiety of lipase is bound to Asn-166. The non-essential tyrosine specifically blocked by diisopropylphosphorofluoridate is Tyr-49 in a cluster of asparagine and glutamine residues. The existence of a highly hydrophobic sequence (206--217) at the C terminus of the CNII fragment is noteworthy.