Perfil bioquímico, inclusive proteinograma, do soro lácteo de búfalas primíparas e pluríparas sadias ao longo da lactação

Resumo: Para avaliar o perfil bioquímico, inclusive proteínas, do soro lácteo de búfalas Murrah primíparas e pluríparas sadias foram analisadas amostras de leite de 30 fêmeas bubalinas durante uma lactação completa. Os animais foram distribuídos em três grupos: G1 - 10 búfalas primíparas, G2 - 10 búfalas pluríparas com duas a três lactações e G3 - 10 búfalas pluríparas com mais de três lactações. O período de lactação foi dividido em: fase inicial (I: primeiro ao terceiro mês de lactação), fase intermediária (T: quarto ao sexto mês de lactação) e fase final (F: sétimo ao nono mês de lactação). Antes da colheita das amostras de leite foram realizados o exame físico da glândula mamária, o teste da caneca de fundo escuro e o California Mastitis Test (CMT). Após a assepsia dos quartos mamários, foram colhidas mensalmente, durante uma lactação completa, amostras de 20mL de leite de cada quarto mamário, em frascos plásticos esterilizados e sem conservante, para a realização do isolamento microbiológico, determinação do perfil bioquímico e fracionamento proteico por meio de eletroforese em gel de poliacrilamida contendo dodecil sulfato de sódio (SDS-PAGE), e amostras de 30mL de leite de cada quarto mamário, em frascos plásticos esterilizados contendo conservante bronopol, para contagem de células somáticas (CCS). Das 1.042 amostras de leite colhidas dos três grupos experimentais durante a lactação, 923 amostras de leite apresentaram reação negativa ao CMT e isolamento microbiológico negativo e foram selecionadas para as análises do perfil bioquímico e fracionamento proteico em SDS-PAGE. Notou-se influência da ordem de parto e da fase da lactação no perfil bioquímico e no proteinograma do soro lácteo de búfalas da raça Murrah sadias. As búfalas primíparas (G1) apresentaram maior atividade das enzimas gamaglutamiltransferase (GGT: 2.346U/L) e fosfatase alcalina (ALP: 181U/L) e maiores concentrações de fósforo (P: 56,6mg/dL), potássio (K: 32,0mg/dL) e α-lactoalbumina (458mg/dL). As fêmeas com duas a três lactações (G2) apresentaram maior CCS (70.700 células/mL) e maiores concentrações de proteína total (1,55g/dL), albumina (100mg/dL), magnésio (Mg: 8,80mg/dL), cloretos (Cl: 176mg/dL), ferro (Fe: 10,7μg/dL), sódio (Na: 178mMol/L) e lactoferrina (59,5mg/dL). As fêmeas com mais de três lactações (G3) apresentaram maiores concentrações de cálcio total (Ca: 41,8mg/dL), cálcio ionizado (Cai: 2,92mMol/L), imunoglobulina A (IgA: 1,32mg/dL), albumina sérica (99,1mg/dL), imunoglobulina G (IgG: 49,7mg/dL) e b-lactoglobulina (1.068mg/dL). Durante a lactação foi observado aumento da CCS, aumento das atividades das enzimas GGT e ALP, aumento das concentrações de proteína total, albumina, P, Mg, Cl, Na, lactoferrina, albumina sérica, IgG, α-lactoalbumina e redução das concentrações de Ca, Fe, Cai, K, IgA e b-lactoglobulina no soro lácteo das búfalas. Os resultados obtidos podem ser utilizados como referências para a espécie bubalina e auxiliar no diagnóstico e no prognóstico de doenças de ocorrência comum na fase de lactação.

Effects of intramammary infection on whey proteinograms of sheep during lactation

The study aimed to identify potential biomarkers of mammary gland infection in Santa Inês sheep. Commercial flocks of sheep provided the same hygiene, sanitary, and nutritional management under semi-intensive production systems were monitored during the lactation stage-and assessed 15, 30, 60, and 90 days after delivery (through the end of lactation and weaning). The California Mastitis Test (CMT) was performed on the mammary glands. Milk was collected for bacterial examination and protein analysis. Bacterial culture and biochemical characterization of the samples were performed. Forty-two milk samples from healthy glands (negative CMT and bacterial testing) and 43 milk samples from infected glands (positive CMT and bacterial testing) taken at the predefined time points were assessed. A rennin solution was used to obtain the whey. The proteins analysis was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which allowed for the quantification of nine whey proteins produced in healthy glands: serum albumin, lactoferrin, IgA, IgG heavy-chain (IgG HC), IgG light-chain (IgG LC), total IgG (IgG HC + IgG LC), α-lactalbumin, β-lactoglobulin, protein with MW 15.000 Da, protein with MW 29.000 Da and eleven whey proteins secreted by infected glands, including haptoglobin and α-1-acid glycoprotein. A comparison of whey proteins between healthy and infected glands showed increases (P<0.05) in the secreted and total contents of all proteins, except for IgG LC and α-lactoalbumin. The most significant changes were observed in α-1-acid glycoprotein, lactoferrin and haptoglobin, which showed three-, five-, and seven-fold increases in secretion, respectively. This study showed that haptoglobin, α-1-acid glycoprotein, lactoferrin, albumin, and the IgA and IgG immunoglobulins may serve as potential biomarkers for mammary gland infection in sheep.

Automaattisilla järjestelmillä lähetettävä suoramarkkinointi

Nykyisin yritykset hyödyntävät hyvin paljon sähköisiä viestimiä suoramarkkinointi-tarkoitukseen. Laki ja hyvä tapa rajoittavat kuitenkin sähköisen suoramarkkinoinnin lähettämistä yksityisille kuluttaja-asiakkaille. Sähköistä suoramarkkinointia harjoittavan yrityksen on noudatettava kohdemaassa voimassa olevia lakeja. Suomessa sähköisen viestinnän tietosuojalaki (SVTSL) on sähköisillä viestimillä lähetettävän mainonnan osalta tärkein lainsäädäntö. SVTL antaa parhaimman kuvan siitä, mitä kaikkea yrityksen on huomioitava harjoittaessaan kuluttajiin kohdistuvaa sähköistä mainontaa. Lainsäädäntöjen lisäksi huomattavan tärkeää on noudattaa viranomaisten, kuten kuluttajaviraston (kuluttaja-asiamiehen) antamia kannanottoja sekä linjauksia. Sähköisen suoramarkkinoinnin osalta Suomessa on käytössä niin sanottu opt in -menettely, jonka mukaan potentiaalisia kuluttaja-asiakkaita saa lähestyä sähköisillä suoramarkkinointiviesteillä vain, mikäli he ovat antaneet siihen suostumuksen. Sähköisen viestinnän tietosuojalaissa on opt in -menettelystä säädös, ja sitä voidaan pitää tärkeimpänä lähtökohtana sähköisiä mainoksia lähetettäessä. Lain mukainen lupapyyntö voidaan esittää monella eri tavalla. Yleisimmin lupa pyydetään heti myynnin yhteydessä valmiilla kaavakkeella. Lisäksi yritykset voivat pyytää lupaa www-sivuilla olevilla verkkolomakkeilla, asiakaspalveluun liittyvillä puheluilla sekä muissa asiakaspalvelutilanteissa. Lain mukaan lupapyynnön tulee olla muodoltaan aktiivinen ja nimenomainen. Käytännössä lupapyyntö ei saa olla valmiiksi täytetty lomake, josta asiakas joutuu esimerkiksi poistamaan rastin, mikäli ei halua vastaanottaa suoramarkkinointiviestejä. Lupapyynnön yhteydessä yrityksen on kerrottava asiakkaalle, mitä mahdollinen suoramarkkinointiaineisto sisältää ja kuinka usein sitä mahdollisesti tullaan lähettämään. Lupapyynnön laajuudesta ei ole mainittu lainsäädännössä. Konsernia koskevissa tapauksissa lupapyyntö voi koskea pelkkää lupaa pyytävää yritystä tai mahdollisesti muita yhteistyössä toimivia konsernin sisäisiä yrityksiä. Tärkeintä on, että kuluttaja on ajan tasalla ja tietoinen siitä, mitä kaikkea lupapyyntö sisältää. Yritysten on hyvä tiedostaa, että kuluttaja-asiakkaita koskeva kielto-oikeus on jatkuvasti voimassa, vaikka asiakas olisi antanut luvan suoramarkkinointiviestien lähettämiseen, ja että asiakkaita on muistutettava jokaisen viestin yhteydessä kyseisestä oikeudesta. Kielto-oikeus on toteutettava niin että asiakas voi milloin tahansa ilmoittaa kieltäytyvänsä vastaanottamasta suoramarkkinointiviestejä. Asiakkaalla pitää olla mahdollisuus maksuttomaan kielto-oikeuteen niin verkossa kuin puhelimitse. Suoramarkkinointiviesti ei ole sisällöltään kovin tarkkaan määritelty, mutta yleisiä sääntöjä on säädetty tiettyjen muotoseikkojen osalta. Ensinnäkin viestistä on tultava selkeästi esille, että kyseessä on suoramarkkinointitarkoituksessa lähetetty viesti, eikä yksityisten henkilöiden välinen viestintä. Lisäksi sähköiseen mainokseen tai suoramarkkinointiviestiin on lisättävä lähettäjän henkilöllisyys sekä voimassa olevat yhteystiedot, jotta asiakas voi esimerkiksi päivittää omat yhteystietonsa. Jokaisen viestin yhteydessä asiakkaalle on annettava selkeät ohjeet siitä, kuinka hän voi käyttää kielto-oikeuttaan esimerkiksi ilmoittamalla voimassaoleva sähköpostiosoite, johon suoramarkkinointikiellot voidaan lähettää. Lapsia ja nuoria voidaan pitää kuluttaja-asiakkaille lähetettävän sähköisen suoramarkkinoinnin erityistapauksena. Lain mukaan lapsille ja nuorille ei saa lähettää sähköistä markkinointiaineistoa ilman vanhempien lupaa, eikä heitä saa houkutella pelien ym. avulla luovuttamaan tietoja itsestään tai perheestään. Toinen kuluttaja-asiakkaiden erityistapaus on niin sanottu kerro kaverille -markkinointi eli K2K-markkinointi. Siinä alkuperäinen mainos on lähetetty yrityksen toimesta ja tarkoituksena on saada kuluttaja mainostamaan kyseistä yritystä tai tuotetta ystävilleen. K2K-markkinointi on tuottanut ongelmia, sillä mikäli kuluttaja A haluaisi lähettää kuluttaja B:lle yrityksen lähettämiä markkinointiviestejä, niin B:n pitäisi antaa opt in -menettelyn mukaisesti yritykselle ensin lupa kyseiseen toimintaan. Kuluttaja-asiamies on antanut K2K-markkinoinnista kannanottonsa. Kannanoton mukaan yritys tarvitsee kolmansien henkilöiden luvan viestien lähettämiseen, mikäli asiakkaat lähettävät yrityksen kirjoittamia valmiita viestejä. Jos kyseessä on tavallinen kuluttajien välinen viestintä, niin lupaa ei luonnollisesti tarvitse pyytää. EU-lainsäädännön pohjalta on säädetty poikkeussääntö koskien yrityksen olemassa olevia asiakkaita. Poikkeussäännön mukaan yritykset saavat lähettää ''vanhoille'' asiakkailleen sähköisiä mainoksia ilman erillistä ennakkosuostumusta. Sääntöä on rajoitettu siltä osin, että asiakkaille saa lähettää mainoksia koskien samaa tai samankaltaisia tuotteita tai palveluita, joita ostaessa asiakas on luovuttanut yritykselle yhteystietonsa. Yrityksen on lain mukaan toimittava opt in -menettelyn mukaisesti, mikäli se kokee tarpeelliseksi lähettää asiakkailleen sähköisiä mainoksia täysin uusista tuotteista. Ottaen huomioon sähköisillä järjestelmillä lähetettävää suoramarkkinointia koskevat lainsäädännöt sekä kannanotot, niin voidaan todeta, että yritysten kannalta viisain tapa toimia on permission based -toimintamalli eli kysytään lupa ja sitten vasta toimitaan. Näin säilytetään hyvä maku sähköisessä suoramarkkinoinnissa ja vältytään turhilta asiakasreklamaatioilta.

Purification and characterisation of a lectin from the red marine alga Pterocladiella capillacea (S.G. Gmel.) Santel. & Hommers.

A lectin present in the marine red alga Pterocladiella capillacea was purified and characterised by extraction of soluble proteins (crude extract) in 20 mM Tris-HCl buffer, pH 7.5. Among the analysed erythrocytes (human blood group A, B and O and the animals ox, goat, chicken and rabbit) the lectin agglutinated specifically rabbit erythrocytes. The hemagglutinating activity assay showed that the lectin was not dependent on divalent cations and was shown to be inhibited by the glycoproteins avidin and mucin. The purification procedure was conduced by precipitation of the crude extract with 80% saturation ammonium sulfate (F0/80) followed by affinity chromatography on guar-gum column. The lectin of P. capillacea was purified 14.5 fold and had a recovery of 27.4% of the original total specific activity present in the crude extract. The absence of carbohydrate suggested that the lectin is not a glycoprotein. The molecular mass of P. capillacea lectin, determined by gel filtration, was 5.8 kDa. SDS-PAGE in the presence of ß-mercaptoethanol gave one band, indicating that the native lectin is a monomeric protein. The activation energy of denaturation process (D G') was calculated to be 106.87 kJ . mol-1 at 70 ºC.

Purificação e caracterização parcial de uma lectina da alga marinha vermelha Vidalia obtusiloba C. Agardh

A lectina da alga marinha vermelha Vidalia obtusiloba foi purificada através da combinação de precipitação com sulfato de amônio, cromatografia de troca iônica em DEAE-celulose e cromatografia de afinidade em goma de guar reticulada. A lectina preferencialmente aglutinou eritrócitos do grupo humano O, nativos e tratados com bromelaina. A atividade hemaglutinante revelou que a lectina era dependente de cátions divalentes (Ca++ ou Mn++) e inibida por N-acetil-galactosamina, D-galactosamina, alfa-lactose and D-galactose e pela glicoproteína mucina de estômago de porco. A massa molecular da lectina, estimada por filtração em gel, foi de 78,9 kDa, enquanto por SDS-PAGE, em presença de beta-mercaptoetanol, a lectina exibiu duas subunidades protéicas diferentes com Mr de 59,6 e 15,2 kDa, sugerindo que a lectina é uma proteína dimérica. Focalização isoelétrica revelou a presença de uma proteína ácida simples, com um ponto isoelétrico entre 4 e 5. A lectina purificada exibiu um conteúdo de carboidrato de 43,2% e uma predominância dos aminoácidos Asp/Asn, Glu/Gln e Leu. A energia de ativação (deltaG') para a desnaturação da lectina foi calculada como sendo 25,4 kcalm.mol-1 a 90 ºC. Ensaios imunoquímicos usando um anti-soro de coelho produzido contra a lectina purificada de V. obtusiloba mostraram que foi possível detectar a presença da lectina em diferentes etapas do processo de purificação. Western blotting de géis de SDS-PAGE mostraram imunocoramento apenas da maior das subunidades da lectina.

Correlation between high molecular weight gluten subunits composition and bread-making quality in Brazilian wheat

Bread-making quality is one of the most important targets in the genetic improvement of wheat. Although extensive analyses of quality traits such as farinography, sodium dodecyl sulfate (SDS) sedimentation, alveography, and baking are made in breeding programs, these analyses require high amounts of seeds which are obtained only in late generations. In this experiment the statistical correlations between the high molecular weight subunit of glutenin and bread-making quality measured by alveograph, farinograph and SDS sedimentation were evaluated. Seventeen wheat genotypes were grown under the same conditions, each producing about 1 kg of seeds for the evaluations. The high molecular weight (HMW) glutenin subunits were analyzed by SDS-PAGE. Statistical correlations were highly significant between HMW glutenin subunits and alveograph and SDS sedimentation. These results indicate the possibility of manipulating major genes for wheat seed quality by coupling traditional breeding with non-destructive single seed analysis. Only half seed is necessary to perform the SDS-PAGE analysis. Therefore, the other half seed can be planted to generate the progeny. Seed yield and SDS sedimentation were statistically correlated, indicating the possibility of simultaneous selection for both traits

Single-step purification of crotapotin and crotactine from Crotalus durissus terrificus venom using preparative isoelectric focusing

We describe the isolation of crotoxin, a presynaptic B-neurotoxin, as well as its subunits B (crotactine) and A (crotapotin) from lyophilized Crotalus durissus terrificus venom by a single-step preparative isoelectric focusing procedure. From 98 mg of dried venom protein 20.1 mg of crotactine and 13.1 mg of crotapotin were recovered in the first step of focalization and 4.2 mg in a second run. These values correspond to 35.7% of the total venom protein applied. Crotactine separated in the 9.3-7.0 pH range (tubes 1-6) and crotapotin in the 1.8-2.8 pH range (tubes 15-19) and both were homogeneous by SDS-PAGE and N-terminal amino acid analysis. Crotactine, a 12-kDa protein, presented hemolytic and phospholipase A2 activity. Thus, using isoelectric focusing we simultaneously purified both toxins in high yields. This method can be used as an alternative for the purification and characterization of proteins from other snake venoms under conditions in which biological activity is retained

A 28-fold increase in secretory protein synthesis is associated with DNA puff activity in the salivary gland of Bradysia hygida (Diptera, Sciaridae)

When the first group of DNA puffs is active in the salivary gland regions S1 and S3 of Bradysia hygida larvae, there is a large increase in the production and secretion of new salivary proteins demonstrable by [3H]-Leu incorporation. The present study shows that protein separation by SDS-PAGE and detection by fluorography demonstrated that these polypeptides range in molecular mass from about 23 to 100 kDa. Furthermore, these proteins were synthesized mainly in the S1 and S3 salivary gland regions where the DNA puffs C7, C5, C4 and B10 are conspicuous, while in the S2 region protein synthesis was very low. Others have shown that the extent of amplification for DNA sequences that code for mRNA in the DNA puffs C4 and B10 was about 22 and 10 times, respectively. The present data for this group of DNA puffs are consistent with the proposition that gene amplification is necessary to provide some cells with additional gene copies for the production of massive amounts of proteins within a short period of time (Spradling AC and Mahowald AP (1980) Proceedings of the National Academy of Sciences, USA, 77: 1096-1100).

Changes in protein profile during coagulation of latex from Carica papaya

We describe the changes in peptide composition by SDS-PAGE analysis of latex from Carica papaya collected at various times after incision of the unripe fruit. The data show that during latex coagulation several peptides are processed in an orderly fashion.

Radioactive fucose as a tool for studying glycoprotein secretion

The efficiency and reliability of radioactive fucose as a specific label for newly synthesized glycoproteins were investigated. Young adult male rabbits were injected intravitreally with [3H]-fucose, [3H]-galactose, [3H]-mannose, N-acetyl-[3H]-glucosamine or N-acetyl-[3H]-mannosamine, and killed 40 h after injection. In another series of experiments rabbits were injected with either [3H]-fucose or several tritiated amino acids and the specific activity of the vitreous proteins was determined. Vitreous samples were also processed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and histological sections of retina, ciliary body and lens (the eye components around the vitreous body) were processed for radioautography. The specific activity (counts per minute per microgram of protein) of the glycoproteins labeled with [3H]-fucose was always much higher than that of the proteins labeled with any of the other monosaccharides or any of the amino acids. There was a good correlation between the specific activity of the proteins labeled by any of the above precursors and the density of the vitreous protein bands detected by fluorography. This was also true for the silver grain density on the radioautographs of the histological sections of retina, ciliary body and lens. The contribution of radioautography (after [3H]-fucose administration) to the elucidation of the biogenesis of lysosomal and membrane glycoproteins and to the determination of the intracellular process of protein secretion was reviewed. Radioactive fucose is the precursor of choice for studying glycoprotein secretion because it is specific, efficient and practical for this purpose

Clinical and molecuar characterization of Brazilian patients with growth hormone gene deletions

Genomic DNA from 23 patients with isolated growth hormone (GH) deficiency (12 males and 11 females: heights -4.9 ± 1.4 SDS) was screened for GH gene deletions by restriction endonuclease analysis of polymerase chain reaction amplification products. Three unrelated patients had typical features of severe GH deficiency and deletions (6.7 kb in two and 7.6 kb in one) of the GH gene. The two patients with 6.7-kb deletions developed growth-attenuating anti-GH antibodies whereas the patient with the 7.6-kb deletion continued to grow with GH replacement therapy. Our finding that 3/23 (~13%) Brazilian subjects had GH gene deletions agrees with previous studies of severe isolated GH deficiency subjects in other populations. Two of three subjects (67%) with deletions developed blocking antibodies despite administration of exogenous GH at low doses. Interestingly, only 1/10 of cases with affected relatives or parental consanguinity had GH-1 gene deletions

Western blot detection of infectious bursal disease virus infection

In order to evaluate the use of a Western blot methodology for the diagnosis of infectious bursal disease virus (IBDV) infection, chickens were experimentally infected with IBDV strains and tested for the presence of viral antigens and antibodies by a blocking Western blot test (bWB). The viral proteins obtained from the bursa of Fabricius (BF) were transferred to a nitrocellulose membrane after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the chicken sera obtained by heart puncture were used for the detection of these proteins. In order to eliminate nonspecific reactions, we used a rabbit anti-chicken serum (blocking tool). By the use of the bWB test, two distinct viral proteins of 43-kDa (VP2) and 32-kDa (VP3) were detected. We suggest the use of this methodology for the detection of IBDV infection in animals suspected of having IBDV reinfection and a chronic subclinical form of the disease. With the use of the rabbit anti-chicken sera for blocking, this method is practical, sensitive and less time consuming

Inhibition of Mayaro virus replication by prostaglandin A1 and B2 in Vero cells

The effect of prostaglandins (PGA1 and PGB2) on the replication of Mayaro virus was studied in Vero cells. PGA1 and PGB2 antiviral activity was found to be dose-dependent. However, while 10 µg/ml PGB2 inhibited virus yield by 60%, at the same dose PGA1 suppressed virus replication by more than 90%. SDS-PAGE analysis of [35S]-methionine-labelled proteins showed that PGA1 did not alter cellular protein synthesis. In infected cells, PGA1 slightly inhibited the synthesis of protein C, while drastically inhibiting the synthesis of glycoproteins E1 and E2.

A single-step purification of bothropstoxin-1

Bothrops venoms are complex mixtures of components with a wide range of biological activities. Among these substances, myotoxins have been investigated by several groups. Bothropstoxin-1 (Bthtx-1) is a phospholipase A2-like basic myotoxin from Bothrops jararacussu. The purification of this component involves two chromatographic steps. Although providing a pure material, the association of these two steps is time consuming and a single-step method using high performance chromatography media would be useful. In the present study, we describe a single-step purification method for Bthtx-1. Bothrops jararacussu venom was dissolved in 1 ml buffer. After centrifugation, the supernatant was injected into a Resource-S cation exchange column connected to an FPLC system and eluted with a linear salt gradient. The complete procedure took 20 min, representing a considerable time gain when compared to a previously described method (Homsi-Brandenburgo MI et al. (1988) Toxicon, 26: 615-627). Bthtx-1 purity and identity, assessed by SDS-PAGE and N-terminal sequencing, resulted in a single band with a molecular mass of about 14 kDa and the expected sequence of the first 5 residues, S-L-F-E-L. Although the amount of protein purified after each run is lower than in the previously described method, we believe that this method may be useful for small-scale purifications.

Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase

The aminopeptidase activity of Phaseolus vulgaris seeds was measured using L-Leu-p-nitroanilide and the L-aminoacyl-ß-naphthylamides of Leu, Ala, Arg and Met. A single peak of aminopeptidase activity on Leu-ß-naphthylamide was eluted at 750 µS after gradient elution chromatography on DEAE-cellulose of the supernatant of a crude seed extract. The effluent containing enzyme activity was applied to a Superdex 200 column and only one peak of aminopeptidase activity was obtained. SDS-polyacrylamide gel electrophoresis (10%) presented only one protein band with molecular mass of 31 kDa under reducing and nonreducing conditions. The aminopeptidase has an optimum pH of 7.0 for activity on all substrates tested and the highest Vmax/KM ratio for L-Leu-ß-naphthylamide. The enzyme activity was increased 40% by 0.15 M NaCl, inhibited 94% by 2.0 mM Zn2+, inhibited 91% by sodium p-hydroxymercuribenzoate and inhibited 45% by 0.7 mM o-phenanthroline and 30 µM EDTA. Mercaptoethanol (3.3 mM), dithioerythritol (1.7 mM), Ala, Arg, Leu and Met (70 µM), p-nitroaniline (0.25 mM) and ß-naphthylamine (0.53 mM) had no effect on enzyme activity when assayed with 0.56 mM of substrate. Bestatin (20 µM) inhibited 18% the enzyme activity. The aminopeptidase activity in the seeds decayed 50% after two months when stored at 4oC and room temperature. The enzyme is leucyl aminopeptidase metal- and thiol group-dependent.