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1.
This study starts by isolating and characterizing the first protein from Labramia bojeri seeds, which belong to the Sapotaceae family. The purified lectin analyzed by SDS-PAGE with and without beta-mercaptoethanol shows two protein bands (M(r) = 19 and 20 kDa), which cannot be resolved. Protein bands have shown similar characteristics as molecular masses, determined by gel filtration and native gel; N-terminal sequences presented a difference in their isoelectric points. We have suggested that those protein bands might be variants of the protein named Labramin. The sequence database search has shown that the N-terminal sequence of Labramin presented a high degree of homology to Kunitz-type trypsin inhibitor (82-52%) despite no trypsin inhibition activity detection. The lectin-like form from Labramin was better inhibited by glycoproteins and has also presented growth inhibition of the fungus Colletotrichum lindemuthianum and the yeast Saccharomyces cerevisiae, but it has not presented an apparent effect on Fusarium oxysporum.  相似文献   

2.
Protease inhibitors from potato juice of cv. Elkana were purified and quantified. The protease inhibitors represent ca. 50% of the total soluble proteins in potato juice. The protease inhibitors were classified into seven different families: potato inhibitor I (PI-1), potato inhibitor II (PI-2), potato cysteine protease inhibitor (PCPI), potato aspartate protease inhibitor (PAPI), potato Kunitz-type protease inhibitor (PKPI), potato carboxypeptidase inhibitor (PCI), and "other serine protease inhibitors". The most abundant families were the PI-2 and PCPI families, representing 22 and 12% of all proteins in potato juice, respectively. Potato protease inhibitors show a broad spectrum of enzyme inhibition. All the families (except PCI) inhibited trypsin and/or chymotrypsin. PI-2 isoforms exhibit 82 and 50% of the total trypsin and chymotrypsin inhibiting activity, respectively. A strong variation within the latter activities was shown within one family and between protease inhibitor families.  相似文献   

3.
Glycoprotein showing inhibitory activity against mast cell degranulation and hyaluronidase activity was purified from the hot water extract of mint plant (Perilla frutescens Britton). The purified inhibitor gave a single band detected with Coomassie brilliant blue staining and periodic acid-Schiff staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The molecular mass was estimated to be 6.0 kDa on SDS-PAGE. The inhibitor did not become inactivated when boiled for 30 min or digested with trypsin, V8 protease, or proteinase K but was inactivated by NaIO(4) oxidation. The inhibitor prevented mast cell degranulation and hyaluronidase activity (IC(50) = 0.42 mg/mL) in a dose-dependent manner. The inhibitor also inhibited the protein kinase C activity. It is possible to purify and characterize a glycoprotein with putative pharmacological properties from mint plants.  相似文献   

4.
Our research on several proteins indicates that accurate molecular weights cannot be determined by Superose 12 column chromatography. In support of this statement, we present data on molecular weights of purified red kidney bean alpha-amylase inhibitor (RKB alphaAI) and white kidney bean alpha-amylase inhibitor (WKB alphaAI) to document this problem. The molecular weight of purified RKB alphaAI determined by Sephadex G-100 gel filtration, polyacrylamide gel electrophoresis, Superose 12 gel filtration and cDNA were 49.0, 51.0, 22.9, and 49.805 kDa (not glycosylated), respectively. The molecular weights of WKB alphaAI by several methods were as follows: Sephadex G-100 gel filtration, 51.0 kDa; Superose 12 gel filtration in 0.2 M NaCl buffer, 23.1 kDa; polyacrylamide gel electrophoresis (PAGE), 51.0 kDa; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 45.0 kDa; multiangle laser light scattering (MALLS), 49.940 kDa; laser-assisted time-of-flight mass spectrometry (LATOFMS), 56.714 kDa; and cDNA sequence (with 12.2% carbohydrate), 55.9 kDa. The data indicate there is ionic interaction between proteins and the matrix of Superose 12 in low ionic strength buffers and hydrophobic interaction at higher ionic strength buffers. Researchers should be cautious when using Superose 12 columns for molecular weight determinations.  相似文献   

5.
A chitosanolytic enzyme was purified from a commercial ficin preparation by affinity chromatographic removal of cysteine protease on pHMB-Sepharose 4B and cystatin-Sepharose 4B and gel filtration on Superdex 75 HR. The purified enzyme exhibited both chitinase and chitosanase activities, as determined by SDS-PAGE and gel activity staining. The optimal pH for chitosan hydrolysis was 4.5, whereas the optimal temperature was 65 degrees C. The enzyme was thermostable, as it retained almost all of its activity after incubation at 70 degrees C for 30 min. A protein oxidizing agent, N-bromosuccinimide (0.25 mM), significantly inhibited the enzyme's activity. The molecular mass of the enzyme was 16.6 kDa, as estimated by gel filtration. The enzyme showed activity toward chitosan polymers exhibiting various degrees of deacetylation (22-94%), most effectively hydrolyzing chitosan polymers that were 52-70% deacetylated. The end products of the hydrolysis catalyzed by this enzyme were low molecular weight chitosan polymers and oligomers (11.2-0.7 kDa).  相似文献   

6.
Polyphenol levels in wines are affected by the wine-making process. Resveratrol is one polyphenol which has been the subject of a commendable amount of recent research. In this work, we found that resveratrol is immediately degraded by tyrosinase. A novel tyrosinase was purified from Carignan grapes. The purification process included salting out and separation on a cation-exchange column, followed by gel filtration. Tyrosinase was purified in a homogeneous form by SDS-PAGE and was characterized: its specific activity toward 3-(3,4-dihydroxyphenyl)-L-alanine (DOPA) increased by a factor of 24 with an overall recovery of 3% of initial activity. The apparent molecular mass of the purified tyrosinase was 40 kDa as determined by SDS-PAGE, and 42 kDa as determined by gel filtration. Its activity was optimal at pH 6 and at 25 degrees C. The enzyme exhibited high activity toward phenylenediamine, epicatechin, pyrogallol, DOPA, and resveratrol. Tyrosinase activity was inhibited by KCN, thiourea, and SO(2). Resveratrol levels were stable following the removal of proteins from the juice, suggesting that early spraying of grapes with SO(2) is an important factor affecting the final amount of resveratrol in wine.  相似文献   

7.
The hepatopancreas of squid (Illex illecebrosus) extract contains a wide range of carboxypeptidase (CP) activities based on hydrolysis of N-CBZ-dipeptide substrates. SDS-PAGE zymograms with N-CBZ-Phe-Leu substrate revealed three activity zones (CP-I, 23 kDa; CP-II, 29 kDa; CP-III, 42 kDa). CP-I was purified 225-fold with 86.20% recovery based on N-CBZ-Ala-Phe activity by chromatography on DEAE-cellulose, gel filtration, and chromatofocusing. The purified enzyme had broad specificity toward N-CBZ-dipeptides; however, it preferred substrates with a hydrophobic amino acid at the C terminus. CP-I had greatest activity with N-CBZ-Ala-Phe (specific activity = 7104 units/mg of protein, K(m) = 0.40 mM, and physiological efficiency = 22863). CP-I had a pI of 3.4 and is a metalloprotease that is activated by Co(2+) and partially inhibited by Pefabloc, a serine protease inhibitor. With N-CBZ-Ala-Phe and Gly-Phe, it had optimum activity at pH 8 and 70 degrees C. The amino acid composition of squid CP-I is similar to that of CP A from other species.  相似文献   

8.
The effect of proteolytic enzymes, associated with Fusarium head blight, on wheat storage proteins and dough functionality was studied. Fusarium damaged kernels (FDK) and sound kernels were hand-picked from F. graminearum Schwabe and F. avenaceum (Fr.) Sacc. infected samples of bread and durum wheat. Scanning electron microscopy revealed significant degradation of endosperm protein in FDK. Storage proteins from FDK and sound kernels were analyzed by SDS-PAGE, RP-HPLC, and SE-HPLC. Total storage protein was lower in FDK but no significant qualitative differences in protein were detected by either RP-HPLC or SDS-PAGE. SE-HPLC was used to follow the hydrolysis of wheat storage protein by proteolytic enzymes found in FDK and a pure culture of F. graminearum. Selective inhibition of proteolytic activity by p-chloromercuribenzoate, and not soybean trypsin inhibitor or iodoacetic acid, suggests that the F. graminearum protease is an alkaline protease. Farinograph and extensigraph curves showed that the presence of FDK decreased dough consistency and resistance to extension. The presence of FDK in flour resulted in a substantial reduction in loaf volume. The loss of dough functionality and loaf volume potential was attributed to the presence of fungal proteases.  相似文献   

9.
This paper describes the purification and characterization of a novel protein from the seeds of Pouteria torta (family Sapotaceae). The protein was purified by a combination of gel filtration, ion-exchange, and reverse phase chromatographies. SDS-PAGE of the purified protein resulted in a single protein band of 14 kDa in the presence and absence of DTT. The lectin-like activity of pouterin was best inhibited by glycoproteins such as fetuin, asialofetuin, heparin, orosomucoid, and ovoalbumin. Pouterin inhibited the growth of the fungi Fusarium oxysporum and Colletotrichum musae and of the yeast Saccharomyces cerevisiae. The incorporation of pouterin into an artificial diet (final concentration = 0.12%, w/w) caused 50% mortality in larvae of the insect Callosobruchus maculatus, whereas 0.08% pouterin produced an ED50.  相似文献   

10.
A lectin fraction from Chardonnay grape juice has been isolated by affinity chromatography on a column of p-aminophenyl beta-D-glucoside-derivatized agarose. The lectin fractions agglutinate rabbit and human erythrocytes without serological specificity. None of the usual monosaccharides, glycosides, or glycoproteins inhibit the hemagglutinating activity. Erythroagglutination is only inhibited by nitrophenyl glycosides, p-nitrophenyl beta-D-glucoside being the strongest inhibitor. In SDS-PAGE in the presence of 2-mercaptoethanol and gel filtration HPLC, the lectin fraction gave a single band or peak corresponding to M(r) 13.2-11.9 kDa, thus indicating it to be a monomer. Three bands were observed by isoelectric focusing with pI values of 4.1, 4. 4, and 4.9. The isolectins seem to be glycoproteins since they are bound on a concanavalin A-Sepharose column.  相似文献   

11.
The soluble potato proteins are mainly composed of patatin and protease inhibitors. Using DSC and both far-UV and near-UV CD spectroscopy, it was shown that potato proteins unfold between 55 and 75 degrees C. Increasing the ionic strength from 15 to 200 mM generally caused an increase in denaturation temperature. It was concluded that either the dimeric protein patatin unfolds in its monomeric state or its monomers are loosely associated and unfold independently. Thermal unfolding of the protease inhibitors was correlated with a decrease in protease inhibitor activities and resulted in an ionic strength dependent loss of protein solubility. Potato proteins were soluble at neutral and strongly acidic pH values. The tertiary structure of patatin was irreversibly altered by precipitation at pH 5. At mildly acidic pH the overall potato protein solubility was dependent on ionic strength and the presence of unfolded patatin.  相似文献   

12.
Proteins associated with starch synthase (SS) activities were identified in immature mungbeans (Vigna radiata L. cv KPS1). Seed soluble extract was separated by native-PAGE and subjected to in situ activity staining. The gel zymogram located starch-enzyme complex bands. The soluble extract was also partitioned by preparative-IEF and screened for SS activity using radioactive assay. IEF fractions eluted within pH 4-6 revealed enriched SS activity of 145-fold. Parallel comparison of the protein profiles among the activity stained enzyme complex and the active isoelectric focused fractions on SDS-PAGE depicted three SS-activity-related proteins with molecular size of 32, 53, and 85 kDa. The 85 kDa protein, however, was identified to be methionine synthase by MALDI-TOF analysis and should be a protein physically associated with the active SS. Polyclonal antibodies raised from eluted native enzyme complex neutralized up to 90% activity and antigenically recognize the other 53 and 32 kDa proteins on Western blot. Antibodies raised from the two individual denatured proteins were able to neutralize SS activities near 60% separately, indicating that the 53 kDa and 32 proteins associated with SS activity are potentially involved in starch biosynthesis during mungbean seed development.  相似文献   

13.
Mung bean trypsin inhibitor (MBTI) of the Bowman-Birk family was purified to homogeneity with a molecular mass of approximately 9 kDa on tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 8887.25 Da as determined by matrix-assisted laser desorption/ionization-quadrupole ion trap-time-of-flight mass spectrometry (MALDI-QIT-TOF MS). Using blue scad myofibrillar proteins as targets, it was found that, in the absence of MBTI, proteolysis of myofibrillar proteins, especially myosin heavy chain (MHC), could be identified after incubation at 55 °C for 2 h, while in the presence of MBTI, with a final concentration of 25 ng/mL, proteolysis of these proteins was greatly suppressed even after incubation for 3 h. Although cysteine proteinase inhibitor E-64 was also effective in preventing protein degradation, inhibitors for metallo- and asparatic proteinases did not reveal obvious inhibitory effects. Our present results strongly suggested that the naturally occurring legume bean seed protein MBTI can be used as an effective additive in preventing marine fish blue scad surimi gel softening, which is quite possibly caused by myofibril-bound serine proteinase (MBSP).  相似文献   

14.
A cysteine protease inhibitor (CPI) in chum salmon ( Oncorhynchus keta) plasma (CSP) was detected after performing inhibitory activity staining against papain under nonreducing condition. The CPI was purified from CSP by affinity chromatography with a yield and purification ratio of 0.94% and 30.36-fold, respectively. CSP CPI had a molecular mass of 70 kDa based on the results of SDS-PAGE and Sephacryl S-100 gel filtration. CSP CPI was a glycoprotein based on the periodic acid-Schiff (PAS) staining of the SDS-PAGE gel and classified as a kininogen. CSP CPI was stable in the pH range of 6.0-9.0 with maximal stability at pH 7.0. CSP CPI presented thermal stability at temperatures below 50 degrees C and exhibited maximal activity at temperatures of 20-40 degrees C. CSP CPI was determined to be a noncompetitive inhibitor against papain, with an inhibitor constant (Ki) of 105 nM.  相似文献   

15.
The phyla Leguminosae contains protease inhibitors, lectins, chitinases, and glycohydrolases as major defense proteins in their seeds. Electrophoretic analysis of the seed proteins of tamarind ( Tamarindus indica L.), an agri-waste material, indicated the unusual presence of two major proteins comparable to overexpression of recombinant proteins. These proteins were identified by amino-terminal analysis to be (1) Kunitz-type trypsin inhibitor and (2) class III endochitinase (34000 Da). These two proteins were purified to apparent homogeneity by a single-step chitin bead affinity chromatography and characterized. The Kunitz inhibitor was specific toward inhibiting trypsin with a stoichiometry of 1:1. The 33000 +/- 1000 Da protein, accounting for >50% of the total seed protein, is an acidic glycoprotein exhibiting a very low endotype hydrolytic activity toward chitin derivatives. SDS-PAGE followed by densitometry of tamarind seed germination indicates the disappearance of the chitinase with the concomitant appearance of a cysteine endopeptidase. On the basis of its abundance, accumulation without any pathogenesis-related stimulus, temporal regulation, amino acid composition, and very low enzyme activity, this 34000 Da protein designated "tamarinin" physiologically serves as the major storage protein.  相似文献   

16.
Biochemical characteristics of patatin proteins purified by ion-exchange and affinity chromatography from tubers of 20 potato cultivars were studied to evaluate their genotype differences with respect to utility groups, table potato cultivars (TPCs) and processing potato cultivars (PPCs). Both groups of cultivars showed similar values of protein content in dry matter (3.98-7.39%) and of patatin relative abundance (5.40-35.40%). Three mass levels (～40.6, 41.8, and 42.9 kDa) of purified patatins were found by MALDI-TOF MS within all cultivars. Differences among mass levels corresponding with the mass of sugar antenna (～1.2 kDa) confirmed the previous concept of different glycosylation extentsin patatin proteins. It was showed that the individual types of patatin varying in their masses occur in the patatin family in a ratio specific for each of the cultivars, with the lowest mass type being the major one. Electrophoretic analyses demonstrated wide cultivar variability in number of patatin forms. Especially 2D-PAGE showed 17-23 detected protein spots independently on the utility group. Specific lipid acyl hydrolase (LAH) activity of purified patatins from the individual tested cultivars varied between 0.92 and 5.46 μmol/(min mg). Patatin samples within most of the TPCs exhibited higher values of specific LAH activity than samples of PPCs. It may be supposed that individual patatin forms do not have similar physiological roles.  相似文献   

17.
Trypsin from tongol tuna (Thunnus tonggol) spleen was purified to 402-fold by ammonium sulfate precipitation, followed by a series of chromatographic separations. The molecular mass of trypsin was estimated to be 24 kDa by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin appearing as a single band on native PAGE showed the maximal activity at pH 8.5 and 65 degrees C. It was stable in a wide pH range of 6-11 but unstable at the temperatures greater than 50 degrees C. The enzyme required calcium ion for thermal stability. The activity was strongly inhibited by 1.0 g/L soybean trypsin inhibitor and 5 mM TLCK and partially inhibited by 2 mM ethylenediaminetetraacetic acid. Activity was lowered with an increasing NaCl concentration (0-30%). The enzyme had a Km for Nalpha-p-tosyl-L-arginine methyl ester hydrochloride of 0.25 mM and a Kcat of 200 s-1. The N-terminal amino acid sequence of trypsin was determined as IVGGYECQAHSQPHQVSLNA and was very homologous to other trypsins.  相似文献   

18.
In the present study, foam-forming and -stabilizing properties of potato proteins were studied using whipping and sparging tests. The soluble potato proteins are mainly composed of patatin and protease inhibitors. The whipping tests showed that less foam was formed from untreated patatin than from the protease inhibitors, but patatin foam was much more stable. The foam-forming properties of patatin could be strongly improved by partial unfolding of the protein. Whipping tests, at both low (0.5 mg/mL) and high (10 mg/mL) protein concentration, also indicated that foams made with an ethanol-precipitated protein isolate were more stable than those made with beta-casein and beta-lactoglobulin. More generally, it can be concluded that when proteins are used as a foaming agent, a high concentration is required, because the protein available is inefficiently used. Also, there are several variables that may all, in different ways, affect both foam formation (amount of foam, bubbles size distribution) and foam stability. These variables include the type and concentration of protein, solvent conditions (pH, I), and the method used to make the foam.  相似文献   

19.
The jojoba, Simmondsia chinensis, is a characteristic desert plant native to the Sonoran desert. The jojoba meal after oil extraction is rich in protein. The major jojoba proteins were albumins (79%) and globulins (21%), which have similar amino acid compositions and also showed a labile thrombin-inhibitory activity. SDS-PAGE showed two major proteins at 50 kDa and 25 kDa both in the albumins and in the globulins. The 25 kDa protein has trypsin- and chymotrypsin-inhibitory activities. In vitro digestibility of the globulins and albumins resembled that of casein and soybean protein concentrates and was increased after heat treatment. The increased digestibility achieved by boiling may be attributed to inactivation of the protease inhibitors and denaturation of proteins.  相似文献   

20.
Dipeptidyl peptidase I (DPP I; EC 3.4.14.1) was purified from porcine skeletal muscle after several steps such as heat treatment, ammonium sulfate fractionation, gel filtration chromatography, and HPLC anion exchange chromatography. The purified enzyme showed a native molecular mass of approximately 200 kDa on Sephacryl S-200 column chromatography. Two protein bands of 65 and 42 kDa were obtained by SDS-PAGE, indicating its oligomeric nature. Maximum activity was reached at pH 5.5 and 55 degrees C. DPP I shared some common substrate specificities, both on synthetic derivatives and on real peptides, with porcine muscle DPP III. The enzyme required reducing agents for full activation, although the halide requirement was not proved. DPP I was inhibited by the assayed cysteine peptidase inhibitors except p-CMB. The serine peptidase inhibitor 3, 4-DCI also inhibited the enzyme as did the divalent cations Co(2+), Mn(2+), and Zn(2+). On the basis of its properties, DPP I may contribute to the generation of dipeptides during the processing of meat and/or meat products, including cooked ham.  相似文献   

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