摘要:

ABSTRACTIn a search for biologically active materials from the classified barley flour produced in Japan an aminopeptidase activity was identified. In this paper, the purification of aminopeptidase is described. The activity of the enzyme was monitored using L‐leucine‐p‐nitroanilide as the substrate. After extraction using 20 mM sodium acetate buffer, pH 5.5, from the 95–75% classification flour, ammonium sulfate fractionation was performed between 30 and 50% saturation. The aminopeptidase was then purified about 160‐fold to homogeneity as assessed by HPLC using the following sequential chromatography steps: hydrophobic interaction chromatography, Sephacryl S‐200HR gel chromatography, DEAM‐ion exchange chromatography, hydroxylapatite chromatography, Sephacryl S‐100HR gel chromatography. The molecular weight of this enzyme was estimated as 62 kDα by size exclusion HPLC. The enzyme had a Kmvalue of 0.22 mM and α

ISSN:0145-8884    

DOI:10.1111/j.1745-4514.1995.tb00544.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

ABSTRACTOxy‐ and metmyoglobin were purified from beef muscle using ion‐exchange chromatography on Mono‐Q. Their reactivity against six monoclonal antibodies (MAbs) to beef myoglobin was compared in a competitive ELISA using biotinylated beef oxymyoglobin. Four MAbs reacted with higher affinity for oxymyoglobin than for metmyoglobin. A similar higher reactivity of oxymyoglobin versus metmyoglobin was obtained with a sandwich ELISA using peroxidase‐labelled rabbit anti‐serum against beef myoglobin. Such a result has never been reported for myoglobins of any species. These MAbs should be valuable tools for analyzing conformational changes of oxymyoglobin solutions when subjected to chemical or physical t

ISSN:0145-8884    

DOI:10.1111/j.1745-4514.1995.tb00545.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

ABSTRACTThe activities of cathepsins B, D, H and L were compared in crude muscle extracts from four species of fish: Pacific whiting (PW), (Merluccius productus); arrowtooth flounder (ATF), (Atheresthes stomias); Alaska pollock (AP), (Theragra chalcogramma); and Pacific cod (PC), (Gadus macrocephalus). Both PW and ATF are known to undergo softening during post‐mortem handling and cooking while AP and PC do not. Cathepsin B and L activities were both higher in extracts of PW and ATF than in AP and PC. Cathepsins B and L activities were both much higher in PW than in ATF. Cathepsin H activity was highest in AP followed by ATF with PC and PW having the lowest activities. Cathepsin D activity was extremely low in all four species.The heat stability of the various cathepsin activities showed cathepsin B to be the most heat labile and was inactivated by 50C heating for 15 min. Cathepsin H activity was inactivated at 60C, while cathepsin L required 70C for inactivation. Cathepsins B and L are the most likely responsible for softening in PW and ATF during holding and subsequent processing. However, during cooking, cathepsin L likely causes the most damage since it requires 70C for inactivation. The difference in heat stability of cathepsins B and L can be used to differentiate between their activitie

ISSN:0145-8884    

DOI:10.1111/j.1745-4514.1995.tb00546.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

ABSTRACTA cysteine proteinase was isolated from immature fruits of Pseudananas macrodontes (Morr.) Harms, a species taxonomically close to pineapple grown in the northeast of Argentina. The protease is active at pH 7.0 to 10.5, and rather unstable at temperatures over 45C. Crude extracts were purified by acetone fractionation followed by chromatofocusing at the pH range 4.5 to 6.5. Two main proteolytic fractions were obtained: fraction I (Mr= 20.6 kDa, pI = 5.9) and fraction II (Mr= 19.3 kDa, pI = 5.3), both homogeneous by gel‐filtration and SDS‐PAGE criteria. This new plant protease (“macrodontin”) could be valuable in processes carried out in neutral‐weak alkaline media. The thermal behavior of the crude enzyme is also a useful property, since it could be easily inactivated in foodstuffs by heating 10 min at 75C. Comparison of macrodontin and alcalase proteolytic behavior on soy concentrates is

ISSN:0145-8884    

DOI:10.1111/j.1745-4514.1995.tb00547.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

ABSTRACTThe fluorescence emission intensity from rapeseed globulin (cruciferin) increased in the presence of anilinonaphthalene‐8‐sulfonate (ANS) at pH 2.0 but not at pH 10. Fluorescence titration studies showed that at pH 7 cruciferin binds 22 (±0.6) moles of ANS per mole of protein with an average dissociation constant (Kd) of 1.9 (±0.1) × 10−5M. At pH 2.0 the number of ligand binding sites (n) decreased to 14 (±0.2) moles of ANS bound per mole of cruciferin. However, the ANS binding affinity increased by about five times (Kd= 3.6 (±1.1) × 10−6M). The fluorescence emission spectrum maxima (Λmax) for the cruciferin‐ANS complex showed a blue shift at pH 2 when compared to Λmaxvalues at pH 7–10. These results are consistent with a loss of the quaternary and tertiary structures of cruciferin and the exposure of surface hydrophobic ANS binding sites at low pH. Cruciferin‐ANS binding parameters at pH 10 were not significantly different from values at pH 7; n = 22 and Kd= 2.7 (±0.2) × 10−5M. Based on these ANS fluorescence measurements cruciferin is stable

ISSN:0145-8884    

DOI:10.1111/j.1745-4514.1995.tb00548.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY