Effect Of Limited Proteolysis Research Articles

The effect of limited proteolysis by different proteases on the formation of whey protein fibrils

Four proteases: trypsin, protease A, pepsin, and protease M were selected to modify whey protein concentrate (WPC) at a low degree of hydrolysis (0.1, 0.2, and 0.3%) before adjusting to pH 2.0 and heating at 90°C to gain insight into the influence of proteolysis on fibril formation. The kinetics of fibril formation were performed on native and modified WPC using the fluorescent dye thioflavin T in conjunction with transmission electron microscopy and far-UV circular dichroism spectroscopy for the morphological and secondary structural analyses. The change in surface hydrophobicity and content of free sulfhydryl groups were also observed during the formation of fibrils for the native and modified WPC. The content of aggregation and thioflavin T kinetic data indicated that the ability of fibril formation was apparently different for WPC modified by the 4 proteases. Whey protein concentrate modified by trypsin aggregated more during heating and the fibril formation rate was faster than that of the native WPC. Whey protein concentrate modified by the other proteases showed slower aggregation with worse amyloid fibril morphology. Compared with the native WPC, the structure of WPC changed differently after being modified by proteases. The state of α-helix structure for modified WPC played the most important role in the formation of fibrils. Under the mild conditions used in this work, the α-helix structure of WPC modified by trypsin caused little destruction and resulted in fibrils with good morphology; the content of α-helices for WPC modified by other proteases decreased to 36.19 to 50.94%; thus, fibril formation was inhibited. In addition, it was beneficial for the modified WPC to form fibrils such that the surface hydrophobicity increased and the content of free sulfhydryl groups slightly decreased during heating.

Improvement of the Antimicrobial and Antioxidant Activities of Camel and Bovine Whey Proteins by Limited Proteolysis.

The compositions and structures of bovine and camel milk proteins are different, which define their functional and biological properties. The aim of this study was to investigate the effects of enzymatic hydrolysis of camel and bovine whey proteins (WPs) on their antioxidant and antimicrobial properties. After enzymatic treatment, both the antioxidant and the antimicrobial activities of bovine and camel WPs were improved. The significantly higher antioxidant activity of camel WPs and their hydrolysates as compared with that of bovine WPs and their hydrolysates may result from the differences in amounts and/or in accessibilities of antioxidant amino acid residues present in their primary structures and from the prevalence of alpha-lactalbumin and beta-lactoglobulin as proteolytic substrates in camel and bovine whey, respectively. The results of this study reveal differences in antimicrobial and antioxidant activities between WP hydrolysates of bovine and camel milk and the effects of limited proteolysis on these activities.

Effects of limited proteolysis and high-pressure homogenisation on structural and functional characteristics of glycinin

Glycinin was treated by limited proteolysis and/or high-pressure homogenisation in this work. The combined treatments significantly improved the solubility of glycinin, especially in acidic aqueous solution. Moreover, the modified glycinin had better emulsification capability and lower surface hydrophobicity. Electrophoresis analysis indicated more aggregates were formed after above treatments. But the laser scanning test showed the particle size of glycinin decreased. Compared with limited proteolysis, the combined treatments further increased the storage modulus and postponed the onset of glycinin gelation. The results of scanning electron microscopy showed that a fibrous curly structure was present in native glycinin. It was not found in modified glycinins. High-pressure homogenisation slightly shortened the onset of glycinin gelation, while limited proteolysis apparently postponed this behaviour. All the treatments formed weaker gels than native glycinin.

The effect of limited proteolysis on canola protein gelation

The objective of this study was to improve canola protein gelation properties through limited enzymatic hydrolysis with trypsin, ficin and bromelin either alone or in combination with the cross-linking enzyme transglutaminase (TG). Proteolysis alone was found to reduce gel strength to below that of the control. This was accompanied by a decrease in molecular weight, observable through SDS-PAGE analysis. Micrographs corroborated the rheological data and showed that protease application brought about a loss of structure. In contrast, limited proteolysis was shown to be a suitable pretreatment to cross-linking with TG, a frequently used cross-linking enzyme. Previous research suggests and this study corroborates that opening the protein structure prior to TG treatment, can enhance its effectiveness. Gels, treated with both a protease and TG, were significantly stronger than those treated with TG alone. It was concluded that while limited proteolysis does not improve canola protein gelation on its own, it can significantly improve gelation properties when combined with TG.

Effect of adsorption characteristics of a modified cellulase on indigo backstaining

AbstractThe effect of limited proteolysis (digestion) of a commercial cellulase preparation (Ecostone® L350) on backstaining with indigo was investigated. The influence of protease (papain) concentration on limited proteolysis of cellulase preparation was studied, applying different ratios of papain/cellulase (w/w). Changes in adsorption on Avicel cellulose of the non‐digested compared with the papain‐digested Ecostone® L350 were examined using the Langmuir adsorption isotherm. The non‐digested Ecostone® L350 exhibited stronger interaction to Avicel cellulose compared with the digested form, while the maximum efficiency of cellulase adsorption to Avicel cellulose decreased after digestion. When papain‐digested Ecostone® L350 was applied on cotton fabrics during the dyeing procedure with indigo, a reduction of indigo backstaining was obtained compared with the non‐digested Ecostone® L350. Copyright © 2004 Society of Chemical Industry

The spinach plasma membrane Ca2+ pump is a 120‐kDa polypeptide regulated by calmodulin‐binding to a terminal region

The spinach (Spinacia oleracea L.) leaf plasma membrane Ca2+‐ATPase is regulated by calmodulin (3‐fold stimulation) and limited proteolysis (trypsin; 4‐fold stimulation). The plasma membrane Ca2+‐ATPase was identified as a 120‐kDa polypeptide on western immunoblots using two different antibodies. During trypsin treatment the 120‐kDa band diminished and a new band appeared at 109 kDa. The appearance of the 109‐kDa band correlated with the increase in enzyme activity following trypsin treatment. The stimulations by calmodulin and trypsin were not additive, suggesting that the 109‐kDa polypeptide represents a Ca2+‐ATPase lackin a terminal fragment involved in calmodulin regulation. This was confirmed by 125I‐calmodulin overlay studies where calmodulin labeled the 120‐kDa band in the presence of Ca2+, while the 109‐kDa band did not bind calmodulin. The effects of calmodulin and limited proteolysis on ATP‐dependent accumulation of 45Ca2+ in isolated inside‐out plasma membrane vesicles were studied, and kinetical analyses performed with respect to Ca2+ and ATP. Calmodulin increased the Vmax. for Ca2+ pumping 3‐fold, and reduced Km for Ca2+ from 1.6 to 0.9 µM. The Km for ATP (11 µM) was not affected by calmodulin. The effects of limited proteolysis on the affinities for Ca2+ and ATP were similar to those obtained with calmodulin. Notably, however, limited proteolysis increased the Vmax. for Ca2+ pumping to a higher extent than calmodulin, indicating incomplete calmodulin activation, or removal of an additional inhibitory site by trypsin.

Effect of limited proteolysis on the stability and enzymatic activity of human placental S-adenosylhomocysteine hydrolase.

Human placental S-adenosylhomocysteine (AdoHcy) hydrolase was subjected to limited papain digestion. The multiple cleavage sites in the enzyme were identified to be Lys94-Ala95, Tyr100-Ala101, Glu243-Ile244, Met367-Ala368, Gln369-Ile370, and Gly382-Val383. Despite multiple cleavage sites in the backbone of the protein, the digested enzyme was able to maintain its quaternary structure and retain its full catalytic activity. The enzyme activity of the partially digested AdoHcy hydrolase was essentially identical to that of the native enzyme at several pH values. The thermal stabilities of the native and partially digested enzymes were only slightly different at all temperatures tested. The stability of both native and partially digested enzymes were examined in guanidine hydrochloride and equilibrium unfolding transitions were monitored by CD spectroscopy and tryptophan fluorescence spectroscopy. The results of these experiments can be summarized as follows: (1) CD spectroscopic analysis showed that the overall secondary and tertiary structures of the partially digested enzyme are essentially identical with those of the native enzyme; and (2) tryptophan fluorescence spectroscopic analysis indicated that there are small differences in the environments of surface-exposed tryptophan residues between the partially digested enzyme and the native enzyme under unfolding conditions. The differences in the free energy of unfolding, delta(delta Gu) [delta Gu(native)-delta Gu(digested)], is approximately 1.3 kcal/mol. When NAD+ was removed from the partially digested enzyme, the secondary and tertiary structures of the apo form of the digested AdoHcy hydrolase were completely lost and the enzymatic activity could not be recovered by incubation with excess NAD+. These results suggest that AdoHcy hydrolase exists as a very compact enzyme with extensive intramolecular bonding, which contributes significantly to the overall global protein stabilization. Identification of the surface-exposed peptide bonds, which are susceptible to papain digestion, has provided some constraints on the spatial orientations of subunits of the enzyme. This information, in turn, has provided supplemental data for X-ray crystallographic studies currently ongoing in our laboratories.

Effect of Limited Proteolysis on the Enzymatic Phosphorylation of Soy Protein

The effects of limited proteolysis on the enzymatic phosphorylation of commercial soy protein isolate were evaluated for several proteases. Proteases were chosen on the basis of their ability to hydrolyze peptide bonds between specific amino acids and included trypsin (basic), chymotrypsin (aromatic), endoproteinase Glu-C from Staphylococcus aureus, strain V8 (acidic), and Pronase E (nonspecific). Hydrolyzed soy isolate was phosphorylated with the catalytic subunit of bovine cardiac muscle protein kinase, and phosphorylation was followed by incorporation of 32P into the protein. Phosphorylation decreased with increased proteolysis for all proteases. Decreased phosphorylation was most pronounced for Pronase E-hydrolyzed protein and least pronounced for trypsin-hydrolyzed soy isolate. The solubility−pH profile of the trypsin-hydrolyzed, phosphorylated soy isolate was compared to those of both the untreated and the hydrolyzed profiles. There was no significant difference in solubility among the profiles over the pH 3−6 range. Keywords: Polypeptide; modification; functionality; hydrolysis; interaction

Effect of limited proteolysis with ?-chymotrypsin on semen with an abnormal sperm penetration assay and possible application for in vitro fertilization or intrauterine insemination

This study was designed to assess the impact of limited proteolysis with alpha-chymotrypsin on the sperm penetration assay (SPA) of infertile patients and to identify a group whose results would normalize with this pretreatment. Further, the application of this treatment to semen of these patients during in vitro fertilization (IVF) and intrauterine insemination (IUI) was reported. Three groups were identified. In one, SPA was abnormal and improved to normal with pretreatment; in the second, SPA was abnormal and did not normalize; and in the third, SPA was normal and improved significantly (included in this group are three known fertile controls). Chymotrypsin pretreatment and repeat SPA are advocated for patients with abnormal SPA. If normalization occurs, pretreatment of semen for IVF or IUI is a therapy to be considered.

Outer envelope membranes from chloroplasts are isolated as right-side-out vesicles

Outer envelope membranes were isolated from purified chloroplasts of pea leaves. The sidedness of the vesicles was analyzed by (i) aqueous polymer-two phase partitioning, (ii) the effect of limited proteolysis on the outer-envelope proteins (OEP) 86 and OEP 7 in intact organelles and isolated membranes, (iii) fluorescence-microscopy and finally (iv) binding of precursor polypeptides to isolated outer-membrane vesicles. The results demonstrate that purified outer envelope membranes occur largely (>90%) as right-side-out vesicles.

Effect of limited proteolysis in the 8th loop of the barrel and of antibodies on porcine pancreas amylase activity

The porcine pancreatic alpha-amylase is a (beta/alpha)8-barrel protein, containing domains A and B (peptide sequence 1-403) and a distinct C-domain (peptide sequence 404-496). Separation of the terminal C-domain from the A and B domains has been attempted by limited proteolysis in the hinge region. Subtilisin was found to hydrolyse amylase between residues 369 and 370 situated in the loop between the eighth beta-strand and alpha-helix. The cleaved amylase was isolated by chromatofocusing and found to retain about 60% of the activity of the native enzyme, while the isolated fragments were inactive. Antigen binding fragments prepared from polyclonal antibodies to native amylase and the CNBr-fragment P1 (peptide sequence 395-496) respectively, were tested for influence on the enzyme activity. Antibodies directed against P1 had no effect whereas antibodies against the peptide sequence 1-394 and amylase respectively inhibited hydrolysis of substrates having four or more glucose residues but not of shorter oligomaltosides. Crystallographic analysis revealed that changes in the region of residue 369 might affect the conformation of the active site as well as of a second binding site. This site, located on the enzyme surface, is proposed to be required for the hydrolysis of larger substrates.

Subunit interactions control protein phosphatase 2A. Effects of limited proteolysis, N-ethylmaleimide, and heparin on the interaction of the B subunit

Protein phosphatase 2A consists of a heterotrimeric complex composed of a catalytic subunit (C) and two associated subunits (A and B). Limited tryptic digestion of the heterotrimeric ABC form resulted in the selective degradation of the Mr = 55,000 B subunit to a 48-kDa polypeptide. The cleavage sites were determined to be within a 3-7-kDa region of the COOH terminus. Proteolysis led to dissociation of the B subunit from the enzyme complex and correlated with an increase in cardiac myosin light chain, smooth muscle myosin light chain peptide, and Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) phosphatase activity. Purification of the digestion products and native gel electrophoresis indicated that dissociation of the B subunit was responsible for the increase in phosphatase activity. Kinetic analyses with several substrates revealed that dissociation of the B subunit resulted in a 2-7-fold increase in Vmax and a 1.6-5 fold increase in Km. Proteolytic dissociation of the B subunit increased the sensitivity of protein phosphatase 2A to inhibition by okadaic acid. Inhibition of the trypsinized enzyme was very similar to that observed for the purified AC form of protein phosphatase 2A. Incubation of the ABC complex with N-ethylmaleimide resulted in dissociation of the C subunit and generation of an AB complex. Selective release of the C subunit indicated that the B subunit interacts directly with the A subunit and that one or more free sulfhydryls are required to maintain the heterotrimeric structure of protein phosphatase 2A. Treatment of the enzyme with heparin resulted in an increase in specific activity that was due to the release of the B subunit from the complex. These results provide evidence that the B subunit binds directly to the A subunit to modulate enzyme activity and substrate specificity and that the COOH-terminal region of this protein is important for interaction with the AC complex. Dissociation of the B subunit by polyanionic substances related to heparin may represent a mechanism for regulating the activity of this enzyme.

Structure‐function relationships in the polypeptide cardiac stimulant, anthopleurin‐A

Selective proteolysis of the polypeptide cardiostimulant anthopleurin-A by trypsin introduces a single break in the polypeptide backbone on the C-terminal side of Arg14. The resulting derivative is devoid of any cardiostimulant activity. The structural changes which accompany this loss of activity have been examined by one- and two-dimensional 1H-NMR spectroscopy. It is shown that the overall backbone folding of anthopleurin-A is conserved on digestion, with some structural changes occurring for residues which are adjacent to the site of cleavage by trypsin. Thus, although previous NMR studies on anthopleurin-A indicate that the region surrounding Arg14 is devoid of any ordered structure, it appears that some degree of structural integrity is required to allow the essential side chains to adopt the conformation necessary to produce a cardiostimulant effect.

The effect of limited proteolysis on GTP-dependent Ca2+ efflux and GTP-dependent fusion in rat liver microsomal vesicles

1. Limited proteolytic digestion of rat liver microsomes (microsomal fractions) with trypsin (5 micrograms/ml), proteinase K (1.0 microgram/ml) and Pronase (20 micrograms/ml final concns.) resulted in abolition of GTP-dependent vesicle fusion. 2. Vesicle fusion could be partially restored to microsomes which had undergone limited tryptic digestion, by the addition of untreated microsomal vesicles. 3. GTP-dependent Ca2+ efflux from rat liver microsomes was also observed to be inhibited by limited proteolysis with trypsin and proteinase K. 4. Limited proteolysis of rat liver microsomes had no effect on subsequent GTP-dependent phosphorylation of polypeptides of Mr 17,000 and 38,000, and thus it is unlikely that the phosphorylation of these proteins is involved in GTP-dependent Ca2+ efflux and GTP-dependent vesicle fusion. 5. GTP binding by Gn proteins [proteins which bind GTP after transfer to nitrocellulose, as defined by Bhullar & Haslam (1986) Biochem. J. 245, 617-620] was inhibited by pre-treatment of microsomes with trypsin, proteinase K and Pronase at concentrations similar to those which abolished GTP-dependent Ca2+ efflux and vesicle fusion. 6. We suggest that one or more of the Gn proteins may be involved in the molecular mechanisms of GTP-dependent vesicle fusion and Ca2+ efflux in rat liver microsomes and that limited proteolytic digestion may be a useful tool in further investigation of these processes.

Localization of the actin-binding sites of Acanthamoeba myosin IB and effect of limited proteolysis on its actin-activated Mg2+-ATPase activity.

Acanthamoeba myosin IB contains a 125-kDa heavy chain that has high actin-activated Mg2+-ATPase activity when 1 serine residue is phosphorylated. The heavy chain contains two F-actin-binding sites, one associated with the catalytic site and a second which allows myosin IB to cross-link actin filaments but has no direct effect on catalytic activity. Tryptic digestion of the heavy chain initially produces an NH2-terminal 62-kDa peptide that contains the ATP-binding site and the regulatory phosphorylation site, and a COOH-terminal 68-kDa peptide. F-actin, in the absence of ATP, protects this site and tryptic cleavage then produces an NH2-terminal 80-kDa peptide. Both the 62- and the 80-kDa peptides retain the (NH+4,EDTA)-ATPase activity of native myosin IB and both bind to F-actin in an ATP-sensitive manner. However, only the 80-kDa peptide retains a major portion of the actin-activated Mg2+-ATPase activity. This activity requires phosphorylation of the 80-kDa peptide by myosin I heavy chain kinase but, in contrast to the activity of intact myosin IB, it has a simple, hyperbolic dependence on the concentration of F-actin. Also unlike myosin IB, the 80-kDa peptide cannot cross-link F-actin filaments indicating the presence of only a single actin-binding site. These results allow the assignment of the actin-binding site involved in catalytic activity to the region near, and possibly on both sides of, the tryptic cleavage site 62 kDa from the NH2 terminus, and the second actin-binding site to the COOH-terminal 45-kDa domain. Thus, the NH2-terminal 80 kDa of the myosin IB heavy chain is functionally similar to the 93-kDa subfragment 1 of muscle myosin and most likely has a similar organization of functional domains.

Pretreatment of human platelet membranes with trypsin abolishes GTP but not Na+ effects on alpha 2-adrenoreceptor-agonist interactions.

The affinity of many types of membrane-bound receptors coupled negatively to adenylate cyclase is regulated by divalent and monovalent cations and by guanine nucleotides (GTP). We used alpha 2-adrenoreceptors of human platelets as a model system to find out the effect of limited proteolysis with trypsin on the regulation of the alpha 2-adrenoreceptor-agonist interactions by GTP and Na+. We found that partial proteolysis of the membranes with trypsin for 3 min at 35 degrees C reduced specific [3H]yohimbine binding to platelet membranes to 40-50% of control. The following characteristics of the receptors remaining after proteolysis were similar to those of untreated membranes: affinity for the agonist and antagonists, stereospecificity, and kinetic properties. Trypsin also did not modify the ability of the receptor's change from a high to low affinity state in the presence of Na+. These findings suggested that the capability of the receptors to recognize the ligand and their ability to undergo a conformational change in the presence of the agonist were retained despite a reduction in the total number of receptors by trypsin. However, the modulation of the receptor--agonist interactions by GTP or Mg2+ was lost in the trypsin-pretreated membranes, while the modulation by Na+ remained intact. It is suggested that the loss of GTP or Mg2+ effects on receptor--ligand interactions produced by trypsin may be due to trypsin-induced disruption of subunits (alpha i, beta gamma) interactions of Gi protein.

Effects of limited proteolysis on broad bean polyphenoloxidase

AbstractPartially purified broad bean polyphenoloxidase was treated with a variety of proteases. Enzyme activity increased after treatment with chymotrypsin, pepsin, subtilisin, thermolysin, trypsin, and Staphylococcus aureus V8 protease but this activation was masked when assayed in the presence of sodium dodecylsulfate. The enzyme was resistant to proteolysis when treated with chymotrypsin, elastase, pepsin, thermolysin, and V8 protease. Proteinase K, subtilisin and trypsin treatment resulted in partial degradation of the enzyme but only trypsin generated an active enzyme form with a slightly smaller molecular weight. Electrophoresis, followed by enzyme staining and or Western blotting, showed that trypsin treatment converted the active 45 kd enzyme into a 43 kd enzyme within 20 min. Treatment with proteinase K, subtilisin and trypsin also produced inactive higher molecular weight forms of purified polyphenoloxidase that were identified by Western blotting. The use of antibodies coupled with Western blotting also identified inactive, active, and latent polyphenoloxidase after protease treatment, and suggests that immunological methods can be used to establish the types and amounts of polyphenoloxidase present in a given tissue.

Bovine milk xanthine oxidase: effect of limited proteolysis on kinetics and structure

Comparaison des caracteristiques de deux echantillons de xanthine oxydase, l'une preparee selon la methode habituelle de purification par voie proteolytique, l'autre par une methode douce non proteolytique que l'on decrit

Effect of limited proteolysis on the functional properties of cottonseed flour.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTEffect of limited proteolysis on the functional properties of cottonseed flourE. H. Rahma and M. S. Narasinga RaoCite this: J. Agric. Food Chem. 1983, 31, 2, 356–358Publication Date (Print):March 1, 1983Publication History Published online1 May 2002Published inissue 1 March 1983https://doi.org/10.1021/jf00116a043RIGHTS & PERMISSIONSArticle Views72Altmetric-Citations11LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (363 KB) Get e-Alerts Get e-Alerts

Medium-chain fatty acid synthesis by goat mammary-gland fatty acid synthetase. The effect of limited proteolysis

Fatty acid synthetase from goat mammary gland was subjected to limited proteolysis by trypsin and elastase. Both proteolytic enzymes selectively cleaved the chain-terminating thioester hydrolase component from the enzyme complex, leaving all other partial activities intact in the core peptides. Trypsin, but not elastase, caused extensive degradation of the released thioester hydrolase. The released thioester hydrolase could be purified to homogeneity by gel filtration. The molecular weight was estimated as 29 000 and the enzyme showed only significant hydrolytic activity toward long-chain acyl-CoA esters. The core peptides retained the ability to synthesize medium-chain acyl-CoA esters in the presence of 2,6-di-O-methyl-alpha-cyclodextrin. The results conclusively show that the terminating thioester hydrolase of goat mammary-gland fatty acid synthetase is not involved in termination of medium-chain-length fatty acid synthesis by this enzyme.