Proteolytic Hydrolysis Research Articles

Linkage of sugar chains to a fragment peptide of FGF-5S by a chemoenzymatic strategy and changes in the rate of proteolytic hydrolysis.

Various O-linked and N-linked sugar chains were linked enzymatically to a fragment peptide (Leu-Ser-Gln(or Asn)-Val-His-Arg) of FGF-5S. First, galactose was linked with beta-(1-->3)-linkage to GalNAc-linked peptide by a transglycosylation using beta-galactosidase from Bacillus circulans (recombinant). Then sialic acid was linked with the aid of sialyltransferase from rat liver (recombinant) to give NeuAcalpha-(2-->3)-Galbeta-(1-->3)-GalNAc-linked hexapeptide. Further, a sialylated 2-chain biantennary sugar chain was linked by a transglycosylation using endo N-acetyl-beta-D-glucosaminidase from Mucor hiemalis (endo M, recombinant). The activity of DNA synthesis in a fibroblast cell line was increased by this glycosylation. The resistance of the obtained glycopeptides towards proteolytic hydrolysis by rat serum and by five proteases was compared with that of original peptide. The resistance was remarkably enhanced by the glycosylation.

Characterization of Oat Endoproteinases that Hydrolyze Oat Globulins

ABSTRACTDuring the germination of oats, the major seed storage proteins (globulins) are hydrolyzed by endoproteinases. We have used two methods to characterize these endoproteinases. A qualitative PAGE method that used oat globulins as gel‐incorporated substrates was used to determine which enzymes hydrolyzed the globulins. The proteolytic hydrolysis products were studied by hydrolyzing the globulins in vitro with the endoproteinases and analyzing the products by SDS‐PAGE. Class‐specific proteinase inhibitors were used to show that the globulin hydrolyzing enzymes were cysteine‐class proteinases. The proteinases were active at pH 3.8. Using the gel analysis method, a little activity was present at the beginning of seed germination, but the major activity only appeared on the sixth day of germination. Extracts from four‐day germinated oats contained cysteine proteinases that hydrolyzed the globulins in vitro to form a polypeptide of intermediate size (MW ≈34,500). Cysteine proteases from an eight‐day germinated sample totally hydrolyzed the globulins in <1 hr. Very little hydrolysis occurred at pH 6.2, the pH of germinated oats endosperm tissue. The fact that hydrolysis occurred quickly at pH 3.8 implies that there is probably pH compartmentalization within the endosperm, with some areas of the seed having a low pH value where the globulins can be degraded.

Stability of apolipoprotein(a) isoform phenotype to postmortem conditions

Studies, such as the one entitled Pathobiological Determinants of Atherosclerosis in Youth or PDAY, have investigated the relationship of atherosclerotic lesions with lipid and lipoprotein risk factors. However, it is possible that proteolytic hydrolysis during the postmortem period could alter protein components of lipoproteins. Apo(a) is the defining protein of Lp(a), a lipoprotein associated with risk of cardiovascular disease. Apo(a) proteins are highly polymorphic for size and we simulated, using baboon tissues, the postmortem conditions for the PDAY study in order to determine their effects on apo(a) phenotypes. The bulk of the postmortem samples (93%) were correctly phenotyped and showed little or no evidence of degradation. Thus, apo(a) isoform size phenotypes can be determined in postmortem samples, although with caution.

Detection of Sewage Organic Chlorination Products That Are Resistant to Dechlorination with Sulfite

Most of the 36 billion gal of treated sewage wastewater discharged daily into the environment in the United States is disinfected via chlorination. To minimize toxicity. dechlorination with sulfite or sulfur dioxide is often performed. Although dechlorination is considered instantaneous and complete, several studies have found residual toxicity of chlorinated/dechlorinated effluent to aquatic life. The authors investigated chlorination/dechlorination of the organic nitrogen components of sewage wastewater using both iodometric titration and a novel LC method. For LC, a postcolumn reaction with iodide rendered submicromolar chloramine concentrations detectable with amperometry. Using a gradient-elution LC separation, the retention and dechlorination behavior of a suite of model amines was determined, representing primary and secondary aliphatic, peptide, and protein-N. Chlorination/dechlorination experiments on freshly collected, tertiary-treated wastewater showed a fraction of the organic N-chloramines are dechlorinated slowly by sulfite with half-lives of >20 min. Chromatographic retention and kinetic behavior of the sewage N-chloramines was consistent with the behavior of the model peptides and proteins. Proteolytic hydrolysis markedly increased the peptide fraction observed upon chlorination of the wastewater. These results suggest that peptides and proteins contribute to slow dechlorination of sewage and may be a factor in the toxicity noted for chlorine-disinfected wastewater.

Immobilization of jack bean urease on hydroxyapatite: urease immobilization in alkaline soils

Jack bean urease was adsorbed and immobilized on hydroxyapatite, a stable calcium phosphate mineral commonly found in alkaline soils. The stability and kinetic properties of the resulting complex were investigated to derive a model that explains the stabilisation of urease in alkaline soil. The adsorption isotherms revealed: (1) an increase of the affinity of urease for hydroxyapatite with increasing ionic strength at constant pH and (2) a decrease of affinity with increasing pH at constant salt concentration. The optimum pH for enzymatic activity increased from 7 to 8 upon adsorption. The Michaelis–Menten parameters for free urease ( V max=230.7 U mg −1; K m=7.45 m m) and for immobilized urease ( V max=152.9 U mg −1; K m=6.89 m m) showed a moderate decrease of enzyme specific activity, but little change of substrate affinity. Urease adsorption resulted in a 2-fold increase in enzyme stability with time and a 7-fold increase in resistance to proteolytic hydrolysis. The results presented indicate that hydroxyapatite could have a significant role in the stabilisation of extracellular urease in alkaline soils.

The Heme Prosthetic Group of Lactoperoxidase: STRUCTURAL CHARACTERISTICS OF HEME l AND HEME l-PEPTIDES

The heme prosthetic group from the bovine milk enzyme lactoperoxidase (LPO), termed heme l, is isolated through an approach that combines proteolytic hydrolysis and reverse-phase high performance liquid chromatographic separation of the resulting digest. Application of different proteases yields either a peptide-bound heme (with trypsin and chymotrypsin) or a peptide-free heme (with proteinase K). Both heme l and heme l-peptide species were investigated by paramagnetic 1H NMR spectroscopy, electrospray mass spectrometry, and peptide sequence analysis. Paramagnetic 1H NMR experiments on the low spin bis(cyano)-Fe(III)heme l complex conclusively define the heme l structure as a 1,5-bis(hydroxymethyl) derivative of heme b. The electrospray mass spectrum of heme l confirms the two-site hydroxyl functionalization on this heme. Paramagnetic 1H NMR spectra of the high spin bis(dimethyl sulfoxide)-Fe(III) complexes of the isolated heme species provide information regarding peptide content. Sequence analyses of peptides released from two heme l-peptide species by base hydrolysis suggest that heme-protein ester linkages in lactoperoxidase occur between the two hydroxyl groups of heme l and the carboxylic side chains of glutamate 275 and aspartate 125. These results confirm the earlier reported structural proposal (Rae, T. D., and Goff, H. M. (1996) J. Am. Chem. Soc. 118, 2103-2104).

In Vitro Stimulation of C1s Proteolytic Activities by C1s-Presenting Autoantibodies from Patients with Systemic Lupus Erythematosus

Anti-C1s autoantibodies (IgG forms), which recognize the conjunction of C1s heavy chain and light chain (C1s-presenting autoantibodies) from patients with systemic lupus erythematosus (SLE), have been found to stimulate C1s enzymatic activities. This is due to acceleration of the proteolytic hydrolysis of the synthetic substrate C1-1 by C1s, enhancement of the complex formation of C1s with its natural pseudosubstrate, C1 inhibitor (C1 inh), and promotion of proteolytic activation of its natural substrate, C4. Seven of fifteen samples from patients with SLE were found to contain such autoantibodies. The hydrolysis of the synthetic substrate C1-1 catalyzed by C1s in 25 to 27 min in the presence of anti-C1s autoantibodies was equivalent to the hydrolysis of C1-1 catalyzed by C1s alone or C1s with control IgG from healthy sera in 110 min, approximately fourfold faster than the reaction in the absence of anti-C1s autoantibodies. Densitometry scanning data showed that the formation of the C1s-C1 inh complex in the presence of anti-C1s autoantibodies was three to four times greater than that with control IgG. It was also noticed that the autoantibodies convert almost all of the latent forms of C1s to an active form that binds to C1 inh. Another group of Western blots showed that C1s cleaved C4 alpha-chain three times faster in the presence of autoantibodies than of control IgG. It is likely that the overconsumption of complement components is common in the pathogenesis of tissue damage occurring in SLE.

Proteolytic processing of the Aplysia egg-laying hormone prohormone.

By using matrix-assisted laser desorption/ionization time-of-flight MS, individual peptidergic neurons from Aplysia are assayed. A semiquantitative method is developed for comparing single-cell profiles by using spectral normalization, and peptides are localized to specific cells by mass spectrometric cell mapping. In addition to all previously identified products of the egg-laying hormone (ELH) gene, other peptides are formed from proteolytic hydrolysis of Leu-Leu residues within ELH and acidic peptide (AP). AP exhibits further processing to yield AP1-20 and AP9-27. These peptides appear to be colocalized in vesicles with ELH, transported to specific neuronal targets, and released in a Ca2+-dependent manner. A differential peptide distribution is observed at a specific target cell, and a low-frequency variation of AP, [Thr21]AP, is detected in a single animal.

Artificial cell containing superoxide dismutase--selection of folding aids for stabilisation of SOD.

Superoxide dismutase (abbreviated as SOD) has been vigorously studied in the fields of radical chemistry and related life science. One of practical problems is how to keep its activity in certain adverse conditions causing denaturation. Artificial cell containing SOD can be prepared by polymer encapsulation or nanocapsulation which has been found to be effective to improve the stability of SOD. For construction of an ideal artificial cell system, some folding aids or aggregation inhibitors were utilised to enhance SOD stability. In this study, three groups of biopolymers are selected as folding aids or aggregation inhibitors for stabilisation of SOD, i.e. albumin, carbohydrates and glycoproteins. Results indicate that the thermostability of SOD is affected by different sort of albumin while some carbohydrates such as cyclodextrins are found to be able to enhance SOD stability. In addition, it is firstly found that selected glycoproteins such as alpha-macroglobulin and ovalbumin are several types of effective folding aids for stabilisation of SOD. They can protect SOD against denaturation even at very high temperature(over 100 degrees C). The stability was tested by the measurement of SOD activity loss using autooxidation method in different adverse conditions such as high temperature, extreme pH medium, proteolytic hydrolysis and long shelf life storage. The possible stabilisation mechanism of using cyclodextrins and glycoproteins as folding aids were discussed.

Physicochemical characterization and differentiation of the components of the cutaneous basement membrane zone

Many of the techniques used in the diagnosis and characterization of the acquired blistering diseases of the skin and of their target antigens require the use of tissue that has been modified by heat, chemicals, or by proteolytic digestion. The effect these treatments may have on the blistering disease antigens is poorly understood and has seldom been taken into account in the interpretation of the results. Likewise, their effect on the immunodetection and expression of the ubiquitous proteins of the basement membrane zone and extracellular matrix has rarely been investigated. We have addressed this problem by probing tissue split after heat, chemical treatment and proteolytic digestion with an extensive panel of antibodies to the hemidesmosome-plasma membrane-anchoring filament complex, the extracellular matrix and the anchoring fibrils. The results showed that chemical modification by sodium chloride, calcium chloride and ethylenediaminetetraacetic acid, and incubation in 0.15 mol/L NaCl at 56 degrees C for 1 min did not adversely affect the expression of the alpha 6 beta 4 integrin, laminin-5, the LH39 antigen, laminin-1, collagen type IV or collagen type VII. The methods that involved proteolytic digestion had the greatest detrimental effect on these basement membrane components, with pepsin having a damaging effect on the greatest number of antigens, and the LH39 antigen being the most sensitive to proteolytic degradation. This project has highlighted the susceptibility of the basement membrane zone and extracellular matrix proteins to proteolytic hydrolysis. It has demonstrated a way of differentiating between specific proteins, and has shown its potential for the differential diagnosis of the autoimmune blistering disease antigens.

Proteolysis of Milk Proteins During Involution of the Bovine Mammary Gland

The role of proteolytic hydrolysis of milk proteins in the mammary gland during involution is unknown. The objectives of this study were to determine the activities of plasmin, plasminogen, and plasminogen activator in mammary gland secretions collected during involution and to identify peptides generated by proteolysis of casein and lactoferrin in those secretions. Mammary secretions were collected from Holstein cows on d 7, 14, and 21 of involution and on d 7 postcalving. Activities of plasmin, plasminogen, and plasminogen activator were determined on the defatted, filtered aqueous phase of mammary secretions. Activities of plasmin, plasminogen, and plasminogen activator were significantly higher on d 7, 14, and 21 of involution than were those on d 7 postcalving. Protein fragments resulting from hydrolysis were detected by SDS-PAGE in samples collected on d 7, 14, and 21 of involution, but few protein fragments were observed in samples collected on d 7 postcalving when plasmin activity was low. Immunoblot analysis showed that a number of peptides observed during involution were generated from αs-casein (CN), β-CN, κ-CN, or lactoferrin. The appearance of peptides from proteins of mammary secretions during early involution was generally correlated with increased plasmin activity. Elevated plasmin activity during mammary involution may be primarily responsible for the observed concurrent hydrolysis of milk proteins in mammary secretions.

Stability of protease inhibitors based on the Bowman-Birk reactive site loop to hydrolysis by proteases.

Bowman-Birk proteinase inhibitor proteins contain two inhibitory regions, each of which is encapsulated within nine-residue disulfide-linked loops. It is known that short cyclic peptides that retain the nine-residue disulfide-bridged motif have inhibitory activity, and can be used as models of the natural inhibitor protein. Two factors are important in determining the effectiveness of such inhibitor peptides: the value of the inhibition constant, and rate at which the inhibitor peptide is hydrolyzed by the proteinase. In this paper we report a study of the inhibitory properties and stability towards proteolytic hydrolysis of a family of synthetic peptides derived from the trypsin reactive site loop of the Bowman-Birk inhibitors. The addition of a single amino acid residue to each end of the nine-residue disulfide-linked loop is found to reduce the rate at which the peptide is hydrolyzed. In addition, changing the P2' residue from Asn-->Ile gives inhibitors with considerably enhanced stability to proteolysis, as well as reduced values of Ki. The implications of these factors for the design of inhibitors based on this loop motif is discussed.

Effect of proteolytic modification and methionine enrichment on the nutritional value of soya albumins for rats

Proteolytic modification, particularly when combined with methionine enrichment, significantly reduced the lectin content and modified the trypsin inhibitor activity of a soya albumin preparation. During both peptic hydrolysis and enzymatic peptide modification, the amount of soya bean agglutinin was reduced. This reduction was verified by the decrease in the intensity of protein staining and immunoblots of the peptic hydrolysate and of the product produced by enzymatic peptide modification, and confirmed by haemagglutination activity measurements and ELISA technique. The functional activity of Kunitz trypsin inhibitor was also partially abolished whereas that of the Bowman-Birk inhibitor was not significantly altered by the enzymatic modifications. In line with the reduction in some of the antinutrients, the nutritional performance of rats fed a diet containing this enzymatically modified product was significantly improved in comparison with that of the control animals fed the original soya albumin but was poorer than the lactalbumin-fed controls. Enzymatic peptide modification of soya albumin fractions by transpeptidation and covalent methionine enrichment is suitable to improve their nutritional value partly by compensating for their methionine deficiency and partly by modifying the structure of the soya antinutrients. Changes in the structure by proteolytic hydrolysis and synthesis of peptide chains can lead to alteration in protein conformation, resulting in modified biological activity and increased nutritional value.

Purification and Characterization of a Cellulose-Binding (beta)-Glucosidase from Cellulose-Degrading Cultures of Phanerochaete chrysosporium

Extracellular (beta)-glucosidase from cellulose-degrading cultures of Phanerochaete chrysosporium was purified by DEAE-Sephadex chromatography, by Sephacryl S-200 chromatography, and by fast protein liquid chromatography (FPLC) using a Mono Q anion-exchange column. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of FPLC-purified (beta)-glucosidase indicated the presence of three enzyme forms with molecular weights of 96,000, 98,000, and 114,000. On further fractionation with a microcrystalline cellulose column, the 114,000-molecular-weight (beta)-glucosidase, which had 82% of the (beta)-glucosidase activity, was bound to cellulose. The (beta)-glucosidases with molecular weights of 96,000 and 98,000 did not bind to cellulose. The cellulose-bound (beta)-glucosidase was eluted completely from the cellulose matrix with water. Cellulose-bound (beta)-glucosidase catalyzed p-nitrophenylglucoside hydrolysis, suggesting that the catalytic site is not involved in cellulose binding. When the cellulose-binding form was incubated with papain for 20 h, no decrease in the enzyme activity was observed; however, approximately 74% of the papain-treated glucosidase did not bind to microcrystalline cellulose. SDS-PAGE analysis of the nonbinding glucosidase produced by papain indicated the presence of three bands with molecular weights in the range of 95,000 to 97,000. On the basis of these results, we propose that the low-molecular-weight (96,000 and 98,000) non-cellulose-binding (beta)-glucosidase forms are most probably formed from the higher-molecular-weight (114,000) cellulose-binding (beta)-glucosidase via extracellular proteolytic hydrolysis. Also, it appears that the extracellular (beta)-glucosidase from P. chrysosporium might be organized into two domains, a cellulose-binding domain and a catalytic domain. Kinetic characterization of the cellulose-binding form is also presented.

Synthesis of a mixture of cyclic peptides based on the Bowman-Birk reactive site loop to screen for serine protease inhibitors.

A peptide mixture containing 21 peptide sequences has been constructed to test the Bowman-Birk inhibitor reactive-site loop motif as the basis of inhibition for a range of serine proteases. The 21 peptides are all based on an 11 amino acid sequence designed from a Bowman-Birk like inhibitor reactive-site loop. Variation has been introduced at the P1 site of the loop, which has been randomised to include all the natural L-amino acids (except for cysteine), plus the non-natural L-amino acids ornithine and norleucine, The mixture of peptides was screened for specific binding to immobilised porcine pancreatic elastase, subtilisin BPN', alpha-chymotrypsin, trypsin, anhydro-alpha-chymotrypsin and anhydrotrypsin. Five peptides from the mixture bind to alpha-chymotrypsin, two of which also bind to anhydro-alpha-chymotrypsin, and two peptides bind trypsin, neither of which binds to anhydro-trypsin. The competitive inhibition constants (K(i)) and the rates of proteolytic hydrolysis of the individual peptides with their respective enzymes were determined. The rates of hydrolysis were found to vary widely and show little correlation with the K(i) values. In the case of the alpha-chymotrypsin inhibitors, the peptides with the lowest K(i) (0.1-0.05 mM) were the only peptides that bound to anhydro-alpha-chymotrypsin. However, no peptides bound to anhydrotrypsin, suggesting a fundamental difference in the way that alpha-chymotrypsin and trypsin are inhibited by these cyclic peptides.

Proteolytic hydrolysis of muscle proteins of harp seal (Phoca groenlandica)

Protein hydrolysates were prepared from mechanically separated seal meat (MSSM) using commercially available microbial enzymes Alcalase and Neutrase. The enzyme in each case was inactivated by acidification with 4 N HCl to pH 4.0. Heme groups were removed, and the hydrolyzed proteins were bleached with charcoal, neutralized to pH 7.0, and dehydrated. The yield of hydrolysate (YH) and the degree of hydrolysis (DH) using Alcalase, under optimum conditions, were 92.75% and 19.5%, respectively. The YH for Neutrase-assisted hydrolysis of MSSM was 72.85%. The dehydrated seal protein hydrolysate (SPH) contained 77.3% crude proteins, 0.74% lipid, and 20.67% minerals, mainly sodium chloride

The topology of the S protein in the yeast-derived hepatitis B surface antigen particles.

Hepatitis B surface antigen particles are highly immunogenic and have been shown to provide a suitable support for the presentation of foreign epitopes. More information about the topology of their constitutive protein, the S (small envelope) protein, is a prerequisite to any rational attempt to replace region of this protein with foreign epitopes without modifying the assembly of the particle. The topology of the S protein within the lipid membrane was investigated here by combining extensive proteolysis of the external protein domains with proteinase K and (FTIR-ATR). The proteolytic hydrolysis of the S protein and the identification of the digestion products allowed characterization of the membrane-protected regions of the protein. FTIR spectra of the digested hepatitis B particles revealed that the peptides associated with the particles are rich in alpha-helix structure. The kinetic of 2H/H exchange provided evidence that a large fraction of the native S protein is poorly accessible to the aqueous medium.

Effects of bile salts on brush-border and cytosolic proteolytic activities of intestinal enterocytes

The effects of bile salts, i.e., sodium deoxycholate, sodium glycocholate, sodium glycodeoxycholate, sodium taurocholate, and sodium taurodeoxycholate, on the activities of individual rat intestinal brush-border membrane peptidases and cytosolic insulin-degrading activities were examined. At 2 mM, bile salts had no effects on aminopeptidases P and W, but did inhibit other brush-border peptidases though no single bile salt was able to inhibit every peptidase. Even at concentrations higher than the CMC, inhibition of brush-border insulin degradation is incomplete while that of cytosolic insulin degradation is complete at low concentrations. The results suggest that bile salts, inhibiting brush-border membrane and cytosolic proteolytic hydrolysis, may be useful for reducing intestinal degradation of peptide drugs; and that bile salts have stronger inhibitory effects on soluble than on membrane-bound insulin-degrading activity.

Characterization of heme c peptides by mass spectrometry

Liquid secondary ion mass spectrometry, also called fast atom bombardment mass spectrometry, has been used to determine proteolytic hydrolysis sites for peptides derived from c-type cytochromes that contain covalently attached heme c. An unexpected fragmentation occurs that breaks the covalent thioether bonds between the heme and the peptide, and the heme fragment further rearranges to give rise to an intense ion at m/z of 617 that corresponds to protonated iron protoporphyrin IX. The observation of this ion fragment can be used to unambiguously identify the presence of authentic heme c in peptides or peptide mixtures at the subnanomole level.

Calpain activity increases in hepatocytes following addition of ATP. Demonstration by a novel fluorescent approach.

Our aim was to measure calpain protease activity during increases in cytosolic free calcium (Ca2+i) after addition of extracellular ATP. The calpain protease substrate t-butoxycarbonyl-Leu-Met-7-amino-4-chloromethylcoumarin was synthesized. Nonfluorescent t-butoxycarbonyl-Leu-Met-7-amino-4- chloromethyl-coumarin diffuses into the cell where it is conjugated to glutathione forming t-butoxycarbonyl-Leu-Met-7-amino-4-methylcoumarin glutathione conjugate (Boc-Leu-Met-MAC-SG). The nonfluorescent, membrane impermeant Boc-Leu-Met-MAC-SG accumulates in the cell. Intracellular proteolytic hydrolysis of Boc-Leu-Met-MAC-SG releases and unquenches the fluorescence of MAC-SG. Intracellular fluorescence of MAC-SG was quantitated in single, cultured rat hepatocytes using digitized video fluorescent microscopy. Enhancement of intracellular fluorescence generation by increases in Ca2+i and inhibition by a calpain inhibitor indicated the probe was a calpain substrate. After addition of ATP, calpain protease activity increased to 156 +/- 13% of basal concurrent with a 3-fold rise of Ca2+i for 2-4 min. Thereafter, Ca2+i decreased to values of 1.5-fold above basal and protease activity returned to normal. Incubation of cells in Ca(2+)-free buffer abolished the rise in Ca2+i and calpain protease activity. Calpain protease activity increases concomitantly with increases of Ca2+i supporting the hypothesis that calpain proteases participate in Ca(2+)-mediated signal transduction.