Gel formation of peptides produced by extensive enzymatic hydrolysis of beta-lactoglobulin

The purpose of the present study was to identify which peptides were responsible for enzyme-induced gelation of extensively hydrolyzed beta-lactoglobulin with Alcalase in order to gain insight into the mechanism of gelation. Dynamic rheology, aggregation measurements, isoelectrofocusing as well as chromatography and mass spectrometry were used to understand the gel formation. A transparent gel was formed above a critical concentration of peptides while noncovalently linked aggregates appear with increasing time of hydrolysis. Extensive hydrolysis was needed for gelation to occur as indicated by the small size of the peptides. Isoelectrofocusing was successful at separating the complex mixture, and 19 main peptides were identified with molecular weight ranging from 265 to 1485 Da. Only one fragment came from a beta-sheet rich region of the beta-lactoglobulin molecule, and a high proportion of peptides had proline residues in their sequence.     

Protein modification during antiviral heat bioprocessing

Heat treatment is routinely used in the preparation of therapeutic protein biopharmaceuticals as a means of viral inactivation. However, in undertaking virucidal heat treatments, a balance must be found between the bioprocessing conditions, virus kill, and the maintenance of protein integrity. In this study, we utilize a simple model protein, hen egg-white lysozyme, to investigate the relationship between antiviral bioprocess conditions (protein formulation and temperature) and the extent and type of protein modification. A variety of industrially relevant wet- and dry-heat treatments were undertaken, using formulations that included sucrose as a thermostabilizing excipient. Although there was no evidence of lysozyme aggregation or crosslinking during any of the heat treatments, using liquid chromatography-electrospray ionization-mass spectroscopy (LC-ESI-MS) and peptide mapping we show that protein modifications do occur with increasingly harsh heat treatment. Modifications were predominantly found after wet-heat treatment, the major covalent modification of lysozyme under these conditions being glycation of Lys(97), by either glucose or fructose derived from hydrolyzed sucrose. The extent of sucrose hydrolysis was itself dependent on both the duration of heat treatment and formulation composition. Advanced glycation end products (AGEs) and additional unidentified products were also present in protein samples subjected to extended heat treatment. AGEs were derived primarily from initial glycation by fructose and not glucose. These findings have implications for the improvement of bioprocesses to ensure protein product quality.     

Partial purification and properties of a nucleoside hydrolase from Crithidia

An enzyme catalyzing the hydrolysis of nucleosides was found to occur in Crithidia fasciculata and was partially purified (30- to 40-fold) by treatment with either streptomycin sulfate or MnCl₂, ammonium sulfate fractionation, acidification and neutralization, passage through Sephadex G-200, and isoelectric focusing. The specific activity of these preparations was about 6 μmnoles of uridine hydrolyzed per mg protein per min. Specificity for the puriue or pyrimidine base was very broad; uridine gave the maximum rate of hydrolysis. Deoxyribosides were not hydrolyzed. The enzyme is relatively stable to heat and to acidification and can be stored frozen. Hydrolysis of uridine is inhibited by borate ions and by adenosine, inosine, and guanosine, but not by cytidine or xanthosine.     

Effects of ribonucleic acid removal methods on proteolytic activity and protein solubility in Candida utilis

Candida utilis was grown under controlled conditions and nucleic acids were removed from whole cells and homogenates by alkaline hydrolysis techniques, en-zymatic methods, and washing with buffer. Homogenization released hydrolytic enzymes and proteolytic activity increased with incubation at elevated temperatures under acidic conditions. Slight proteolysis occurred in all incubated samples and this may contribute to protein insolubilization. Very little protein was lost during incu-bation when compared to similar processes using bakers' yeast. This can be due to lower levels of protease activities in C. utilis. Alkaline hydrolysis methods resulted in hydrolysis of some proteins and irreversible insolubilization of the protein. These methods also destroyed any residual enzymatic activities. Heat denaturation studies suggest that protein insolubilization occurs at neutral pH when heat treatments equivalent to or greater than 85° C for 15 min are used. SDS-PAGE methods were used to characterize and monitor changes in protein. Eighteen proteins and/or sub-units were present at levels of 1% or greater. Results may help to explain changesin functional properties of sample preparations which accompany RNA removal.     

Quantification of glutamine in proteins and peptides using enzymatic hydrolysis and reverse-phase high-performance liquid chromatography

An enzymatic method for hydrolyzing bovine milk proteins was developed. Purified milk proteins (alpha-lactalbumin, beta-lactoglobulin, and beta-casein) were hydrolyzed in 0.1 M Hepes buffer (pH 7.5) containing pronase E, aminopeptidase M, and prolidase at 37 degrees C for 20 h. Free glutamine and other amino acids were derivatized with phenylisothiocyanate and separated using a C18 Pico-Tag column. Amino acids were eluted from the column with an aqueous sodium acetate-acetonitrile gradient with detection at 254 nm. Glutamine recoveries from hydrolyzed alpha-lactalbumin, beta-lactoglobulin, and beta-casein were 78 +/- 4, 98 +/- 3, and 101 +/- 3% of the theoretical values, respectively. The recoveries of most amino acids were comparable with those obtained using acid hydrolysis, except for the recoveries of proline and acidic amino acids. These peptide bonds appeared to be resistant to enzymatic hydrolysis and also to inhibit the hydrolysis of adjacent amino acids. Free glutamine was found to be very stable (97% recovery) under the enzymatic hydrolysis conditions.     

Hydrolysis of native poly(hydroxybutyrate) granules (PHB), crystalline PHB, and artificial amorphous PHB granules by intracellular and extracellular depolymerases.

Native poly(hydroxybutyrate) (PHB) granules, purified PHB and artificial amorphous PHB granules were examined as putative substrates for hydrolysis by the intracellular depolymerase system of Rhodospirillum rubrum and the extracellular depolymerase of Pseudomonas lemoignei. The R. rubrum depolymerizing system requires pretreatment of granules with a heat stable 'activator' fraction; the activator can be replaced by mild trypsin treatment. Artificial granules were prepared with a cationic detergent, cetyltrimethylammonium bromide (CTAB) and an anionic detergent, (sodium cholate). Cholate and CTAB PHB granules were hydrolyzed by both enzyme systems; however, some differences were noted. Cholate granules were hydrolyzed in the absence of the R. rubrum activator fraction. Activator was required for the hydrolysis of CTAB granules but could be replaced by heparin in the extracellular depolymerase system but not in the intracellular depolymerase system. A Triton X-114 extract of native PHB granules inhibited the hydrolysis of trypsin-activated granules by the intracellular depolymerase. The inhibition was reversed by the activator fraction. Detergent extracts of granules activated with the R. rubrum activator were unable to inhibit the hydrolysis of trypsin-activated granules. These data suggest that the activator acts to modify an inhibitor present on native granules.     

Effects of the embryonic axis and phytohormones on proteolysis of the storage protein in buckwheat seed

The role of the embryonic axis in regulation of proteolysis of the main storage protein was studied in buckwheat ( Fagopyrum esculentum ) seed. Polyacrylamide gel electrophoresis (PAGE) analysis revealed that removal of the embryonic axis had no effect on the first stage of hydrolysis, that is proteolytic modification, of 13S globulin. This modification took place in the growing seedlings also in the presence of cycloheximide, i.e. it was due to an enzyme present in dry seed. However, in isolated cotyledons the 13S globulin was not degraded completely. Incubation of isolated cotyledons with cytokinins, gibberellic acid and indoleacetic acid could not replace the excised embryonic axis. At the same time, proteolysis of the 13S globulin in the growing seedlings was strongly inhibited by casein hydrolyzate. It is suggested that a complete proteolysis of the modified storage protein is regulated by the concentration of hydrolysis products at the site of hydrolysis. The embryonic axis serves, most probably, as a site of efflux of the products of protein hydrolysis in the cotyledons during seedling growth and thus regulates the course of proteolysis.
Abscisic acid (10–100 μ M ) was without effect on modification of the 13S globulin, but suppressed the complete proteolysis of the protein by inhibiting, apparently, the synthesis of the cysteine proteinase in the growing seedlings.     

Meprin-A and -B. Cell surface endopeptidases of the mouse kidney

The proteinase meprin-A is a disulfide-linked tetramer of 90-kDa glycoprotein subunits. It is expressed at high levels in kidney brush border membranes of random bred and certain inbred strains of mice. Some mouse strains (e.g. C3H/He) do not express meprin-A subunits, but do produce a similar but less well characterized metalloendopeptidase, meprin-B. In the present study, meprin-B was purified from C3H/He mouse kidneys to electrophoretic homogeneity, and the relationship between it and meprin-A was investigated. The papain-solubilized form of meprin-B was similar to meprin-A in amino acid composition, molecular mass, secondary, and quaternary structure. However, immunoblots indicated that the enzymes have some common and some distinct epitopes. Lectin blots indicated both enzymes have high mannose and/or complex biantennary oligosaccharides, but there are differences in the complex-type glycosylation. Peptide maps and sequencing of cyanogen-bromide fragments of the enzymes revealed some different amino acid sequences. Thermal inactivation studies indicated that meprin-B was much less stable than meprin-A; the half-life for inactivation at 58 degrees C for meprin-A was 50 min, whereas for meprin-B it was less than 3 min. Both enzymes hydrolyzed azocasein and insulin B chain, but limited proteolysis of the enzymes with trypsin activated meprin-B 5-20-fold, whereas meprin-A was activated 2-fold at most. Analysis of hydrolysis products of the oxidized insulin B chain revealed some common and some distinct sites of cleavage. Bradykinin was a good substrate for meprin-A, while it was not hydrolyzed by meprin-B. A synthetic peptide, YLVC(SO3-)GERG, derived from insulin B chain was hydrolyzed faster by meprin-B than meprin-A, and neither enzyme was activated by trypsin treatment against this substrate. Taken together, the data indicate that the two metalloendopeptidases have many similarities but are distinct enzymes.     

Stimulation by phospholipid vesicles of proteolysis of egg white lysozyme by chymotrypsin

Egg white lysozyme was rapidly and extensively hydrolyzed by chymotrypsin in the presence of negatively charged phospholipid vesicles. The extent of hydrolysis of lysozyme by chymotrypsin depended on the amount of phospholipid present. The optimum amount of phospholipid varied with the amounts of both lysozyme and chymotrypsin. The proteolysis was strongly inhibited at high ionic strength. The amidolytic activity of chymotrypsin against a synthetic substrate was inhibited by phospholipid. Purified phosphatidic acid and phosphatidylethanolamine from egg yolk induced susceptibility of lysozyme to chymotrypsin, whereas synthetic dimyristoyl phosphatidylcholine did not. The extent of the hydrolysis was smaller with phosphatidic acid and phosphatidylethanolamine than with phospholipid mixture, indicating that vesicles of phospholipid mixture were more effective than those of phosphatidic acid or phosphatidylethanolamine in enhancing the proteolysis of lysozyme by chymotrypsin.     

Susceptibility to trypsinolysis of esterified milk proteins

Methyl-, ethyl- and propyl-esters of beta-lactoglobulin, alpha-lactalbumin and beta-casein were prepared and then hydrolyzed with trypsin in various conditions. Resulting hydrolysates were analysed by SDS electrophoresis and RP-HPLC. The degree of hydrolysis of esterified samples was generally lower than those of the non-modified proteins. The highest degrees of hydrolysis were obtained at pH 7--8 with native and esterified protein samples. beta-Lactoglobulin propyl ester and beta-casein methyl ester yielded the lowest degrees of hydrolysis. Ethyl- and propyl-esters of beta-casein showed high resistance towards tryptic attack, even after 20 h of hydrolysis. SDS electrophoretic patterns of tryptic hydrolysates of native proteins showed bands corresponding to low molecular weights. Tryptic hydrolysates of esterified proteins showed bands with higher sizes. RP-HPLC profiles of tryptic hydrolysates of esterified samples showed peaks with longer elution times than those obtained with native proteins, indicating the presence of more hydrophobic peptide populations. A peptic pre-treatment improved tryptic action on esterified proteins. It resulted in a better resolution of RP-HPLC profiles and in a complete disappearance of the protein after 20 h tryptic hydrolysis.     

β-Lactoglobulin/polysaccharide interactions during in vitro gastric and pancreatic hydrolysis assessed in dialysis bags of different molecular weight cut-offs

The effects of gum arabic, low methylated (LM) pectin or xylan at levels of 0 and 50 wt.% on beta-lactoglobulin (β-lg) digestibility were studied as well as the interactions between the two macromolecules during in vitro hydrolysis. The proteolysis was performed in a system involving a two-step hydrolysis: either pepsin alone, or pepsin followed by a trypsin/chymotrypsin (T/C) mixture in dialysis bags with molecular weight cut-offs (MWCO) 1000 or 8000 Da. Digestibility was estimated by the N release and by a SDS-PAGE electrophoresis of retentates from the two dialysis bags after hydrolysis. Turbidimetric measurements monitored the structural evolution of mixtures during the two-step hydrolysis. Results showed that β-lg was almost resistant to peptic digestion and that polysaccharides increased the N release despite a reduction of pepsin activity. This is due to the formation of electrostatic complexes between polysaccharides and β-lg, which reduced β-lg aggregation, increasing its solubility. The polysaccharides reduced significantly the β-lg T/C digestibility as determined using a dialysis bag with a MWCO 1000 Da, without a modification of their enzymatic activities. No significant effect of polysaccharides on the β-lg digestibility was detected using the dialysis bag with a MWCO 8000 Da. The electrophoresis pattern did not show differences in the profile of retentates in relation with the dialysis bag used. This suggests that non-specific interactions could occur during the second step of hydrolysis between polysaccharides and amino acids or peptides smaller than 8000 Da.     

Resistance of α-globulin fromSesamum indicum L. to proteases in relationship to its structure

α-Globulin, the high-molecular-weight protein fraction fromSesamum indicum L., was hydrolyzed to low-molecular-weight protein and peptides by pepsin, while its resistance to hydrolysis by group-specific enzymes, trypsin or α-chymotrypsin, was very high. The protein showed definite structural changes after proteolysis, especially after peptic hydrolysis, as evidenced from various biophysical data. The sedimentation velocity pattern of α-globulin hydrolyzed by trypsin or α-chymotrypsin indicated reduction in the percentage of 11S component, while the pepsinhydrolyzed sample was devoid of any 11S component, indicating the absence of a native protein molecule. The fluorescence emission spectra of the various hydrolyzed α-globulin showed a red shift in the fluorescence emission maximum. The red shift was maximum with α-globulin hydrolyzed by pepsin and minimum with the trypsin-hydrolyzed sample. The far-ultraviolet-circular dichroic measurements indicated that most of the ordered structure of α-globulin was absent after pepsin hydrolysis, while after trypsin and chymotrypsin hydrolysis conformational changes were less.     

Multiple steps during the formation of beta-lactoglobulin fibrils

In this study, the heat induced fibrilar aggregation of the whey protein beta-lactoglobulin is investigated at low pH and at low ionic strength. Under these circumstances, tapping mode atomic force microscopy results indicate that the fibrils formed have a periodic structure with a period of about 25 nm and a thickness of one or two protein monomers. Fibril formation is followed in situ using light scattering and proton NMR techniques. The dynamic light scattering results show that the fibrils that form after short heating periods (up to a few hours) disintegrate upon slow cooling, whereas fibrils that form during long heating periods do not disintegrate upon subsequent slow cooling. The NMR results show that even after prolonged heating an appreciable fraction of the protein molecules is incorporated into fibrils only when the beta-lactoglobulin concentration is above approximately 2.5 wt %. The data imply multiple steps during the heat induced formation of beta-lactoglobulin fibrils at low pH and at low ionic strength: (partly) denatured protein monomers are either incorporated into fibrils or form instead a low molecular weight complex that is incapable of forming fibrils. Fibril formation itself also involves (at least) two steps: the reversible formation of linear aggregates, followed by a slow process of "consolidation" after which the fibrils no longer disintegrate upon slow cooling.     

Beta-lactoglobulin/polysaccharide interactions during in vitro gastric and pancreatic hydrolysis assessed in dialysis bags of different molecular weight cut-offs

The effects of gum arabic, low methylated (LM) pectin or xylan at levels of 0 and 50 wt.% on beta-lactoglobulin (beta-lg) digestibility were studied as well as the interactions between the two macromolecules during in vitro hydrolysis. The proteolysis was performed in a system involving a two-step hydrolysis: either pepsin alone, or pepsin followed by a trypsin/chymotrypsin (T/C) mixture in dialysis bags with molecular weight cut-offs (MWCO) 1000 or 8000 Da. Digestibility was estimated by the N release and by a SDS-PAGE electrophoresis of retentates from the two dialysis bags after hydrolysis. Turbidimetric measurements monitored the structural evolution of mixtures during the two-step hydrolysis. Results showed that beta-lg was almost resistant to peptic digestion and that polysaccharides increased the N release despite a reduction of pepsin activity. This is due to the formation of electrostatic complexes between polysaccharides and beta-lg, which reduced beta-lg aggregation, increasing its solubility. The polysaccharides reduced significantly the beta-lg T/C digestibility as determined using a dialysis bag with a MWCO 1000 Da, without a modification of their enzymatic activities. No significant effect of polysaccharides on the beta-lg digestibility was detected using the dialysis bag with a MWCO 8000 Da. The electrophoresis pattern did not show differences in the profile of retentates in relation with the dialysis bag used. This suggests that non-specific interactions could occur during the second step of hydrolysis between polysaccharides and amino acids or peptides smaller than 8000 Da.     

Lysophospholipase-catalyzed hydrolysis of lysophospholipids in Mycoplasma gallisepticum membranes.

Mycoplasma gallisepticum strains have a membrane-bound lysophospholipase which hydrolyzes lysophospholipid generated in these membranes by treatment with an external phospholipase. This paper studies the hydrolysis of the membranous lysophospholipids by an enzyme residing in the same membrane (intramembrane utilization) or in adjacent membranes (intermembrane utilization). To study intermembrane hydrolysis, the phospholipids of M. gallisepticum were labeled with [3H]oleic acid. Membranes were prepared, heated at 65 degrees C, and subsequently treated with pancreatic phospholipase A2. This resulted in membranes whose enzyme was heat inactivated, but which contained lysophospholipid. When these membranes were mixed with M. gallisepticum cells or membranes, the lysophospholipid was hydrolyzed by the membranous lysophospholipase. To study intramembrane hydrolysis, [3H]oleyl-labeled membranes of M. gallisepticum were treated with pancreatic phospholipase A2 at pH 5.0. At this pH, lysophospholipid was generated but not hydrolyzed. Adjustment of the pH to 7.4 resulted in hydrolysis of the lysophospholipid by the membranous lysophospholipase. These procedures permitted measuring the initial rates of intramembrane and intermembrane hydrolysis of the lysophospholipid, showing that the time course and dependence on endogenous substrate concentration were different in the intramembrane and intermembrane modes of utilization. They also permitted calculation of the molar concentration of the lysophospholipid in the membrane and its rate of hydrolysis, expressed as moles per minute per cell or per square centimeter of cell surface.     

Influence of the Embryonic Axis on Protein Hydrolysis in Cotyledons of Cucurbita maxima

Four-day time course studies of the hydrolysis of cotyledonal storage protein were conducted on intact seeds, seed cotyledons detached from their embryonic axes and on detached cotyledon pairs germinated in the presence of three excised embryonic axes of Cucurbita maxima Duch., cv. Chicago Worted Hubbard. Detached cotyledons germinated alone showed little hydrolysis of the storage protein. However, the amount of protein hydrolysis of the detached cotyledon pairs germinated in the presence of three excised embryonic axes was comparable to the amount hydrolyzed in the cotyledons of intact germinating seeds. Visual growth differences among these treatments were also evident. The size and yellow color intensity of the fourth day treatments were shown to increase in the following order: detached cotyledon pairs alone, intact seedlings, detached cotyledon pairs in the presence of three excised axes. The growth of the hypocotyl and radical was also modified by removal of the cotyledons. These findings suggest that storage protein degradation and cotyledonal growth are controled by the axis. They also indicate that the cotyledons have some influence on the growth of the axes. Time-course studies were made on the hydrolysis of storage protein in the cotyledons of squash and on the distribution of the hydrolytic products during the germination of light- and dark-grown plants. The storage protein was not hydrolyzed during the first 24 hours. It was hydrolyzed at a uniform rate from 1 to 5 days and at a slightly decreased rate from 5 to 7 days. Most of the hydrolytic products were transported to the axial tissue. Proteinase activity in the cotyledons rapidly increased during germination to a maximum level at 2 to 3 days. This was followed by a decline to about the initial value after 7 days.     

Characterization of the catalytic subunit of an anion pump

The ArsA protein, the 63-kDa catalytic subunit of an oxyanion-translocating ATPase, was purified by successive chromatography using Q-Sepharose, red agarose, and phenyl-Sepharose to a specific activity in excess of 1 mumol of ATP hydrolyzed per min per mg of protein. ATPase activity was dependent on the presence of the oxyanionic substrates. Inhibitors of other classes of ion-translocating ATPases had no effect on ArsA ATPase activity, including N,N'-dicyclohexyl-carbodiimide, azide, vanadate, and nitrate. The apparent Km for ATP was determined to be 0.13 mM. The optimal pH range for ATP hydrolysis was 7.5 to 7.8. ATPase activity required Mg2+ at a molar ratio of 2 ATP:1 Mg2+. Limited proteolysis by trypsin was used to study conformational changes produced upon binding of substrates to the ArsA protein. In the absence of substrates, the ArsA protein was rapidly cleaved by trypsin to a major product of 30 kDa. ATP was partially protected from trypsin digestion, while the anionic substrate antimonite alone had no effect on proteolysis. Combination of the two substrates nearly completely protected the ArsA protein from proteolysis. Proteolytic cleavage correlated with loss of anion-stimulated ATPase activity and substrate protection from cleavage correlated with retention of activity. These results demonstrate that ATP and antimonite together produce a conformational change which is different from that of the ArsA protein in the presence of either substrate alone and suggest interaction between the oxyanion and ATP binding sites.     

Identification and purification of membrane and soluble forms of the major surface protein of Leishmania promastigotes

A major integral membrane glycoprotein of 63 kDa (p63), present at 500,000 copies/cell, was found on the surface of Leishmania major LEM 513 promastigotes. This protein was labeled either by surface iodination of the cells or by metabolic incorporation of [35S]methionine. Peptide maps of the proteins labeled by the two procedures were identical. Protein p63 was purified in three steps: extraction and phase separation in the nonionic detergent Triton X-114, chromatography on DEAE-cellulose, and finally chromatography on a Mono-Q column. The carbohydrate content as well as the concanavalin A receptor activity were characterized. A hydrophilic form of p63 was generated during the purification of the protein. This form was not derived by proteolysis from the amphiphilic protein found in the membrane, but may have been generated by the hydrolysis of a lipid containing myristyl residue(s) anchoring the protein in the membrane.     

Neurite outgrowth in PC12 cells. Distinguishing the roles of ubiquitylation and ubiquitin-dependent proteolysis

Nerve growth factor (NGF)-induced neurite outgrowth from rat PC12 cells was coincident with elevated (>/=2-fold) levels of endogenous ubiquitin (Ub) protein conjugates, elevated rates of formation of 125I-labeled Ub approximately E1 (Ub-activating enzyme) thiol esters and 125I-labeled Ub approximately E2 (Ub carrier protein) thiol esters in vitro, and enhanced capacity to synthesize 125I-labeled Ub-protein conjugates de novo. Activities of at least four E2s were increased in NGF-treated cells, including E2(14K), a component of the N-end rule pathway. Ubiquitylation of 125 I-labeled beta-lactoglobulin was up to 4-fold greater in supernatants from NGF-treated cells versus untreated cells and was selectively inhibited by the dipeptide Leu-Ala, an inhibitor of Ub isopeptide ligase (E3). However, Ub-dependent proteolysis of 125I-labeled beta-lactoglobulin was not increased in supernatants from NGF-treated cells, suggesting that neurite outgrowth is promoted by enhanced rates of synthesis (rather than degradation) of Ub-protein conjugates. Consistent with this observation, neurite outgrowth was induced by proteasome inhibitors (lactacystin and clasto-lactacystin beta-lactone) and was associated with elevated levels of ubiquitylated protein and stabilization of the Ub-dependent substrate, p53. Lactacystin-induced neurite outgrowth was blocked by the dipeptide Leu-Ala (2 mM) but not by His-Ala. These data 1) demonstrate that the enhanced pool of ubiquitylated protein observed during neuritogenesis in PC12 cells reflects coordinated up-regulation of Ub-conjugating activity, 2) suggest that Ub-dependent proteolysis is a negative regulator of neurite outgrowth in vitro, and 3) support a role for E2(14K)/E3-mediated protein ubiquitylation in PC12 cell neurite outgrowth.