Extent Of Proteolysis Research Articles

Microbiology and biochemistry of Fossa (pit) cheese

A microbiological and biochemical characterization of the Fossa (pit) cheese is reported. The cheeses analysed showed di!erences for the protocol of production and type of cheese-milk used (bovine or ovine). The total number of mesophilic bacteria and the number of specic microbial groups di!ered among the cheeses. Lactococci used as starters were found at very low numbers. Non-starter lactic acid bacteria (NSLAB) such as Lactobacillus plantarum, Lb. curvatus and Lb. paracasei subsp. paracasei were found at high numbers (5.8}7.8 log cfu g~1). Cheeses produced from pasteurized or raw milks and using lactococci as starters or not showed similar microbiological and biochemical characteristics. High concentrations of NaCl-in-moisture (ca. 11.5%) and a low value of a 8 (ca. 0.850) negatively in#uenced the microbial content and proteolysis. The ratio pH 4.6-soluble N/total N (23.6}39.1%) and the concentration of free amino acids (11.37}41.06 mg g~1) were very high and varied with cheeses. Fossa cheeses which contained the highest number of NSLAB also had the highest concentration of amino acids. The principal amino acids were glutamic acid, valine, leucine and lysine. Urea-PAGE electrophoresis of the pH 4.6 insoluble and soluble fractions di!erentiated the cheeses. Apart from the variations in the protocol of production, the RP-HPLC of the ethanol-soluble fraction showed a peptide prole which was common to all the cheeses. Especially cheeses which had the highest concentrations of free amino acids and the highest number of NSLAB, also contained the highest aminopeptidase, dipeptidase and iminopeptidase activities. Fossa cheeses also showed a moderate lipolysis which varied among the samples: total free fatty acids ranged from 578 to 1676 mg kg~1. The highest concentrations were found independently of the milk used. The principal fatty acids were butyric, caproic, palmitic and oleic acids. Sensory evaluation of the Fossa cheeses showed di!erences especially related to the extent of proteolysis. ( 2000 Elsevier Science Ltd. All rights reserved.

Ripening Profiles of Goat Cheese Produced from Milk Treated with High Pressure

ABSTRACT:Goat cheeses were made from pasteurized (72 °C, 15 s) and high‐pressure (HP)‐treated milk (500 MPa, 15 min, 20 °C). At 45 days of ripening, cheeses made from both types of milk were similar in moisture, quality, electrophoretic profiles, water‐soluble nitrogen, and total free fatty acid contents. Cheeses made from HP‐treated milk had higher pH and salt, matured more quickly, as determined by formation of total free amino acids, and developed strong flavors. Reverse‐phase high‐performance liquid chromatography showed differences between the peptide profiles of the cheeses. Differences in small peptides and free amino acids indicated a higher extent of proteolysis in cheeses made from HP‐treated milk.

Morphologic criteria and detection of apoptosis.

Apoptosis is an organized, energy dependent process, which leads to cell death. Its definition is based on distinct morphological features [10] and demonstration of internucleosomal DNA degradation [27], executed by selectively activated DNAses [4, 22]. The morphologic hallmarks of apoptosis include chromatic margination, nuclear condensation and fragmentation, and condensation of the cell with preservation of organelles. The process is followed by fragmentation of the cell into membrane-bound apoptotic bodies, which undergo phagocytosis by nearby cells without associated inflammation [10, 11]. Apoptosis characteristically occurs in insolated single cells. The duration of apoptosis is estimated to be from 12 to 24 hours, but in cell culture visible morphologic changes are accomplished in less than two hours [10, 16]. Non-apoptotic cell death, a prototype of which is cell death due to ischemia (oncosis), is characterized by depletion of intracellular ATP stores, swelling of the cell with disruption of organelles and rupture of the plasma membrane [15]. Groups of necrotic cells and inflammation are found in tissues [10, 15]. The significance of apoptosis has mostly been studied using the TUNEL assay that detects DNA strand breaks in tissue sections and allows quantification of apoptotic cells by light microscopy [6]. Common experience seems to be that the TUNEL assay is prone to false positive or negative findings. This has been explained by the dependence of the staining kinetics on the reagent concentration [17], fixation of the tissue [2] and the extent of proteolysis [17]. Active RNA synthesis [12] and DNA damage in necrotic cells [17, 19] may cause non-specific staining. To obtain reliable and reproducible results, TUNEL assay should be carefully standardized by using tissue sections treated with DNAse (positive control of apoptosis). Quantification of apoptosis should include enough microscopic fields and identification of the cell type undergoing apoptosis. The specificity of the results can be substantiated by combining other methods with TUNEL, such as assessment of the pattern of DNA fragmentation or evaluation of the morphological features. Even though there is high variation in the results obtained in consecutive studies under the same circumstances, increasing evidence shows that TUNEL-positive cardiomyocytes and internucleosomal DNA fragmentation are associated with various cardiac diseases, including acute myocardial infarction and heart failure [reviewed in 5, 9]. Some morphological features of apoptosis have been observed in TUNEL-positive cardiomyocytes using light microscopy (Figure 1) or confocal microscopy [20]. Electron microscopic evidence of apoptosis has been found in the degenerating conduction system [7], in experimental heart failure [23], and in human hibernating myocardium [3]. In acutely ischemic myocardium the interpretation of the findings remains controversial, since only non-apoptotic cell morphology has been found in electron microscopy [8, 19]. One explanation might be abortion of the apoptotic program due to the lack of ATP before the morphologic features are fully evident [14]. Another explanation is the possibility that non-apoptotic cell death and apoptosis share common mechanisms in the early phases of the processes [14, 19]. The exact mechanisms of ischemic cell death remain to be clarified and the classification between apoptosis and non-apoptosis cell death to be specified. Recently, caspase activation has emerged as the central molecular event leading to apoptosis, preceding DNA degradation and the development of apoptotic morphology [22, 25]. New methods have been developed to demonstrate caspase activation [1, 13]. Inhibition of caspase may be an efficient way to prevent apoptotic cardiomyocyte death as well as to define and specifically probe the significance of apoptotic cell death in cardiac diseases.

The extent of proteolysis is independent of sarcomere length in lamb longissimus and psoas major.

The objective of this experiment was to determine the effect of sarcomere length on postmortem proteolysis and meat tenderness. Eighteen Dorset market-weight sheep were slaughtered conventionally. The longissimus thoracis et lumborum and psoas major from each carcass were either left intact on the carcass (control), which was chilled at 0 degrees C, or excised from the carcass and chilled in an ice slurry (0 degrees C). At 24 h, control muscles were excised, and all muscles were cut into sections and assigned to 1 or 10 d of postmortem storage at 2 degrees C. Sarcomere length was shorter (P < .01), as intended, in the shortened relative to the control treatment and in longissimus relative to psoas major (1.36 vs 1.69 microm, raw longissimus; 1.45 vs 3.03 microm, raw psoas major). Sarcomere length was not affected (P > .05) by aging time. Western blot analysis of troponin-T and desmin indicated no effect (P > .05) of the shortened treatment compared to the control on the extent of proteolysis. Regardless of aging time or treatment, troponin-T was more degraded (P < .01) in longissimus than in psoas major (38.1 vs 23.5%) and desmin tended to be more degraded (P = .08) in longissimus than in psoas major (50.4 vs 35.1%). Regardless of muscle or treatment, aging 10 d compared to 1 d increased degradation of troponin-T (46.3 vs 15.3%) and desmin (69.3 vs 16.1%). Warner-Bratzler shear force was greater (P < .01) in the shortened treatment than in control (6.9 vs 3.8 kg), greater (P < .01) in longissimus than in the psoas major (6.5 vs 4.2 kg), and greater (P < .01) with 1 d than with 10 d of aging time (6.1 vs 4.6 kg). A muscle x aging time interaction (P < .05) indicated shear force declined more in longissimus than in psoas major during aging. We conclude that sarcomere length did not affect the extent of proteolysis. However, sarcomere length may have an indirect effect on tenderization during aging due to its effect on initial tenderness.

Effect of calpastatin on degradation of myofibrillar proteins by mu-calpain under postmortem conditions.

To improve our understanding of the regulation of mu-calpain activity in situ during postmortem storage of muscle, the effect of different calpastatin levels on proteolysis of myofibrillar proteins by mu-calpain in a system closely mimicking postmortem conditions was studied. Increasing the amount of calpastatin in the incubations limited both the rate and extent of proteolysis of myofibrillar proteins and autolysis of mu-calpain. Excess calpastatin (i.e., a mu-calpain:calpastatin ratio of 1:4) did not inhibit proteolysis completely. Western blot analysis revealed that proteolysis of myofibrillar proteins virtually ceased after 7 d of incubation, despite the presence of partly autolyzed, therefore seemingly active, mu-calpain. A series of incubations of autolyzed mu-calpain revealed that the autolyzed form of this enzyme is unstable at an ionic strength observed in postmortem muscle. The possible significance of these results in terms of the regulation of mu-calpain activity in postmortem muscle is discussed.

Changes in the composition of juice expressed from Camembert cheese during ripening

Cheese can be considered as a paracasein network including fat and aqueous phases. Changes in the composition of the aqueous phase reflect the enzymatic and physico-chemical phe- nomena that occur in cheese during ripening. For the first time, juice was expressed by pressing from Camembert cheese ripened up to 16 d and analysed in addition to the cheese to evaluate the extent of proteolysis together with the solubilisation of minerais. The amount of expressable juice decreased sharply during ripening, i.e. from 67 to 19 % of the total water content for cheeses ripened 0 and 16 d, respectively. In parallel, the levels of pH 4.6-soluble nitrogen and non-protein nitrogen increased in the whole Camembert cheese; the increase was higher in the juice. Simultaneously, the concentration of organic phosphate in the juice increased with concomitant decreases in the concentrations of cal- cium and inorganic phosphate. This last observation suggests that salt precipitates at the surface of the cheese. The expression and subsequent analysis of juice from Camembert cheeses is an appropriate method for following the sequential action of proteolytic enzymes and minerai transfer that occur dur- ing ripening. © InralElsevier, Paris. soft cheese / aqueous phase / proteolysis / mineral/ ripening

Effect of maceration on nitrogen fractions in hay and silage

An intensive mechanical conditioning treatment, referred to as maceration, was applied at mowing to alfalfa or timothy in order to enhance drying rate, reduce wilting time and possibly reduce respiration losses and proteolysis. In 1995, a laboratory trial was conducted using two levels of force (1750 and 3500 Newton) and five levels of conditioning: a control (no conditioning), one passage, three passages, five passages and seven passages through two steel knurled rolls. All forages were dried in a controlled environment at 30 °C and conserved as hay. The level of force did not affect the chemical composition of the forages obtained. However the nitrogen (N) fractions were affected by the level of maceration. As the level of conditioning increased, the soluble N fractions (A and B1) of both forages decreased (P &lt; 0.001). Meanwhile, the slowly degradable N fraction (B3) increased linearly (P &lt; 0.001) in timothy and quadratically (P &lt; 0.003) in alfalfa. The fraction of unavailable N (fraction C) also increased linearly (P &lt; 0.01) with intensity of maceration. In 1996, alfalfa was conditioned in the field at four intensity levels: a control (rubber roll-conditioning), one passage, two passages, and three passages through a full-size mower-macerator with three knurled rolls. The alfalfa dried under poor climatic conditions with alternating rain and sunshine and was conserved as silage at 30% dry matter (DM) after a 40-h wilt or as hay after a 90-h wilt. Neutral detergent fibre (NDF), acid detergent fibre (ADF), and ash contents increased linearly (P &lt; 0.001) with the level of maceration; the increase was greater in hay than in silage. The non protein nitrogen (fraction A) decreased (P &lt; 0.001) while fraction B3 and unavailable N (fraction C) increased (P &lt; 0.001) with level of maceration. The results suggest that maceration decreases the extent of proteolysis during conservation and preserves a higher proportion of the slowly degradable N (escape nitrogen). Key words: Forage, maceration, chemical composition, nitrogen fractions

Influence of naturally acid-soluble proteins from beans ( Phaseolus vulgaris L.) on in vitro digestibility determination

The in vitro digestibility of bean ( Phaseolus vulgaris) protein fractions was studied using a pepsin-pancreatin system. Enzymatic hydrolysis was stopped by adding a strong acid and the extent of proteolysis determined by measurement of free amino groups in the soluble fraction. The in vitro digestibility of bean protein fractions was low when in the native state and was differently affected by denaturation. For phaseolin, the main reserve protein, heating caused a significant increase of susceptibility to hydrolysis, whereas heat had no apparent effect on digestibility of glutelins and albumins (II). For the PIL (protease inhibitor-lectin rich) fraction, which was shown to have a composition similar to total albumins, there was a decrease of digestibility, probably associated to disulfide bond formation upon heating. Results of in vitro digestibility were shown to be strongly dependent on the utilization of a sample blank to account for proteins naturally soluble in the acid used to interrupt hydrolysis, which would otherwise be estimated as digested protein. These proteins are characterized by a high carbohydrate content, probably responsible for their high solubility and low digestibility.

Simple approach to reducing proteolysis during secretory production of human parathyroid hormone in Saccharomyces cerevisiae.

A gene coding for human parathyroid hormone (hPTH) was synthesized and cloned into a yeast expression and secretion vector containing the mating factor alpha pre-pro leader sequence and the galactose-inducible promoter, GAL10. The intact hPTH(1-84) was found to be secreted into the culture medium. As observed in the previous reports on the secretory production of hPTH in yeast, however, the proteolytic cleavage occurred as the culture proceeded, resulting in a significant loss of the intact hPTH. Attempts were therefore made to reduce the extent of proteolysis by simply controlling the culture conditions. The proteolytic cleavage was significantly reduced by the addition of an excess amount of l-arginine (>/=0.2M) to the culture medium, which resulted in a marked improvement in the yield of intact hPTH. To examine whether l-arginine affects the activities of intracellular proteases such as KEX2 endoproteinase or extracellular proteases, the proteolysis experiments were performed by incubating the commercial intact hPTH in a yeast host culture supernatant. The results demonstrated that l-arginine at high concentrations reduced the rate of hPTH proteolysis by inhibiting extracellular proteases.

Evaluation of proteolysis in Parmesan cheese using electrophoresis and HPLC

The proteolytic changes occurring during the ripening of Parmesan cheese were studied using urea-polyacrylamide gel electrophoresis (urea-PAGE) of caseins, HPLC analysis offree amino acids (FAA) and Kjeldahl determination of soluble nitrogen fractions. An electrophoretic ripening index for the evaluation of proteolysis in Parmesan cheese was established. The separation of caseins by alkaline PAGE (12 % T, 2.6 % C, pH 8.9, 5 M urea) was followed by the densitometric analysis of the y- and ~-casein fractions. The relationship between the resulting coefficients (y-Cn/~-Cn) and the age of reference samples of Original ltalian Grana Padano (6-22 months) was linear up to 15 months, allowing an evaluation of the extent of proteolysis and therefore a deduction of the age of the Parmesan samples analysed. Threshold levels (y-Cn/~-Cn) were proposed for the verification of the required age of Parmesan cheese. The coefficients (y-Cn/~-Cn) as weil as the 13-casein content of two addition al series of references and of 117 commercial Parmesan sampI es are presented. Commercial Parmesan samples retailed as a loaf or as prepacked slices were generally found to fulfill the requirements concerning endoproteolytic changes during ripening. However, man y grated Parmesan samples taken from retail outlets in Austria showed poor quality, which was probably due to the adulteration with products with low proteolysis (e.g., cheese rind, very young cheese). HPLC analysis ofFAA was also used to characterize the ripening process. Although FAA content ofreference samples of Grana Padano showed a very high degree of variability, a distinct increase of FAA content could be obser- ved during the ripening period. However, two series of reference samples, which had completely different electrophoretic casein patterns, could not be distinguished by HPLC analysis of the total amount of FAA. As an experiment, two loaves of Parmesan cheese were removed from the ripe- ning room at an age of 4 and 2 months and were subsequently stored at 6 C for 18 months. During the storage period, endoproteolytic changes were slowed down and breakdown of C:XS1-and ~-casein as weil as the accumulation of degradation products proceeded at a reduced rate, and could be detected using urea-PAGE casein patterns, but not by HPLC analysis of FAA content. In conse- quence of these findings, no accurate evaluation of the age of commercial Parmesan samples was pos- sible by means of the total amount of FAA. Since Kjeldahl determination of WSN and HPLC analysis of FAA content give insufficient information about the distinct endoproteolytic changes, which were found to be typical for Parmesan cheese, urea-PAGE of the casein fraction has to be do ne to enable * Oral communication at the IDF symposium 'Ripening and quality of cheeses', Besancon, France,

CHANGES IN THE PERCEIVED TEXTURAL PROPERTIES OF CHEDDAR CHEESE DURING MATURATION

ABSTRACTThe perceived textural attributes of an English Cheddar were measured by a trained panel at various intervals during ripening, including those corresponding to mild and medium maturation times for this variety. The moisture content and pH were also monitored. After the recommended ripening period of 50 weeks the matured cheese was significantly less springy, firmer, harder, crumblier and creamier, indicating that textural attributes are related to the age of the Cheddar. Measures beyond the recommended maturation period revealed further significant differences. Comparisons of the measures taken at mild, medium and full maturity also revealed differences in the textural properties of the different classes of Cheddar. Significant correlations (p ≥0.01) between pH and springiness, crumbliness by fingers and creaminess suggested a strong relationship between these textural attributes and the extent of proteolysis. The results of this study revealed a strong relationship between age and the textural attributes of cheddar when restricted to one particular variety.

Possible implications of milk pasteurization on the manufacture and sensory quality of ripened cheese

Abstract Cheese made from raw milk represents an important proportion of the traditional cheeses, particularly in South European countries. Besides destruction of pathogenic bacteria, the most significant changes in milk relevant to cheesemaking, which are induced by pasteurization are: • a partial elimination of the milk microorganisms which may grow in cheese during ripening, • a partial or total activation or inhibition of the plasmin/plasminogen complex, cathepsin D, lipoprotein lipase and alkaline phosphatase. Enzymes from psychrotrophic bacteria, acid phosphatase and xanthine oxidase, which may be active during ripening, withstand pasteurization., • a slight (7%) denaturation of serum proteins and little or no modification of the cheesemaking properties (coagulation, acidification by lactic acid bacteria). From experimental work carried out on several cheese varieties, comparing pasteurized or microfiltered milk and raw milk cheeses, it was found that facultatively heterofermentative lactobacilli, Micrococcaceae , enterococci, and propionibacteria in Swiss-type cheese, are found at higher levels in raw milk cheese. The main biochemical modification of cheese during ripening concerns the nature and extent of proteolysis. Although there is no clear trend in the breakdown of α s1 - and β -caseins, milk pasteurization leads to a significant decrease of the amount of small peptides and free amino acids and to different HPLC profiles. Experiments carried out with sensory analysis show that, in all cases, pasteurized or microfiltered milk cheeses have received lower flavour intensity scores than raw milk cheeses. From this review, it is concluded that the indigenous milk microflora, with its diversity of species and strains, appears to be mainly responsible of the specific sensory properties of raw milk cheeses.

A comparison of the flavour and texture of Cheddar cheese of factory or farmhouse origin

Diversity in the flavour and texture of Cheddar cheese was studied in 34 samples of factory or farmhouse origin. Sensory profiles were measured using an integrated Design, Data capture and Sensory Profiling Protocol (DDASPP). The main differences in character of the cheese were between the sub-set of farmhouse cheeses manufactured from raw milk and cheese made from pasteurised milk. Raw milk cheese was more intensely flavoured than conventional product, but was notable for elevated ratings for atypical flavours such as rancid, bitter and unclean. In general, farmhouse cheese showed excessively wide variations in composition which were also associated with atypical flavour or texture. Within factory-made products there was little distinction between samples of Scottish, English, Irish and Canadian origin. However, mature samples of New Zealand Cheddar were of a slightly different character. No strong relations between the sensory properties and composition of the cheese could be deduced. Nevertheless, as expected, the extent of proteolysis and moisture in non-fat solids content of the samples were associated with differences in flavour and texture.

Influence of milk type, coagulant, salting procedure and ripening time on the final characteristics of Picante cheese

Abstract Picante da Beira Baixa cheese is a hard, spicy, salty traditional cheese with a minimum ripening time of 120 d that is manufactured in Portugal at a farm level only. The purpose of this work was to study the influence of several manufacturing conditions (viz. mixture of ovine and caprine milks, source of coagulant, level of NaCl addition, and duration of ripening period) on the final characteristics of this cheese following a (replicated) factorial design. Milk type proved to be a statistically significant technological parameter in terms of numbers of viable microorganisms of various genera and the extent of proteolysis, probably as a consequence of higher initial contamination of caprine milk. The type of coagulant had a major effect on proteolysis: values of water-soluble nitrogen for cheeses coagulated with animal rennet were in general lower than those for cheeses coagulated with plant rennet, but much smaller differences were detected between values of non-protein nitrogen: and breakdown of αs- and β-caseins was more extensive in Picante cheeses manufactured with plant rennet. The level of NaCl was a statistically significant parameter for all microbiological, physicochemical and chemical characteristics measured, an observation that is probably due to its capacity to reduce water activity. Ripening time did not have a significant effect on the numbers of viable staphylococci.

Inhibition of IκB-α and IκB-β Proteolysis by Calpain Inhibitor I Blocks Nitric Oxide Synthesis

Lipopolysaccharide (LPS) stimulates the induction of the inducible isoform of nitric oxide synthase (iNOS) in part by inducing the nuclear translocation of the transcription factor nuclear factor-κ B (NF-κB). LPS induces ubiquination and phosphorylation of the IκB inhibitory subunit of NF-κB. Subsequently, the ubiquitin–proteasome multicatalytic enzyme complex catalyzes the proteolytic degradation of IκB with resultant nuclear translocation of NF-κB. Our results demonstrate that the proteasome inhibitor calpain inhibitor I dose-dependently inhibited LPS-induced nitric oxide synthesis in RAW macrophages. The inhibitor was found to block iNOS transcription and protein translation as noted by Northern analysis and Western blotting, respectively. LPS stimulated rapid proteolytic degradation of IκB-α which was inhibited by approximately 50% in the presence of calpain inhibitor I. In contrast, LPS induced the delayed proteolytic degradation of IκB-β which was almost totally inhibited by calpain inhibitor I. Calpain inhibitor I also decreased the LPS-induced nuclear translocation of NF-κB. These results demonstrate that the ubiquitin–proteasome complex has an important role in induction of iNOS in response to stimuli which act via the NF-κB/IκB signal transduction pathway. Furthermore, the results suggest that the ubiquitin–proteasome complex is important in the degradation of IκB-β as well as IκB-α. Finally, we have demonstrated that there is a marked difference in the extent of proteolysis of IκB-α and IκB-β when the ubiquitin–proteasome complex is inhibited with calpain inhibitor I.

Electrophoretic ripening index for the evaluation of proteolysis and the deduction of the age of Parmesan cheese

We have established an electrophoretic method to evaluate proteolysis in Parmesan cheese by means of an objective ripening index. The separation of caseins by alkaline polyacrylamide gel electrophoresis (PAGE) (12% T, 2.6% C, pH 8.9, 5 M urea) is followed by the densitometric analysis of theγ- andβ-casein fractions. The relationship between the resulting coefficients (γ-Cn/β-Cn) and the age of reference samples of Original Italian Grana Padano (6–22 months) was linear up to 15 months, allowing an evaluation of the extent of proteolysis and therefore a deduction of the age of the Parmesan samples analysed. Based on this calibration we propose to use a threshold level of 1.3 (γ-Cn/β-Cn) to verify the required age, i.e. 12 months, of Parmesan cheese, retailed as a loaf or prepacked slices. Commercially grated Parmesan samples may contain ≤20% cheese rind, which has a very low degree of casein breakdown. So a threshold level of 0.8 (γ-Cn/β-Cn) is proposed for all grated Parmesan products. The preparation of reference solutions with defined coefficients (γ-Cn/β-Cn), corresponding to the proteolysis indices of reference samples of different aged Grana Padano is described. The coefficients (γ-Cn/β-Cn) as well as theβ-casein content of two additional series of reference samples and of 49 commercial Parmesan samples taken from retail outlets in Austria and Italy are presented.

Intragranular prolactin phosphorylation and kallikrein cleavage are regulated by zinc and other divalent cations

Rat prolactin (PRL) secretory granules contain enzymes for proteolytic cleavage and serial phosphorylation, but hormone cleavage products and phosphorylated PRL are not detected until just prior to exocytosis. Similarly, although PRL is stored in granules, in part, as high-mol-wt oligomers, PRL is primarily monomeric in the circulation. PRL secretory granules contain zinc, calcium, and magnesium, which inhibit depolymerization and dissolution of granules. Divalent cations also protect cysteine free thiol residues in the carboxy-terminal region of the intragranular hormone. The present studies examined the effect of removal and replacement of divalent cations on kallikrein cleavage and phosphorylation of secretory granule PRL.Kallikrein cleavage was assessed utilizing two experimental protocols. First, granules were treated with or without 3 mM EDTA, free hormone thiols were alkylated, the PRL was cleaved by kallikrein, and the small kallikrein-cleavage peptides were assessed by reversephase HPLC. No differences in hormone cleavage owing to removal of divalent cations were observed at this concentration of EDTA. Second, divalent cations in granules were reduced/removed by 10 mM EDTA/ 3 mM o-phenanthroline (OP), followed by addition of either 5 mM zinc, magnesium, calcium, or additional EDTA. Kallikrein cleavage was then initiated. In this instance, the extent of proteolysis was analyzed by two-dimensional polyacrylamide gel electrophoresis (PAGE) of the larger remnant PRL pieces. After treatment with 10 mM EDTA/3 mM OP, results indicated that cleavage between R174 and R175 (site 1) was unaffected by added cations or additional EDTA. Recovery of site 2 cleaved PRL (L1-K185) and site 3 cleaved PRL (L1-R188) was∼40% reduced by zinc, but unaffected by calcium or magnesium. Additional EDTA resulted in increased recovery of site 2 cleaved PRL, but no change in site 3 recovery, suggesting the presence of tightly bound intragranular zinc around site 2, even after the initial EDTA/OP treatment.Phosphorylation of PRL at S177 was studied using the same protocols. Phosphorylation was increased by added EDTA, even at 3 mM, and decreased by divalent cations, with no marked specificity for zinc observed. An additional experiment studied phosphorylation without exposure to kallikrein. Comparisons between the plus and minus kallikrein experiments showed kallikrein to have no apparent preference for unmodified or phosphorylated PRL.From the kallikrein cleavage and phosphorylation studies and modeling of PRL, we suggest D181 as a likely site for intragranular zinc coordination. When C189 and C197 are present as free thiols in intragranular PRL, these may also contribute to binding. Zinc coordination in this region of the molecule apparently regulates proteolytic processing by kallikrein, as well as contributing to the stability of the hormone storage forms.

Susceptibility of β-Lactoglobulin and Sodium Caseinate to Proteolysis by Pepsin and Trypsin

The susceptibility of β-LG and sodium caseinate to proteolysis by pepsin and trypsin was investigated using SDS or urea-PAGE. The effects were studied of heat, urea, and 2-mercaptoethanol on proteolysis.Native β-LG was resistant to hydrolysis by pepsin or trypsin because of its compact globular structure. Heat treatment of β-LG solutions at 90 to 100°C for 5 or 10min caused changes in the structure or conformation of the protein that rendered it accessible to pepsin and enhanced the extent of proteolysis by trypsin. The susceptibility of β-LG to proteolysis by pepsin was markedly increased in the presence of urea (3 to 6 M), and the effect was reversible after removal of urea by dialysis. Proteolysis by trypsin was also increased by the presence of 2% 2-mercaptoethanol. Sodium caseinate was very accessible to pepsin without pretreatment and was extensively hydrolyzed at pH 1 to 5 in the presence of 5 M urea (which prevented the protein from precipitation in the isoelectric region); optimal pH was about 2. The activity of pepsin on sodium caseinate at pH 2 was not significantly affected by urea concentration up to about 8 M. The results indicated that the changes in conformation and structure of β-LG that were induced by heating, reduction, or urea rendered the protein susceptible to peptic hydrolysis.

Calpain-induced proteolysis of normal human tau and tau associated with paired helical filaments.

The major components of neurofibrillary tangles (NFT) in Alzheimer's disease are bundles of paired helical filaments (PHF) which are primarily composed of highly phosphorylated tau proteins (PHF-tau). To further understand the mechanism of PHF accumulation in NFT, we examined the calpain-induced proteolysis of highly purified and primarily non-aggregated PHF and normal tau proteins with various contents of phosphate isolated from either fetal (F-tau) or adult human brain (N-tau). The extent of proteolysis was determined by decreases in tau immunoreactivity using Western-blot analysis and a panel of site-specific tau antibodies (Alz 50, Tau-2, Tau 14, Tau-1, AT8, E-11, AH-1 and PHF-1). We found that full-size polypeptides of N-tau and F-tau were similarly and rapidly proteolyzed in vitro by calpain (calpain II, 3.3 units/mg protein) during a 10-min incubation at 30 degrees C, and that their half lives (t1/2) were 1.5 min and 1.8 min, respectively. Analysis of immunoblots suggests that full-length polypeptides of tau are first degraded into large fragments similar in size to that generated endogenously, then into smaller fragments. Since both endogenous and in-vitro-generated tau fragments retained N-terminal epitopes, the results suggest that most of the calpain-sensitive sites may be located in the C-terminal half of the tau molecule. In contrast, PHF were extremely resistant to degradation and only a fivefold higher concentration of calpain (16.7 units/mg protein) induced partial proteolysis of PHF. A major calpain-generated fragment was a 45-kDa polypeptide derived from the C-terminal region of PHF-tau, which forms a core of filaments. The results suggest that the inaccessibility of potential calpain-digestion sites in the filament core could contribute to the resistance of PHF to calpain and subsequently lead to the accumulation of PHF in Alzheimer's disease. The results also suggest that hyperphosphorylation of tau may be marginally involved in the resistance of PHF to degradation by calpain. Ultrastructural examination revealed that, in contrast to previous studies with trypsin, calpain did not alter the morphologic appearance of filaments; after incubation with calpain, the majority of PHF remained short and disperse and the number of PHF aggregated into NFT-like clusters was not significantly increased. The results suggest that the role of calpain in promoting the aggregation and clustering of filaments is limited.

The structure of casein aggregates during renneting studied by indirect Fourier transformation and inverse Laplace transformation of static and dynamic light scattering data, respectively

Aggregation of casein micelles after addition of the proteolytic enzyme chymosin has been studied by static and dynamic light scattering at three different concentrations of casein corresponding to dilutions 1:100, 1:500, and 1:1000 of native milk. The static light scattering data have been analyzed by an indirect Fourier transformation method which gives the distance distributions as a function of time. From these curves radius of gyration and an average number of casein micelles in the aggregates have been derived as a function of time. The dynamic light scattering experiments give the hydrodynamic radius as a function of time after the addition of rennet. The initial radius of gyration for the intact casein micelles is 140 nm. The corresponding hydrodynamic radius is also 140 nm. This shows that the casein micelles are not solid spheres. Inspection of a plot of relative mass versus radius of gyration for the aggregates appearing after the addition of chymosin shows that two processes take place. First extended linear aggregates are built up to a relative mass of the aggregates of about 10 and then restructuring of aggregates occurs such that increasingly compact objects are formed. Whereas the first process exhibits a relatively fast growth in size, the aggregates grow slowly in size during the second process. Further evidence of the formation of linear aggregates followed by more dense aggregates was obtained by forming the ratio between the radius of gyration and the hydrodynamic radius. This ratio increases to values of about 2.5 (indicating that linearly extended molecules are present followed) by a decrease to about 1. The log–log plot of mass versus radius of gyration is linear up to relative masses of about 10 with a slope of about 2. This extends up to sizes of 1 μm in diameter. The slope then increases to values indicating branching and thereby the formation of more compact aggregates. For relative masses below 10 and sizes below 1 μm sedimentation is unlikely to occur and information about the mechanism of aggregation can be obtained. The aggregation number as a function of time has been analyzed in terms of Smoluchowski’s equations with a rate constant including both functionality and a changing barrier height as a function of the extent of proteolysis. The functionality obtained from Smoluchowski’s equations is about 2.1.