Preparation and amino acid sequence of human kappa-casein

Human kappa-casein was prepared from whole casein by successive hydroxyapatite and thiol-Sepharose chromatographies. The primary structure of its 99-residue N-terminal fragment has been determined by sequencing peptides obtained by tryptic and chymotryptic digestions of the whole protein. This fragment overlaps the known sequence of the 65-residue C-terminal fragment. The 158-residue sequence of human kappa-casein was compared to those of goat, ewe, cow and rat kappa-caseins. Only 22% of the residues are identical in homologous positions. The rate of divergence of the 93-residue N-terminal segment (para-kappa-casein) appears to be higher than that of the rest of the molecule.     

Kinetics of milk coagulation: III. Mathematical modeling of the kinetics of curd formation following enzymatic hydrolysis of kappa-casein--parameter estimation

A step function model of milk micelle agglomeration is proposed to explain the observed kinetics of milk clotting following rennet addition. The model ties together the primary and secondary phases of coagulation. The basis of the model is that no micelle flocculation takes place until ca. 75% of the kappa-casein in the milk is hydrolyzed, at which time flocculation occurs rapidly and the rate limiting step for the clotting process shifts to the kappa-casein hydrolysis reaction. Using such a model, it is possible to explain the clotting kinetics for both rapidly denaturing enzymes and stable enzyme systems. The average rate of the flocculation reaction can be obtained from clotting time-versus-reciprocal-enzyme-concentration data by extrapolating the data to infinite enzyme concentration. The critical conversion required for imminent flocculation can be found by extrapolating the enzyme concentration to zero. This approach indicates that the critical conversion necessary for gelation is temperature dependent changing from a limiting value of essentially 100% hydrolysis at temperatures below 15 degrees C to only 60% conversion at temperatures above 30 degrees C.     

Kinetic studies on the action of Mucor pusillus, Mucor miehei acid proteases and chymosins A and B on a synthetic chromophoric hexapeptide

The action of two milk-clotting fungal proteases from Mucos pusillus and Mucor miehei and of chymosins A and B on the hexapeptide, Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe, and on kappa-casein were studied. The effects of pH and temperature on the initial rates of hydrolysis of the hexapeptide were examined. Crystalline chymosin and M. pusillus protease exhibited optimal activities around 49 and 55 degrees C, respectively, whereas the optimum temperature for M. miehei protease is higher than 63 degrees C. The optimum pH was about 4.7 for both fungal proteases whereas chymosin A and chymosin B exhibited optimal activities around 4.2 and 3.7, respectively. Kinetic parameters were then determined under optimal conditions and/or at pH 4.7. Fungal proteases had kcat/Km ratios that were similar to each other and that were significantly greater than the ratios obtained for the chymosins. Nevertheless, chymosins had much greater clotting activities towards kappa-casein relative to their proteolytic activities towards the synthetic peptide.     

The regulation of proteolysis in normal fibroblasts as they approach confluence. Evidence for the participation of the lysosomal system.

The effect of the lysosomotropic agent NH4Cl and the proteinase inhibitors leupeptin, Z-Phe-Ala-CHN2 (benzyloxycarbonylphenylalanylalanyldiazomethane) and pepstatin on the degradation of intracellular proteins in Swiss 3T3 mouse and normal human fibroblasts in both the exponential and stationary (confluent) growth phases in nutritionally complete conditions was investigated. Inhibitory effects of all four agents on degradation in both growth states were detected. The increase in proteolysis normally occurring as cells approach confluence could be completely blocked by NH4Cl, by Z-Phe-Ala-CHN2, or by pepstatin in the presence of leupeptin. These results suggest that the lysosomal system is responsible for the regulation of proteolysis at confluence and further confirm its role in 'basal' proteolysis in growing cells.     

Purification and characterization of milk clotting enzyme from goat (Capra hircus)

Chymosin, the major component of rennet (milk clotting enzyme), is an acid protease produced in the fourth stomach of milk-fed ruminants including goat and sheep in the form of an inactive precursor prochymosin. It is responsible for hydrolysis of kappa-casein chain in casein micelles of milk and therefore, used as milk coagulant in cheese preparation. The present investigation was undertaken to purify and characterize goat (Capra hircus) chymosin for its suitability as milk coagulant. The enzyme was extracted from abomasal tissue of kid and purified nearly 30-fold using anion exchanger and gel filtration chromatography. Goat chymosin resolved into three major active peaks, indicating possible heterogeneity when passed through DEAE-cellulose ion exchange column. The purified enzyme had a molecular mass of 36 kDa on SDS-PAGE, which was further confirmed by Western blot analysis. The purified enzyme preparation was stable up to 55 degrees C with maximum activity at 30 degrees C. The milk clotting activity was decreased steadily as pH is increased and indicated maximum activity at pH 5.5. Proteolytic activity of goat chymosin increased with incubation time at 37 degrees C. Goat chymosin was found to be more thermostable than cattle chymosin and equally stable to buffalo chymosin.     

Primary structure of the casein macropeptide of kappa casein of buffalo]

The complete amino acid sequence of Italian water buffalo (Bubalus arnee) caseinomacropeptide, the C-terminal fragment released from kappa-casein by chymosin, has been determined. It contains 64 amino acid residues including one phosphoserine and differs from its bovine (Bos taurus) B counterpart by 10 amino acid substitutions. The sequence of the last 11 amino acid residues of para-kappa-casein is also reported. In relation to the Ala148/Asp substitution which is responsible for the different electrophoretic behaviour of bovine kappa-caseins B and A, water buffalo kappa-casein is homologous to the bovine variant B. It is suggested that a variant Thr136-Ala148 might be the wild type of the Bos genus.     

Effect of temperature on dynamic viscoelasticity during the clotting reaction of fibrin

The dynamic rigidity and loss moduli for fibrinogen-thrombin solution were determined during clotting in the temperature range between 15 and 45 degrees C. The rigidity of fibrin gel decreased with increasing clotting temperature, owing to the dissociation of cross-links. The rate constant of the dissociation of cross-links increased with increasing temperature. The rate constant of the cross-linking reaction increased and then decreased through a maximum with increasing temperature. It is explained by assuming that denaturation of fibrin occurs at high temperature. The irreversible denaturation becomes appreciable at high ionic strength. The activation energy and the enthalpy change for the cross-linking reaction of fibrin is about 35 and 15 kcal/mol, respectively. The enthalpy change for the reversible denaturation is about 46 kcal/mol.     

The kinetics of milk coagulation: IV. The kinetics of the gel-firming process

A mechanistic kinetic model of gel firmness development during milk gel formation is presented. The model correctly accounts for the influence of enzymatic kappa-casein hydrolysis on the rate of firmness development in renneted milk gels. The model used is based on two first-order reactions occurring in series. The first reaction is enzymatically controlled and corresponds to the formation of gel crosslink sites by kappa-casein hydrolysis. The second reaction is nonenzymatic and corresponds to the process of crosslink formation and depletion of active sites. The model successfully predicts gel firmness development in the temperature range 31-45 degrees C for a variety of initial enzyme concentrations.     

Limited hydrolysis of tRNA by phosphodiesterase.

Digestion of tRNA by electrophoretically pure phosphodiesterase is limited to a short sequence of nucleotides at the 3'-terminus. On the average, four percent of all nucleotides can be released from tRNA. The optimum Mg2 concentration is 10mM and the optimum pH 9.2. The mode of action is a random attack by the enzyme on the substrate. The terminal AMP is completely removed at 15 degrees C after short incubation; about 400 mol of AMP were removed per min by 1 mol of enzyme. The following CMP residues are released much more slowly; at 15 degrees C incompletely, and at 37 degrees C more or less completely in 1 h. In about 50% of the tRNA molecules, the fourth nucleotide could be removed in very long incubations or with very high enzyme concentrations.     

Purification and partial characterization of Oenococcus oeni exoprotease

The exoprotease from Oenococcus oeni produced in stress conditions was purified to homogeneity in two steps, a 14-fold increase of specific activity and a 44% recovery of proteinase activity. The molecular mass was estimated to be 33.1 kDa by gel filtration and 17 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These results suggest that the enzyme is a dimer consisting of two identical subunits. Optimal conditions for activity on grape juice were 25 degrees C and a pH of 4.5. Incubation at 70 degrees C, 15 min, destroyed proteolytic activity. The SDS-PAGE profile shows that the enzyme was able to degrade the grape juice proteins at a significantly high rate. The activity at low pH and pepstatin A inhibition indicate that this enzyme is an aspartic protease. The protease activity increases at acidic pH suggesting that it could be involved in the wine elaboration.     

Purification and properties of three endopeptidases from baker's yeast

Three endopeptidases, proteinases A, B, and Y, were purified from baker's yeast, Saccharomyces cerevisiae. Two molecular forms of proteinase A (PRA), Mr 45,000 and 54,000, (estimated on SDS-PAGE) were obtained. Both forms were inhibited by pepstatin and other acid proteinase inhibitors. The enzyme digested hemoglobin most rapidly at pH 2.7-3.2 and casein at pH 2.4-2.8 and 5.5-6.0. The optimum pH for hydrolysis of protein substrates could be shifted to about 5 with 4-6 M urea. Urea also stimulated the enzyme activity by 30-50%. As other acid proteinases, the enzyme preferentially cleaved peptide bonds of X-Tyr and X-Phe type. A proteinase B (PRB) preparation of approximately Mr 33,000 possessed milk clotting activity and showed an inhibition pattern typical for seryl-sulfhydryl proteases. The purified enzyme could be stabilized with 40% glycerol and stored at -20 degrees C without significant loss of activity for several months. The third endopeptidase, designated PRY, of Mr 72,000 when estimated by Sephadex G-100 gel filtration, had properties resembling PRA and PRB. Similar to PRB, it could be inhibited by up to 90% with phenylmethylsulfonyl fluoride and para-chloromercuribenzoate and preferentially hydrolyzed the Leu15-Tyr16 peptide bond of the oxidized beta-chain of insulin. On the other hand, contrary to PRB, it had neither milk clotting activity nor esterolytic activity toward N-acetyl-L-tyrosine ethyl ester and N-benzoyl-L-tyrosine ethyl ester and was stable during storage at -20 degrees C without glycerol. The enzyme also showed a lower pH optimum for hydrolysis of casein yellow than PRB.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of temperature on the flocculation rate of renneted casein micelles

The flocculation rate constant of completely renneted casein micelles in milk ultrafiltrate was measured by Rayleigh light scattering between 20 and 35 degrees C. In this temperature range an apparent energy of activation of 103 kJ mol (+/-11 kJ mol : n = 50) was measured. At 15 degrees C clotting was not longer perceptible. The activation of the flocculation between 20 and 35 degrees C is explained not so much by the height of the energy barrier separating the clotting micelles, as by the very negative temperature coefficient of that barrier. In line with this conclusion it is suggested that renneted micelles adhere through hydrophobic bonding. The flocculation rate constant of renneted casein micelles is independent of micelle size at the four temperature levels studied.     

Degradation of short and long lived proteins in isolated rat liver lysosomes. Effects of pH, temperature, and proteolytic inhibitors

Most previous studies on inhibitors of lysosomal protein breakdown have been performed on isolated or cultured cells or on perfused organs. We have tested various inhibitors of proteolysis on lysosomes isolated from livers of rats injected with [14C]leucine 15 min (short labeling time) and 16 h (long labeling time) before killing. Intact lysosomes were incubated with different inhibitors (leupeptin, propylamine, E-64, pepstatin, and chloroquine) in increasing concentrations. None of these caused more than a 40-75% inhibition of proteolysis irrespective of labeling protocol. Chloroquine was the most effective inhibitor, followed by leupeptin, propylamine, E-64, and pepstatin. When lysosomes were incubated with various combinations of inhibitors, including a weak base and an enzyme inhibitor, a somewhat higher inhibition (86%) was obtained. To assess if lysosomes are active in the degradation of both short and long lived proteins, lysosomes were isolated from livers of rats labeled with [14C]leucine for various time intervals. The highest fractional proteolytic rates were seen for short lived proteins. If the recovery of the isolated lysosomes is taken into consideration, about 80% (short labeling time) and 90% (long labeling time) of the total proteolysis in the homogenate could be accounted for by lysosomes. Isolated Golgi, mitochondrial, and microsomal fractions displayed negligible proteolytic activities. The cytosol contributed one-fifth of the total protein breakdown of short lived proteins, whereas insignificant proteolysis was recovered in the cytosolic fraction following long time labeling. Accordingly, we propose that 1) lysosomal inhibitors do not completely suppress proteolysis in isolated lysosomes and that 2) both short and long lived proteins are degraded in lysosomes.     

Activation mechanism of erythrocyte cathepsin E. evidence for the occurrence of the membrane-associated active enzyme

Activation of the erythrocyte cathepsin E located on the cytoplasmic surface of the membrane in a latent form was studied in stripped inside-out membrane vesicles prepared from human erythrocyte membranes. Incubation of the vesicles at 40 degrees C at pH 4 resulted in increased degradation of the membrane proteins, especially band 3. This proteolysis was selectively inhibited by the inclusion of pepstatin (isovaleryl-Val-Val-statyl-Ala-statine) or H 297 [Pro-Thr-Glu-Phe(CH2-NH)Nle-Arg-Leu] in the incubation mixtures, indicating that cathepsin E, as the only aspartic proteinase in erythrocytes, is responsible for the proteolysis. Two potential active-site-directed inhibitors of aspartic proteinases, pepstatin and H 297, were used to prove the occurrence of the membrane-associated active enzyme. To minimize potential errors arising from non-specific binding, the concentrations of the inhibitors used in the binding assay (pepstatin, 5 x 10(-8) M; H 297, 1 x 10(-5) M) were determined by calibration for purified and membrane-associated cathepsin E. The inhibition of the membrane-associated cathepsin E by each inhibitor, which showed the binding of the inhibitor to the activated enzyme, was temperature- and time-dependent. The binding of each inhibitor to the enzyme on the exposed surface of the membrane at pH 4 was highly specific, saturable, and reversible. The present study thus provides the first evidence that cathepsin E tightly bound to the membrane is converted to the active enzyme in the membrane-associated form, and suggests that this enzyme may be responsible for the degradation of band 3.     

Fluid Gels: a New Feedstock for High Viscosity Jetting

Suspensions of gel particles which are pourable or spoonable at room temperature can be created by shearing a gelling biopolymer through its gelation (thermal or ion mediated) rather than allowing quiescent cooling – thus the term ‘fluid gel’ may be used to describe the resulting material. As agar gelation is thermoreversible this type of fluid gel is able to be heated again to melt agar gel particles to varying degrees then re-form a network quiescently upon cooling, whose strength depends on the temperature of re-heating, determining the amount of agar solubilised and subsequently able to partake in re-gelation. Using this principle, for the first time fluid gels have been applied to a high viscosity 3D printing process wherein the printing temperature (at the nozzle) is controllable. This allows the use of ambient temperature feedstocks and by altering the nozzle temperature, the internal nature (presence or absence of gel particles) and gel strength of printed droplets differs. If the nozzle prints at different temperatures for each layer a structure with modulated texture could be created.     

Apolipoprotein B-100 of plasma low density lipoproteins undergoes proteolysis by contact activation factors when plasma is treated with dextran sulfate-500-MgCl2

Human plasma low density lipoproteins (LDL) isolated by ultracentrifugation showed a single band corresponding to apolipoprotein B-100 (apoB-100) by SDS-gradient gel electrophoresis (GGE). In turn, apoB-100 of LDL precipitated from plasma by dextran sulfate-500 (DS)-MgCl2 exhibited several bands indicative of a degradative process. The degradation was more extensive at 0 degrees C than at either 23 degrees C or 37 degrees C, and appeared to be related to a protease activity that cleaved both the synthetic peptide, Z-Phe-Arg-7-amido-4-methylcoumarin (Z-Phe-Arg-AMC) and apoB-100. Proteolysis was proportional to the DS added to the plasma, was prevented by the kallikrein inhibitor, D-Phe-L-Phe-L-Arg-CHCl2, and was significantly decreased in plasma specimens of patients with either factor XII or prekalikrein deficiency. LDL pre-purified by ultracentrifugation and then precipitated by DS in the absence of plasma exhibited no proteolysis. However, proteolysis was observed when LDL interacted with kallikrein. The two main apolipoproteins of HDL3, apoA-I and apoA-II, were not affected by this proteolytic process. We interpret the results to indicate that the negatively charged surface provided by DS accelerates in plasma the autoactivation of factor XII and the activation of prekallikrein, resulting in an increase of the effective concentration of kallikrein and possibly other proteases and proteolysis of LDL-apoB-100. The higher degree of the DS-induced proteolysis of apoB-100 at 0 degrees C than at 23 degrees C is likely the consequence of enhanced autoactivation of factor XII and a decreased efficiency of plasma inhibitors, such as C1-inhibitor. We speculate that the proteolysis of apoB-100 induced by DS is not limited to this polyanion, but may also be the property of other negatively charged agents, particularly at cold temperatures.     

Kinetics of pepsin-initiated coagulation of kappa-casein.

The kinetics of pepsin initiated coagulation of kappa-casein have been studied at pH 5.8. The primary and secondary phases of coagulation are shown to proceed simultaneously. The theory of enzymatically initiated clotting reactions proposed by Payens (Payens, T.A.J. (1976) Neth. Milk Dairy J. 30, 55--59) has been applied to this clotting system and has been used to obtain rate constants for the secondary phase of coagulation. As expected, clotting rate constants for kappa-casein increase with pepsin concentration. An activation energy of 30.6 kcal/mol has been obtained for the secondary phase of coagulation. Turbidity measurements are a convenient means for studying the secondary phase of coagulation but do not provide an unambiguous means for studying the primary phase of the reaction.     

Resolution and characterisation of multiple isoforms of bovine kappa-casein by 2-DE following a reversible cysteine-tagging enrichment strategy

Visualisation of multiple isoforms of kappa-casein on 2-D gels is restricted by the abundant alpha- and beta-caseins that not only limit gel loading but also migrate to similar regions as the more acidic kappa-casein isoforms. To overcome this problem, we took advantage of the absence of cysteine residues in alpha(S1)- and beta-casein by devising an affinity enrichment procedure based on reversible biotinylation of cysteine residues. Affinity capture of cysteine-containing proteins on avidin allowed the removal of the vast majority of alpha(S1)- and beta-casein, and on subsequent 2-D gel analysis 16 gel spots were identified as kappa-casein by PMF. Further analysis of the C-terminal tryptic peptide along with structural predictions based on mobility on the 2-D gel allowed us to assign identities to each spot in terms of genetic variant (A or B), phosphorylation status (1, 2 or 3) and glycosylation status (from 0 to 6). Eight isoforms of the A and B variants with the same PTMs were observed. When the casein fraction of milk from a single cow, homozygous for the B variant of kappa-casein, was used as the starting material, 17 isoforms from 13 gel spots were characterised. Analysis of isoforms of low abundance proved challenging due to the low amount of material that could be extracted from the gels as well as the lability of the PTMs during MS analysis. However, we were able to identify a previously unrecognised site, T(166), that could be phosphorylated or glycosylated. Despite many decades of analysis of milk proteins, the reasons for this high level of heterogeneity are still not clear.     

Sequential Limited Proteolysis of Myelin Basic Protein by Neutral Protease Activities of Bovine Brain

Acid extracts of delipidated white matter of bovine brain were prepared, and their proteolytic activities toward myelin basic protein (MBP) were evaluated at pH 3 and pH 7. This was done by measuring changes in total protein using a selective dye-binding assay, and by evaluating peptide patterns by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry. At pH 7 greater than 50% of total protein and about 75% of MBP were degraded after 48 h, whereas at pH 3 it was less than 20% altogether. Neutral proteolysis of MBP entailed up to 12 different proteolytic peptide fragments in the molecular weight range of 17.5 to 6 kd. Its enzymatic nature was verified using protease inhibitors, including N-ethylmaleimide, phenylmethylsulfonyl fluoride, o-phenanthroline, and EDTA, as well as pepstatin A and alpha 2-macroglobulin. Both transient changes in percentages of some intermediate peptides and differential effects of individual inhibitors on electrophoretic peptide patterns strongly suggest a sequential type of limited proteolysis. The results also indicate that acid extracts contained several endopeptidases of which a cysteine protease appears to initiate the breakdown of MBP.     

The inhibition of macrophage protein turnover by a selective inhibitor of thiol proteinases.

1. A new inhibitor of thiol proteinases, benzyloxycarbonylphenylalanylalanine diazomethyl ketone (benzyloxycarbonylphenylalanylalanyldiazomethane, Z-Phe-Ala-CHN2) was added to cultured mouse peritoneal macrophages prelabelled with [14C]leucine. The degradation of protein was studied under conditions of basal proteolysis in the presence of 10% pig serum. After a lag of about 6 h a time- and dose-dependent inhibition of protein degradation was observed, up to a maximum of about 40%. 2. The inhibitor entered the cells with kinetics consistent with entry by pinocytosis, giving access to the lysosomal system. 3. Intracellular cathepsin B was almost completely inactivated after 90 min of exposure of the culture to 0.1 mm-inhibitor. 4. The inhibition of proteolysis and of cathepsin B was reversed virtually completely within 24 h, when the inhibitor was removed from the medium. Since the inhibitor forms a covalent bond with the enzyme, the recovery of cathepsin B activity presumably reflects production of new molecules of active enzyme. 5. The inhibitory effects of pepstatin, the carboxyl proteinase inhibitor, were under some circumstances additive with those Z-Phe-Ala-CHN2, and were also largely reversible. 6. It is concluded that thiol proteinases play a major role in lysosomal proteolysis in cultured macrophages.