Kinetics of milk coagulation: I. The kinetics of kappa casein hydrolysis in the presence of enzyme deactivation

The kinetics of the primary phase of the enzymatic coagulation of milk, i.e., kappa-casein hydrolysis, was investigated in the presence and in the absence of concurrent enzyme deactivation processes. For conditions under which the enzyme is stable, the rate of hydrolysis can be described by Michaelis-Menten kinetics, as has been reported by previous investigators. A mathematical model, experimental data, and parameter estimates are provided for kappa-casein hydrolysis in the presence of concurrent deactivation of enzyme. The model accurately describes the experimental results when porcine pepsin was used as the renneting enzyme. The model and the experimental results indicate that samples of milk treated under conditions where deactivation of enzyme is significant can have fractional conversions of kappa-casein ranging from zero to unity and yet contain no active enzyme at the termination of the treatment.     

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.     

Kinetics of milk coagulation: II. Kinetics of the secondary phase: micelle flocculation

A kinetic model for the agglomeration of milk micelles following kappa-casein hydrolysis is described. The key features of the model are: (1) the surface potential of casein micelles is sufficient to explain the colloidal stability of the milk system; (2) the reduction in surface potential following kappa-casein hydrolysis explains the loss of stability; (3) partial hydrolysis leads to limited agglomeration; and (4) the kinetics of agglomeration are compatible with the theory that completely hydrolyzed micelles have only a limited number of interaction sites. The model accurately predicts solution turbidity increase assuming that micelles have only circa 1.2 interaction sites on the average under the experimental conditions of this study.     

Specificity of hydrolysis of bovine kappa-casein by cell envelope-associated proteinases from Lactococcus lactis strains.

The cell envelope-associated proteinases from Lactococcus lactis subsp. cremoris H2 (a PI-type proteinase-producing strain) and SK11 (a PIII-type proteinase-producing strain) both actively hydrolyze the kappa-casein component of bovine milk but with significant differences in the specificity of peptide bond hydrolysis. The peptide bonds Ala-23-Lys-24, Leu-32-Ser-33, Ala-71-Gln-72, Leu-79-Ser-80, Met-95-Ala-96, and Met-106-Ala-107 were cleaved by both proteinase types, although the relative rates of hydrolysis at some of these sites were quite different for the two proteinases. Small histidine-rich peptides were formed as early products of the action of the cell envelope-associated proteinases on kappa-casein, implicating this casein as a possible significant source of histidine, which is essential for starter growth. The major difference between the two proteinase types in their action on kappa-casein was in their ability to cleave bonds near the C-terminal end of the molecule. The bond Asn-160-Thr-161 and, to a lesser extent, the bond Glu-151-Val-152 were very rapidly cleaved by the PIII-type proteinase, whereas hydrolysis of these bonds by the PI-type proteinase was barely detectable (even after 24 h of digestion). Differential hydrolysis of kappa-casein at these sites by the two different proteinase types resulted in the formation of distinctive, high-M(r) products detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The identification of the kappa-casein genotype in Holstein dairy cattle using the polymerase chain reaction

Summary The polymerase chain reaction (PCR) was used to amplify a 99-bp region from the kappa-casein gene of Holstein dairy cattle which contains nucleotide substitutions that are diagnostic of the two major protein variants of kappa-casein. Identity of the amplified product was confirmed by direct sequencing. Digestion of the PCR product with MboII (A-variant specific) or TaqI (B-variant specific) allowed direct determination of the genotype of the animal (homozygous or heterozygous). A total of 58 lactating cows with known kappa-casein phenotype were tested using PCR. In all cases, the measured genotype confirmed the phenotype. We have also tested the genotype of 42 sires that were top ranked for milk yield by the CIAQ (Centre d'insemination artificielle du Quebec). The B-allele of kappa-casein which occurred at a frequency of 0.13 among the proven bulls is associated with superior milk for industrial applications. Identification of the kappa-casein genotype by PCR in bulls and calves would provide a means for rapidly changing the frequency of the B-allele in the breeding population by selection.     

An agarose gel method for the determination of DNA interstrand crosslinking applicable to the measurement of the rate of total and "second-arm" crosslink reactions

A simple agarose gel electrophoresis method for the determination of DNA interstrand crosslinks is described. Following complete denaturation of 32P-end-labeled DNA the presence of an interstrand crosslink results in renaturation to double-stranded DNA. The single- and double-stranded bands separated on an agarose gel can be accurately quantitated by densitometry of the autoradiograph produced from the dried gel. The technique is particularly applicable to detailed time-course experiments of both total crosslink formation and, following removal of free drug, the "second-arm" of the crosslink reaction. The method is illustrated for a number of nitrogen mustard antitumor agents, showing how the moiety attached to a bifunctional reactive group can influence the extent and rate of crosslink formation and, in particular, the conversion of monoadducts to crosslinks. It is sensitive enough to follow the formation of crosslinks by slow and inefficient cross-linking agents such as busulfan which have not previously been measured by physical procedures.     

Amyloid fibril formation by bovine milk kappa-casein and its inhibition by the molecular chaperones alphaS- and beta-casein

Caseins are a unique and diverse group of proteins present in bovine milk. While their function is presumed to be primarily nutritional, caseins have a remarkable ability to stabilize proteins, i.e., to inhibit protein aggregation and precipitation, that is comparable to molecular chaperones of the small heat-shock protein (sHsp) family. Additionally, sHsps have been shown to inhibit the formation of amyloid fibrils. This study investigated (i) the fibril-forming propensities of casein proteins and their mixture, sodium caseinate, and (ii) the ability of caseins to prevent in vitro fibril formation by kappa-casein. Transmission electron microscopy (TEM) and X-ray fiber diffraction data demonstrated that kappa-casein readily forms amyloid fibrils at 37 degrees C particularly following reduction of its disulfide bonds. The time-dependent increase in thioflavin T fluorescence observed for reduced and nonreduced kappa-casein at 37 degrees C was suppressed by stoichiometric amounts of alphaS- and beta-casein and by the hydrophobic dye 8-anilino-1-naphthalene sulfonate; the inhibition of kappa-casein fibril formation under these conditions was verified by TEM. Our findings suggest that alphaS- and beta-casein are potent inhibitors of kappa-casein fibril formation and may prevent large-scale fibril formation in vivo. Casein proteins may therefore play a preventative role in the development of corpora amylacea, a disorder associated with the accumulation of amyloid deposits in mammary tissue.     

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.     

Molecular origin of the sequence-dependent kinetics of reactions between cisplatin derivatives and DNA

Experimental and theoretical studies related to the kinetics of interactions between cisplatin derivatives and DNA are critically reviewed. The reaction between DNA and cisplatin is a multistep reaction, consisting of aquation, electrostatic preassociation, nucleophilic substitution of one aqua ligand by a DNA guanine, and crosslink formation. If the reacting platinum complex is cis-[PtCl(NH₃)₂(H₂O)]⁺, the monoadduct bears one chlorido ligand, and the crosslink formation is preceded by a second aquation step. The kinetics of all these steps, including that of the monoadduct aquation, is significantly modulated by the flanking bases. In the case of the cisplatin derivatives carboplatin and oxaliplatin, recent work has clearly shown that the opening of the dicarboxylate chelate ring is faster than cisplatin hydrolysis; however, the even much faster ring closure keeps the concentration of the ring-opened species very low. Correlation of data from reactions with nucleotides on the one hand and from those with DNA on the other hand indicates that in vitro, the reactions with DNA proceed with the intact dicarboxylates as the reacting species. Thus, for carboplatin and oxaliplatin, the species reacting with DNA are electroneutral, while for cisplatin, the reactive species are cationic. The fact that nevertheless the in vitro sequence-selectivities appear similar for all three complexes suggests that hydrogen bonding between platinum leaving groups and DNA residues is not a factor determining the sequence-selective binding to GG and AG sequences.This review focuses at the studies designed to quantify and explain the physical origin of the influence of DNA sequence on the reaction kinetics.     

The Contractile and Control Sites of Natural Actomyosin

The various contractile and control sites of natural actomyosin gel were studied by comparing the kinetics of ATP hydrolysis with those of gel contraction, measured as an increase in turbidity. Contraction of actomyosin gel seems to require the cooperative reaction of ATP (with Mg) at two different sites. One of these sites catalyzes the hydrolysis of ATP and most probably contributes the driving force for contraction; the binding of ATP to the other site appears to break certain links that retard movement of the gel components. At limiting concentrations of ATP, the rate of contraction seems to depend on the rate of breaking these links as well as on the rate of ATP hydrolysis. But when both sites are saturated, the rate of contraction appears to be limited only by the rate of ATP hydrolysis. In addition to these two contractile sites, there are also two different control sites. At one, the relaxing site, the binding of ATP with Mg inhibits ATP hydrolysis and gel contraction. At the other, the binding of calcium activates contraction by overcoming the inhibitory action of Mg and ATP at the relaxing site. This control system—inhibition by substrate and disinhibition by calcium—can be selectively inactivated by heat and reactivated by dithiothreitol, a disulfide-reducing agent. These observations on the isolated contractile system are discussed in relation to the contraction and relaxation of muscle.     

Nucleotide-free kinesin hydrolyzes ATP with burst kinetics

Bovine brain kinesin binds ADP tightly and contains a stoichiometric amount of ADP at its active site when isolated in the presence of free Mg2+ (Hackney, D. D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6314-6318). EDTA in excess of Mg2+ weakens ADP binding and nucleotide-free kinesin can be prepared by gel filtration with excess EDTA. On addition of ATP, this nucleotide-free enzyme catalyzes the rapid hydrolysis of a stoichiometric amount of ATP in a burst phase followed by much slower continued ATP hydrolysis limited by the release of ADP from the active site. This burst reaction is evident both by formation of [32P]Pi from [gamma-32P]ATP and by formation of [alpha-32P]ADP from [alpha-32P]ATP. At 1.1 nM kinesin active sites, the observed rate of the burst phase increases linearly with ATP over the 1-20 nM range yielding a bimolecular rate of net ATP binding and hydrolysis of 2.5 microM-1 s-1. The intercept at zero ATP is 0.008 s-1 which equals the ADP release rate at 0.008-0.009 s-1. This predicts a Km for ATP of approximately 3.5 nM and measurements of the dependence on ATP concentration of the steady state rate and amount of bound ADP are consistent with a Km of this magnitude.     

The mycotoxin patulin induces intra- and intermolecular protein crosslinks in vitro involving cysteine, lysine, and histidine side chains, and alpha-amino groups

As previous studies have indicated a multiple electrophilic reactivity of patulin (PAT) towards simple thiol nucleophiles, we have methodically investigated the ability of PAT to covalently crosslink proteins in vitro. By means of sodium dodecylsulphate polyacrylamide gel electrophoresis, the formation of PAT-induced intermolecular protein-protein crosslinks was clearly demonstrated for bovine serum albumin containing one thiol group per molecule, but also for the thiol-free hen egg lysozyme. Characterization of the crosslink sites was carried out by (1) modulation of the thiol groups with N-ethylimaleimide and 2-iminothiolane; (2) comparison with various known crosslinking agents, i.e. phenylenedimaleimide, glutardialdehyde, and dimethylsuberimidate, and (3) fluorescence incorporation studies using dansyl-labeled amino acids and a fluorescent glutathione derivative. The thiol group of cysteine was preferred for PAT-mediated crosslink reactions, but the side chains of lysine and histidine, and alpha-amino groups also exhibited reactivity. PAT can act both as a homobifunctional as well as a heterobifunctional crosslinking agent. The initial formation of a monoadduct with a thiol group appears to activate PAT for the subsequent reaction with an amino group, but also leads to rapid loss of further electrophilic properties when no second nucleophile for crosslink completion is available. Studies using microtubule proteins as a protein with experimentally controllable quarternary structure and a proposed cellular target for PAT toxicity emphasized the influence of specific sterical conditions on crosslink formation at low protein concentrations. Non-polymerized microtubule proteins, i.e. tubulin alpha,beta-dimers, formed a defined product with PAT consisting of an intramolecularly crosslinked beta-tubulin, whereas guanosine triphosphate- or paclitaxel-induced polymerization to microtubule-like quarternary structures prior to treatment with PAT gave rise to intermolecular crosslink formation between alpha- and beta-tubulin. In contrast, denaturated tubulin yielded none of those two new protein species, but only unspecific intramolecular crosslinks and highly crosslinked aggregates. Thus, in addition to the amino acid composition, the tertiary and quarternary superstructures of proteins appear to markedly influence their reactivity towards PAT. Under appropriate conditions, the generation of protein crosslinks could easily be observed at concentrations of PAT equal to or even below the concentration of the protein. The relevance of these novel reaction pathways of PAT demonstrated in vitro for its in vivo mechanisms of toxicity remains to be investigated.     

The influence of chromatin compactness on the stoichiometry of the Feulgen-Schiff procedure studied in model films. I. Theoretical kinetics and experiments with films containing isolated deoxyribonucleic acid.

Theoretical considerations on the expected kinetics of the course of the Feulgen-Schiff reaction show that the leveling off of the first part of the Feulgen hydrolysis curve can be explained by the gradual conversion of deoxyribonucleic acid (DNA) to apurinic acid (APA). In addition, depolymerization of DNA caused by the acid used for hydrolysis can account for the decline after a maximum is reached in this curve. With the aid of polyacrylamide model films containing DNA, a detailed study was made both of the process of purine liberation which results in the formation of APA and of the depolymerization processes which cause losses of stainable material. The liberation of purine bases was analyzed by ultraviolet absorbance measurements and by gel chromatography of the neutralized hydrolysing acid. APA concentration was monitored by following the loss of ultraviolet absorbance associated with the purine losses. The depolymerization process was followed by phosphorus determinations. The experimental results were found to be in accordance with the kinetics expected from the theoretical model.     

Isolation and properties of human kappa-casein

Human kappa-casein was isolated from human whole casein by gel filtration with Sephadex G-200 and hydroxylapatite chromatography in the presence of sodium dodecyl sulfate (SDS). The kappa-casein was calcium-insensitive and did stabilize human beta-casein and bovine alpha s1-casein against precipitation by calcium ions. Formation of micelles from human beta- and kappa-caseins, and calcium ions was confirmed by electron microscopic observation. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), a single band was obtained. The formation of para-kappa-caseins by chymosin was confirmed by SDS-PAGE. Two para-kappa-caseins with apparent molecular weights of 13,000 and 11,000 appeared. The molecular weight of intact human kappa-casein was estimated to be approximately 33,000. The human kappa-casein contained about 40% carbohydrate (15% galactose, 3% fucose, 15% hexosamines, and 5% sialic acid) and 0.10% (1 mol/mol) phosphorus. Its amino acid composition was similar to that of bovine kappa-casein except for serine, glutamic acid, and lysine contents.     

Isolation and some effects of functional, low-phenylalanine kappa-casein expressed in the milk of transgenic rabbits

Patients suffering certain metabolic diseases (e.g. phenylketonuria) need a low-phenylalanine diet throughout their lives. Transgenic rabbits were created to express low-phenylalanine kappa-casein in their milk. The aim was to demonstrate for the first time the feasibility of producing a modified milk protein in addition to normal milk proteins. A gene construct containing the coding region of the rabbit kappa-casein gene was modified by site-specific oligonucleotide directed mutagenesis. Four of the five phenylalanine amino acids present in the mature protein were mutated and the gene construct was used to create two transgenic rabbit lines. The transgenic rabbits produced the recombinant kappa-casein at a high level in their milk causing a reduction in the average size of the casein micelles. The low-phenylalanine kappa-casein was digestible with chymosin and it was separated from its native counterpart and from the other milk proteins by a one-step HPLC method on a reversed-phase column. In the future, low-phenylalanine casein produced in transgenic animals could be used as dietary replacements to meet the special requirements of certain consumer groups.     

Restriction fragment length polymorphism analysis of the kappa-casein locus in cattle

The two common genetic variants (A and B) of bovine kappa-casein originate from two point mutations in the codons for the aminoacids in position 136 and 148. These mutations give rise to polymorphic sites for the restriction endonucleases Hin dIII, AluI, HinfI, Mbo II and TaqI. We have examined DNAs of several Italian Friesian cows and bulls of known and unknown genotype by Southern analyses using kappa-casein cDNA probes. Restriction fragment length polymorphisms (RFLPs) specific for the A and B alleles were identified for each of the above enzymes, except for AluI, which has a non-polymorphic site 12bp away from the polymorphic one. We have also found two new polymorphic sites for MboII and TaqI in the non-coding regions. These sites differentiate the A allele into two new variants, named A1 and A2. The RFLP analysis permits the characterization of kappa-casein alleles even in the absence of their expression. This should facilitate selective breeding programmes aimed at increasing the frequency of the kappa-casein B allele whose product improves the cheesemaking properties of milk.     

Detection of the cholera toxin-binding activity of kappa-casein macropeptide and optimization of its production by the response surface methodology

The cholera toxin (CT)-binding activity of purified kappa-casein macropeptide (CMP) from bovine kappa-casein was detected. In addition, a statistical model was developed to optimize the production of CMP. CMP was prepared by chymosin hydrolysis of kappa-casein and a subsequent 3% trichloroacetic acid treatment. CMP was further fractionated in an ion-exchange column by FPLC. CT binding activity was eluted at 0.18 M NaCl and was a single 8.9 kDa peptide without tyrosine and arginine residues. The CT binding activity was rapidly lost by a carbohydrase treatment. The conditions for CMP production with chymosin were optimized by using the response surface methodology (RSM). The estimated optimum levels of the factors were as follows: reaction temperature, 38.5 degrees C; pH, 6.44; and time, 35.9 min. A validation experiment was performed in which CMP was prepared under the predicted parameters, and it was ascertained that the estimated optimum conditions gave better production of CMP than any other conditions.     

The involvement of one of the three histidine residues of cow kappa-casein in the chymosin-initiated milk clotting process.

Cow kappa-casein has been modified by photo-oxidation in the presence of rose bengal and by the chemical reagents diethyl pyrocarbonate, 2-hydroxy-5-nitro-benzyl bromide and iodoacetic acid. Photo-oxidation resulted in the destruction of histidine and tryptophan residues and all of the histidines could be ethoxy-formylated by treatment with diethyl pyrocarbonate. Both procedures caused a loss in the susceptibility of the Phe-Met linkage of kappa-casein to chymosin hydrolysis. Treatment of kappa-casein with 2-hydroxy-5-nitrobenzyl bromide and iodoacetic acid caused the loss of tryptophan and methionine residues respectively but, in both cases, the susceptibility of the modified protein to chymosin hydrolysis remained unaffected. Of the amino acids examined it is concluded that only the histidine residues of cow kappa-casein are important for the hydrolytic action of chymosin and, furthermore, the treatment with diethyl pyrocarbonate suggests that only one of the three histidines plays an essential role.     

DNA interstrand crosslink formation by mechlorethamine at a cytosine-cytosine mismatch pair: kinetics and sequence dependence

Expansion of the triplet repeat DNA sequence d[CGG]n.d[CCG]n is a characteristic of Fragile X syndrome, a human neurodegenerative disease. Stable intrastrand conformations formed by both d[CGG]n and d[CCG]n, and involving G-G and C-C mismatch pairs, respectively, are believed to be of importance in the development of the disease. We have shown previously that C-C mismatch pairs can be crosslinked covalently by mechlorethamine, a nitrogen mustard alkylating agent, and hence this reaction may be of value as a probe for conformers of d[CCG]n. To characterize the mechlorethamine C-C crosslink reaction further, here we report the kinetics and sequence dependence of formation of the crosslink species, using a series of model duplexes. The rate of reaction depends on the base sequence proximal to the C-C mismatch pair. Hence, in 19mer duplexes containing a central d[M4M3M2M1Cn1n2n3n4].d[N4N3N2N1Cm1m2m3m4] sequence, where M-m and N-n are complementary base pairs, the amount of crosslink increased with increasing G-C content of the eight base pairs neighboring the C-C mismatch and with the proximity of the G-C pairs to the C-C mismatch. Molecular dynamics simulations of the solvated duplexes provided an explanation of these data. Hence, for a C-C pair flanked by G-C base pairs the mismatched cytosine bases remain stacked within the duplex, but for a C-C pair flanked by A-T base pairs, the simulations suggested local opening of the duplex around the C-C pair, making it a less effective target for mechlorethamine.