Bovine Milk Enzyme Research Articles

Synthesis and bioevaluation of 2-phenyl-4-methyl-1,3-selenazole-5-carboxylic acids as potent xanthine oxidase inhibitors

A series of 2-phenyl-4-methyl-1,3-selenazole-5-carboxylic acid derivatives (8a–f, 9a–m) were synthesized and evaluated for inhibitory activity against xanthine oxidase in vitro. Structure–activity relationship analyses have also been presented. Most of the target compounds exhibited potency levels in the nanomolar range. Compound 9e emerged as the most potent xanthine oxidase inhibitor (IC50 = 5.5 nM) in comparison to febuxostat (IC50 = 18.6 nM). Steady-state kinetics measurements with the bovine milk enzyme indicated a mixed type inhibition with Ki and Ki′ values of 0.9 and 2.3 nM, respectively. A molecular modeling study on compounds 9e was performed to gain an insight into its binding mode with xanthine oxidase, and to provide the basis for further structure-guided design of new non-purine xanthine oxidase inhibitors related with 2-phenyl-4-methyl-1,3-selenazole-5-carboxylic acid scaffold.

Reduction of bacterial counts and inactivation of enzymes in bovine whole milk using pulsed electric fields

Bacterial count reduction and enzyme inactivation in bovine whole milk by pulsed electric field (PEF) processing combined with pre-heating and intermediate cooling was compared with that induced by thermal treatments (maximum of 63 °C for 30 min and 73 °C for 15 s). Milk was pre-heated to 55 °C for 24 s and PEF-treated in continuous mode at electric field intensities of 15.9–26.2 kV cm−1 for 17–101 μs. PEF treatments reduced the number of inoculated (8.3 log cfu mL−1) Escherichia coli and Listeria innocua to less than 2 log cfu mL−1. PEF treatments at 20.7–26.2 kV cm−1 reduced the total plate count and indigenous alkaline phosphatase activity to a level similar to that measured following thermal treatments. Treatment at 26.1 kV cm−1 for 34 μs, combined with pre-heating, reduced the activity of plasmin, xanthine oxidase and lipolysable fat by 12%, 32% and 82%, respectively, compared with raw milk.

Kinetic and thermodynamic parameters of alkaline phosphatase and γ—glutamyl transferase inactivation in bovine milk

Kinetic and thermodynamic studies were carried out at different time–temperature combinations in order to evaluate the suitability of alkaline phosphatase (ALP) and γ-glutamyl transferase (GGT) as markers for the heat treatment of milk. The average activities of the enzymes in bovine milk were 0.46 ± 0.02 U.mL−1 for ALP and 4.05 ± 0.26 and 4.55 ± 0.24 U.mL−1 for GGT in milk with different fat levels. A detailed comparative kinetic study of ALP inactivation was performed in the temperature range of 50 to 75 °C in raw whole milk and buffer in order to assess the stabilizing effect of milk compounds on ALP. The degree and rate of inactivation of GGT in raw cow’s milk with 1.5% and 2.5% fat were measured in the temperature range of 60 to 85 °C. Kinetic studies showed that the thermal inactivation of ALP and GGT followed first-order kinetics. The influence of temperature on the inactivation rate constant was quantified using the Arrhenius equation and was characterized by activation energy (E a) values of 214.09 ± 12.9 and 222.02 ± 5.6 kJ.mol−1 for inactivation of ALP in buffer and raw milk. For GGT, E a values ranged from 190.8 ± 6.06 to 183.7 ± 8.4 kJ.mol−1 for enzyme inactivation in low-fat milk and in milk containing 2.5% fat, respectively.

Crystal Structures of Urate Bound Form of Xanthine Oxidoreductase: Substrate Orientation and Structure of the Key Reaction Intermediate

Two contradictory models have been proposed for the binding mode of the substrate xanthine to and its activation mechanism by xanthine oxidoreductase. In an effort to distinguish between the two models, we determined the crystal structures of the urate complexes of the demolybdo-form of the D428A mutant of rat xanthine oxidoreductase at 1.7 Å and of the reduced bovine milk enzyme at 2.1 Å, the latter representing a reaction intermediate. The results clearly indicate the catalytically relevant binding mode of the substrate xanthine.

Kinetics of thermal inactivation of alkaline phosphatase in bovine and caprine milk and buffer

The thermal inactivation of alkaline phosphatase (ALP) in raw bovine and caprine milk was investigated in the temperature range 54 to 69 °C. To assess the stabilizing effect of milk compounds on ALP, inactivation experiments were also carried out in 0.1 m potassium phosphate buffer, pH 6.6. Each set of inactivation experiments was fitted simultaneously using kinetic models that were based on either one-step or two-step mechanisms. The parameters of the Arrhenius equation showed that the stabilization effect of milk compounds on ALP had an entropic character. They also indicated a different structure of bovine and caprine milk ALPs, which was reflected by a higher stability of the bovine milk enzyme.

Effects of high pressure treatment on indigenous enzymes in bovine milk: Reaction kinetics, inactivation and potential application

Abstract High pressure-induced inactivation of the indigenous milk enzymes alkaline phosphatase (ALP), γ -glutamyltransferase (GGT) and phosphohexoseisomerase (PHI) was studied in the pressure range 400–800 MPa at temperatures between 5 and 40 °C. With respect to pressure stability the following ranking was observed: ALP>GGT>PHI. PHI was inactivated after pressure treatment at 500 MPa and 20 °C for 10 min. In terms of reaction kinetics, inactivation of GGT followed first-order reaction kinetics in the range of 400–800 MPa whereas a reaction order of 1.5 was found for ALP. Reactivation of pressure-treated ALP was observed at low enzyme activity resulting from severe pressure treatment and 2 h storage at 35 °C. The influence of process temperature on the pressure-induced inactivation of GGT and ALP was limited in the range 5–40 °C.

NADH oxidation and superoxide production by caprine milk xanthine oxidoreductase

Abstract Xanthine oxidoreductase (XOR) was purified from caprine milk where more than 90% exists in demolybdo ‘inactive’ form. The dehydrogenase form of the enzyme, oxidises nicotinamide adenine dinucleotide (reduced) (NADH), in the presence of oxygen, and generates superoxide anion radical (O 2 − ) significantly faster than does the oxidase form. The corresponding forms of human and bovine milk enzymes behaved similarly. The steady-state kinetics of NADH oxidation and O 2 − production, in the absence and presence of NAD + , by the dehydrogenase form of XOR from the three species, are analysed. Allopurinol, oxypurinol and amflutizole blocked the reducing substrates that act at the molybdenum site, and all of which were ineffective in the case of NADH oxidase activity. However, and as expected, diphenyleneiodonium was a powerful inhibitor of NADH oxidation. The possible physiological and pathological significance of reactive oxygen species, especially O 2 − , arising from NADH oxidation by caprine XOR is discussed.

Y-700 [1-[3-Cyano-4-(2,2-dimethylpropoxy)phenyl]-1H-pyrazole-4-carboxylic acid]: a potent xanthine oxidoreductase inhibitor with hepatic excretion.

Y-700 (1-[3-Cyano-4-(2,2-dimethylpropoxy)phenyl]-1H-pyrazole-4-carboxylic acid) is a newly synthesized inhibitor of xanthine oxidoreductase (XOR). Steady-state kinetics with the bovine milk enzyme indicated a mixed type inhibition with K(i) and K(i) ' values of 0.6 and 3.2 nM, respectively. Titration experiments showed that Y-700 bound tightly both to the active sulfo-form and to the inactive desulfo-form of the enzyme with K(d) values of 0.9 and 2.8 nM, respectively. X-ray crystallographic analysis of the enzyme-inhibitor complex revealed that Y-700 closely interacts with the channel leading to the molybdenum-pterin active site but does not directly coordinate to the molybdenum ion. In oxonate-treated rats, orally administered Y-700 (1-10 mg/kg) dose dependently lowered plasma urate levels. At a dose of 10 mg/kg, the hypouricemic action of Y-700 was more potent and of longer duration than that of 4-hydroxypyrazolo(3,4-d)pyrimidine, whereas its action was approximately equivalent to that of 2-(3-cyano-4-isobutoxyphenyl)-4-methyl-5-thiazolecarboxylic acid, a nonpurine inhibitor of XOR. In normal rats, orally administered Y-700 (0.3-3 mg/kg) dose dependently reduced the urinary excretion of urate and allantoin, accompanied by an increase in the excretion of hypoxanthine and xanthine. Y-700 (1 mg/kg) was absorbed rapidly by the oral route with high bioavailability (84.1%). Y-700 was hardly excreted via the kidneys but was mainly cleared via the liver. These results suggest that Y-700 will be a promising candidate for the treatment of hyperuricemia and other diseases in which XOR may be involved.

상업적 응유효소의 탈지유에 대한 단백질 분해 작용

상업적 단백질 분해 효소에 0.02% <TEX>$CaCl_2$</TEX>를 첨가하여 응유 활성화를 시킨 탈지유에 대한 분해 작용의 결과를 요약하면 다음과 같다. 다양한 효소별 가수분해 시간에 따른 가수분해도는 미생물 유래 효소와 trypsin은 pepsin과 papain W-40보다 높은 분해도를 나타냈다. 12% TCA 용액에 가용성인 NPN의 양은 trypsin이 가장 높은 분해도를 나타내었고 rennet과 pepsin이 가장 낮은 분해도를 보였다. 전기영동에 있어서 trypsin과 protease S는 <TEX>$\alpha$</TEX>- lactalbumin을 분해하였고 papain w-40은 <TEX>$\beta$</TEX>- lactoglobulin을 미약하게 분해하였으며 neutrase 1.5는 90분 이후부터 <TEX>$\alpha$</TEX>-lactalbumin과 <TEX>$\beta$</TEX>-lactoglobulin을 분해하였다. Rennet과 비교한 전기영동상에서는 rennet에 의해 분해 되지 않은 <TEX>${\alpha}_s$</TEX>- casein과 <TEX>$\beta$</TEX>-casein을 trypsin과 protease S가 다량 분해하였고 <TEX>$\kappa$</TEX>-casein은 rennet에 비해 papain W-40이 상당 수준의 분해상을 나타내었다. 이상의 결과 가수분해도 및 NPN 양은 trypsin, neutrase 1.5 및 protease S가 다른 효소에 비해 높게 나타났으며, 전기영동상에서는 pepsin과 neutrase 1.5가 rennet과 유사한 경향을 나타내었다. Proteolytic activities of some commercial milk clotting enzymes(rennet, trypsin, pepsin, papain W-40, neutrase 1.5 and protease S) in bovine skim milk containing 0.02% <TEX>$CaCl_2$</TEX> were determined by measuring DH(Degree of Hydrolysis), NPN(Non Protein Nitrogen) and by comparing patterns of SDS-PAGE(Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis). The DH of microbial enzymes(neutrase 1.5 and protease S) and trypsin in bovine skim milk were higher than those of pepsin and papain W-40. The amounts of NPN in the milk treated with trypsin and the other animal enzymes(rennet and pepsin) showed the highest and lowest degrees of proteolysis, respectively. SDS-PAGE showed that trypsin and protease S hydrolyzed <TEX>$\alpha$</TEX>-lactalbumin and papain W-40 hydrolyzed <TEX>$\beta$</TEX>-lactoglobulin slightly, while neutrase 1.5 hydrolyzed both <TEX>$\alpha$</TEX>-lactalbumin and <TEX>$\beta$</TEX>-lactoglobulin after treating for 90 min. Trypsin and protease S easily hydrolyzed <TEX>${\alpha}_s$</TEX>-casein and <TEX>$\beta$</TEX>-casein, which were not hydrolyzed by rennet. Papain W-40 hydrolyzed <TEX>$\kappa$</TEX>-casein more than rennet as shown in SDS-PAGE. Based on the results of the experiments, the DH and NPN of trypsin, neutrase 1.5 and protease S were shown to be higher than those of the other enzymes. The SDS-PAGE patterns of papain W-40 and neutrase 1.5 were similar with that of rennet.

Lactoperoxidase: physico-chemical properties, occurrence, mechanism of action and applications.

Lactoperoxidase (LP) is one of the most prominent enzymes in bovine milk and catalyses the inactivation of a wide range of micro-organisms in the lactoperoxidase system (LP-s). LP-systems are also identified as natural antimicrobial systems in human secretions such as saliva, tear-fluid and milk and are found to be harmless to mammalian cells. The detailed molecular structure of LP is identified and the major products generated by the LP-s and their antimicrobial action have been elucidated for the greater part. In this paper several aspects of bovine LP and LP-s are discussed, including physico-chemical properties, occurrence in milk and colostrum and mechanisms of action. Since the introduction of industrial processes for the isolation of LP from milk and whey the interest in this enzyme has increased considerably and attention will be paid to potential and actual applications of LP-systems as biopreservatives in food and other products.

Predictive modelling of inactivation of bovine milk α-l-fucosidase in a high-temperature short-time pasteurizer

Abstract In response to recent concerns regarding the persistence of food-borne pathogens in cheese and the need to ensure that milk destined for cheesemaking has received the minimal heat-treatment (thermization; 63–65°C for 16 s), the aims of the present study were to examine the bovine milk enzyme α- l -fucosidase (FC) as a possible indicator of thermization and to assess the use of a reflectance colorimetric procedure for the enzyme assay. Standard curves in whole and skim milk were prepared to convert the b ∗ colour units used in reflectance colorimetry to enzyme activity units. The assay procedure was sensitive enough to detect 7.5% raw milk mixed with heated milk and showed that the levels of FC activity in whole and skim milk were not significantly different (P>0.05). The enzyme had a broad pH optimum from 5.0 to 6.0, with a Km of 0.6 m m , and a Vmax of 61.6 nmol mL−1 h−1. Thermal inactivation of FC, determined using a plate heat exchanger, was linear with temperature using holding tubes of nominal times of 3–60 s. The integrated lethal effect was determined in four independent pasteurizer trials conducted at intervals of approximately 3 weeks and a linear model for FC was developed. This model was used with the inter-trial variability and @RISK™ risk analysis simulation software to demonstrate that a log10 reduction in FC activity of 1.2 can be achieved using conditions corresponding to thermization (65°C for 16 s) with 95% confidence. These results suggest that FC can be used as an indicator of heat-treatment or thermization of milk destined for cheesemaking.

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

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

Sensitive enzyme linked immunosorbent assay (ELISA) for xanthine oxidase

The enzyme, xanthine oxidase, has been implicated as a major source of destructive free radicals in ischemia-reperfusion injury associated with a range of pathological states [l]. Enzymic activity has been detected in many mammalian tissues [4] by using, variously, spectrophotometric [51, radiometric[6] and fluorimetric [71 assays. Apart from bovine milk fat globule membrane, which has long provided the major source of purified enzyme [a], liver and intestine generally show the highest levels of activity [4]. This is also true for humans, for whom enzyme activity in other tissues is barely detectable by conventional spectrophotometric assay [9]. Particular attention has, in recent years, has been focussed on human heart, in which the majority of workers have failed to detect significant enzyme activity [lo]. This failure contrasts with the immunofluorescence microscopy studies of Jarasch and co-workers [9] who clearly detected xanthine oxidase in capillary endothelial cells of human heart in common with such endothelial cells in many other bovine and human tissues. We have recently described the purification of xanthine oxidase from human milk [ll]. The human enzyme, surprisingly, shows specific xanthine oxidase activity that is only about 1% that of bovine milk enzyme, an observation that could reflect a predominance of 'inactive', possibly 'desulpho' form in human milk [ll]. We have also succeeded in isolating xanthine oxidase from human heart [11,12]. This enzyme too has very low activity toward conventional substrates which could explain previous failures to detect it [lo]. These wide variations in conventional xanthine oxidase activity, particularly of the human enzyme, point out a need for an immunoassay for detection of xanthine oxidase in tissue. The basis for such an assay is now described. Rabbits were immunised with bovine milk xanthine oxidase (Biozyme Laboratories, Blaenavon, UK, 1OOpg) and the resulting antiserum [13] was affinity purified on a column of bovine xanthine oxidase (Biozyme) coupled to CNBr-activated Sepharose 4B (Sigma Chemical Co., Poole, U.K.). A portion of the resulting polyclonal anti(bovine xanthine oxidase) antibodies were biotinylated as described by Kemeny 1141, for use, together with non-biotinylated antibodies, in the ELISA described below. Non-biotinylated, affinity-purified antibodies [lOOpl, lpl/ml in coating buffer (5OmM carbonate-bicarbonate buffer, pH 9.6, containing 0.01% thiomersal)] were added to each well of a 96-well microtitre plate (Becton Dickinson Labware, Plymouth, UK) and allowed to stand overnight at 4°C. The wells were then washed with phosphate buffered saline containing 0.1% Tween 20 (PBS-T) and incubated with 1% (w/v) casein in PBS-T (PBS-T/casein) for lh at room temperature. Serial two-fold dilutions of a standard solution of bovine xanthine oxidase (Biozyme, 5pg/ml in PBS-T/casein) were added to successive wells and allowed to stand for 2h at room temperature before washing with PBS-T/casein and incubation with biotinylated affinity-purified antibodies (lOOpl, 0.7Fg/ml in PBS-T/casein) f o r 2h at room temperature. The wells were again washed with PBS-T/casein and streptavidinhorse radish peroxidase (Sigma, 100~1, 0.25~g/ml in PBS-T/casein) was added to each well. The plates were allowed to stand overnight at 4C, washed extensively with PBS and incubated with peroxidase substrate (1OOp1) [substrate solution comprises tetramethylbenzidine (Sigma) in dimethylsulphoxide (10mg/ml, 0.25ml) in 0.1M sodium acetate buffer, pH 6.0, (24.75ml) containing 30% hydrogen peroxide (5pl)I. Colour was allowed to develop before stopping the reaction by addition of 1.84M sulphuric acid and measuring A,,, in a Multiscan MCC plate reader (Labsystems, Basingstoke, UK) . A plot of h9, against dilution of stock xanthine oxidase solution gave a standard curve, use of which allowed levels of enzyme as low as 0.5ng/ml consistently to be detected . We thank the Medical Research Council for a studentship to LJP.

Study of the functional domains of β1-4Galactosyl transferase through the expression of the deletion constructs of cDNA in &lt;italic&gt;E. coli&lt;/italic&gt; and mammalian cells: disulfide bond between CYS134 and CYS247 is required for folding and enzyme activity while the transmembrane domain is essential for stable expression in mammalian cells.

β1,4Galactosyltransferase (β1,4GT) belongs to the family of glyeosyltransferases and transfers galactose from UDP-galactose to N-acetylglucosamine to produce N-acetyllactosamine with a β1-4 linkage[1]. Bovine β1,4GT is a 402 residue long protein, which has a general topology similar to other glycosyltransferases and type II transmembrane proteins that consists of a short amino-terminal cytoplasmic tail. a signal anchor domain, a stem region and a carboxyl-terminal catalytic domain. It has been localized in the trans-cisternae of the Golgi apparatus and has also been detected on the plasma membrane of several cell types. To delineate the function of various regions of bovine β1 AGT, we prepared cDNA constructs coding for the 402 residue long protein and its amino-terminal deleted forms in pGEX-2T vector, and expressed them in E. coli as glutathione-S-transferase (GST) fusion proteins. Recombinant proteins were localized in inclusion bodies which were solubilized in 5 M guanidine-HC 1. Renaturation and regeneration of the enzyme activity from the solubilized protein was strictly dependent on the presence of an “oxido-shuffling” reagent, a mixture of 8:1 mM reduced:oxidized glutathione. Renaturedireoxidized fusion proteins, purified on glutathione-affinity columns, were active in β1 AGT and lactose synthetase (LS) assays. After thrombin cleavage of the fusion protein and subsequent removal of the GST domain, the recombinant fj1 AGT has a specific activity of 50-70% as compared to that of bovine milk enzyme. The Km of the recombinant protein for N-acetylglucosamine and UDP-galactose is similar to that of bovine milk β1 AGT. Deletion analyses show that even after removal of the first 130 residues, both β1 AGT and LS activities remain intact.

Purification and partial characterization of xanthine oxidase from human milk

Xanthine oxidase was purified from human milk in yields comparable with those obtained from bovine milk. The freshly purified enzyme appeared homogenous in gel permeation FPLC and SDS-PAGE, consistent with its being a homodimer with total M r290 000 ± 6000. The ultraviolet/visible absorption spectrum differed only by slightly from that of bovine milk enzyme and showed an A 280/ A 450 ratio of 5.13 ± 0.29, indicating a high degree of purity. Xanthine oxidase activities of purified enzyme varied with batches of milk, ranging between 3 and 46 mU/mg protein; values that are some two to three orders of magnitude smaller than those shown by the most highly purified samples of bovine milk enzyme. Direct comparison with commercially-available bovine milk enzyme showed that activities involving xanthine as reducing were 1–6% that of the bovine enzyme, whereas those involving NADH, in contrast, were of the same order for the two enzymes. Anerobic bleaching experiments indicated that less than 2% of the human enzyme was present as a form active with xanthine. These findings, together with the activity data, are consistent with a very high content, possibly greater than 98%, of demolybdo- and/or desulpho-forms of human enzyme, both of which occur, to a lesser extent, in bovine xanthine oxidase. Molybdenum assay indicated that demolybdo-enzyme could only account for some 26% of this indicated component, suggesting that desulpho-enzyme may account for the remainder.

Effects of allopurinol on endotoxin-induced increase in serum xanthine oxidase in the horse

Using a modified bovine milk enzyme kinetic assay, xanthine oxidase activity of serum collected from 34 adult, healthy horses of both sexes was determined. Enzyme activity varied from 0 to 126 mU litre-1 with a mean of 44.95 +/- 21.05 mU litre-1. The optimal pH and temperature for maximal activity were 7.8 and 28 degrees C, respectively. Freezing the serum for four days at -70 degrees C did not destroy the enzyme activity. Various doses (25, 50 and 75 micrograms kg-1, intraperitoneally) of endotoxin (lipopolysaccharide D1 Escherichia coli O26:B6) previously known to have caused moderate to severe systemic clinical signs of endotoxaemia in horses produced a significant dose related increase in serum xanthine oxidase activity. Pretreatment (12 hours) with allopurinol (5 and 50 mg kg-1, intravenously [corrected]) significantly reduced the rise in xanthine oxidase activity in endotoxin (50 micrograms kg-1, intraperitoneally) treated horses. The results of this study suggest that xanthine oxidase catalysed production of superoxide radicals may play a role in the pathogenesis of endotoxaemia and that allopurinol, an alternate substrate, should be further evaluated for its therapeutic potential in endotoxin related systemic diseases in horses.

Purification, properties and cation activation of galactosyltransferase from lactating-rat mammary Golgi membranes.

Galactosyltransferase was purified from Golgi membranes of lactating-rat mammary gland and studied with respect to its physical and enzymic (lactose synthetase) properties. The enzyme occurred in both monomeric (43-46 kDa) and apparently dimeric (90 kDa) forms. It was very unstable except in the presence of phospholipid, detergent, or cations binding to site 2. The amino acid composition and the N-terminal sequence closely resembled that of the human and bovine milk enzymes, particularly in respect to a Pro-Pro-Pro-Pro sequence. Kinetic studies demonstrated a high-affinity Mn2+-binding site (1) essential for activity, and a low-affinity Mn2+-binding site (2) that could also bind spermidine or clupeine. Mn2+ binding at site 2 raised Vmax fivefold. Spermidine binding at site 2 enhanced Mn2+ binding at site 1, and influenced binding of glucose. At physiological glucose concentration, clupeine or spermidine activated nearly as well as 15 mM MnCl2 and are regarded as models of a natural cation activator that remains to be isolated. Evidence is given for an essential histidine residue in the galactosyltransferase. It is proposed that site 1 Mn2+ participates directly in the reaction mechanism, whereas site 2 is a regulator site allosterically activated by a basic protein.

A study of the heat stabilities of a number of indigenous milk enzymes

SummaryHeat stability profiles of a number of indigenous bovine milk enzymes were examined with the object of being able to monitor heat treatments slightly more severe than typical pasteurization conditions by measurements of residual enzyme activity after heating. Assay procedures were limited to simple fluorimetric, or preferably colorimetric, methods that would be most likely to form the basis of a quick, simple and inexpensive test. Both lipoprotein lipase and α-fucosidase were relatively sensitive to heat and were totally inactivated at temperature/time combinations below those of pasteurization, but the latter may be satisfactory for studying temperatures in the range 55–65°C. Rather more heat stable wereN-acetyl-β-glucosaminidase and γ-glutamyl transpeptidase, which may be most appropriate for 65–75°C and 70–80°C respectively. Higher temperature treatments between 80 and 90°C could best be investigated by following α-mannosidase or xanthine oxidase activity.

Immunological similarity of milk sulfhydryl oxidase and kidney glutathione oxidase

A radioimmunoassay for sulfhydryl oxidase, a membrane enzyme, was developed using antibodies raised to the bovine milk enzyme which had been purified by transient covalent affinity chromatography on a cysteinylsuccinamidopropyl-glass matrix. Bovine milk sulfhydryl oxidase and bovine kidney sulfhydryl oxidase (“glutathione oxidase”) appear to be immunologically identical as evidenced by parallel responses in radioimmunoassays. Antibodies raised to the purified milk sulfhydryl oxidase can immunoprecipitate glutathione oxidase activity, but not γ-glutamyltransferase (“transpeptidase”) activity, from bovine kidney preparations.

Proteinases in normal bovine milk and their action on caseins.

Native proteolytic enzymes in good quality normal bovine milk readily hydrolysed the caseins during incubation or storage, producing the gamma-caseins, proteose-peptone components 5 (PP5) and 8-fast (PP8F) and a considerable number of other unidentified fragments, many of which were also subsequently found in the proteose-peptone fraction. The rate of casein hydrolysis was greater in pasteurized than in raw milk, with beta-casein being slightly more susceptible to attack than alpha S1-casein. Measurements of gamma-casein and proteose-peptone formation have been made and it was found that PP5 was an intermediate product that was subject to further proteolysis while PP8F was a stable end-product. With the exception of component 3 (PP3), virtually all constituents of the proteose-peptone fraction increased during storage and appeared to be products of the action of proteolytic enzymes. Further evidence was obtained from the effects of various inhibitors that the principal proteinase of normal milk is plasmin, but slight differences were apparent between the protein breakdown patterns induced by storage and by added plasmin, which was consistent with the presence of more than one proteinase. Incubations in the presence of soya bean trypsin inhibitor to prevent plasmin action clearly revealed that another enzyme(s) was also involved.