Studies on a trace cell lytic activity associated with alpha-lactalbumin

alpha-Lactalbumin (alpha-LA) has been examined with a new and sensitive method for determination of lysozyme activity. Samples of bovine, human, equine, and rat alpha-LA exhibited cell lytic activity, from 2 X 10(-6) to 45 X 10(-6) of the specific activity of hen eggwhite lysozyme. The activity was chromatographically inseparable from bovine and human alpha-LA. Bovine serum albumin and purified beta-lactoglobulin were inactive. The pH profiles and reaction kinetics of bovine and human alpha-LA showed differences from those of the corresponding milk lysozymes, indicating that their lytic activities were not likely to have resulted from trace lysozyme content. Thus, it appears that a weak cell lytic activity is inherent to alpha-LA.     

An improved method of photometric determination of cyclodextrin glucanotransferase activity

The method of spectrophotometric determination of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) activity with the use of phenolphthalein as a colored reagent has been improved. This technique includes an enzymatic reaction at 40 degrees C for 60 min in 2% starch, with subsequent supplementation of the reaction mixture (0.5 ml) with the phenolphthalein reagent (3.0 ml) prepared in 0.1 M potassium carbonate buffer (pH 11.0) according to a special procedure, and measurement of the optical density of the obtained mixture at 553 nm. The activity was calculated using the exponential growth equation that connects a drop in the optical density and the degree of dilution of the enzyme. The described technique is suitable for working in a sufficiently broad range of specific activity of beta-CGTase and does not require precise adjustment of the degree of dilution of solutions analyzed.     

An improved method of photometric determination of cyclodextrin glucanotransferase activity

The method of spectrophotometric determination of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) activity with the use of phenolphthalein as a colored reagent has been improved. This technique includes an enzymatic reaction at 40°C for 60 min in 2% starch, with subsequent supplementation of the reaction mixture (0.5 ml) with the phenolphthalein reagent (3.0 ml) prepared in 0.1 M potassium carbonate buffer (pH 11.0) according to a special procedure, and measurement of the optical density of the obtained mixture at 553 nm. The activity was calculated using the exponential growth equation that connects a drop in the optical density and the degree of dilution of the enzyme. The described technique is suitable for working in a sufficiently broad range of specific activity of β-CGTase and does not require precise adjustment of the degree of dilution of solutions analyzed.     

Isolation of spheroplasts from Escherichia coli 85 for aspartate-ammonia-lyase localization

Conditions were determined for preparation of spheroplasts from E. coli under the action of lysozyme in the presence of EDTA. The preparation took from 10 to 15 min. The degree of conversion to spheroplasts was monitored spectrophotometrically at 660 nm. The spheroplasts formed were unstable in Tris-HCl buffer and immediately lysed, but they were more stable in 1 M sucrose. At lysozyme concentrations above 40 micrograms/ml of the reaction mixture, the cells lysed to a greater extent. The distribution of aspartate ammonia-lyase activity between the precipitate of the spheroplasts and the supernatant allowed the authors to suggest that aspartase should be located in the cytoplasm.     

3'-phosphoadenylylsulfate:galactosylceramide 3'-sulfotransferase. An optimized assay in homogenates of developing brain.

An optimized in vitro assay of 3'-phosphoadenylysulfate:galactosylceramide 3'-sulfotransferase (EC 2.8.2.11, galactosylceramide sulfotransferase, formerly known as galactocerebroside sulfotransferase) activity is presented, that can be used in crude homogenate of brain tissue of various developmental stages. The enzyme activity is determined by measuring the [35S]sulfatides formed by the enzymic transfer of [35S]sulfate from 3'-phosphoadenoside 5'-phospho [35S]sulfate to galactosylceramides. The sulfatide formation at 30 degrees C is linear up to 30 min and up to a protein concentration of 1 mg per 0.5 ml assay volume. The presence of 0.4% Triton X-100 and 50 micrometer exogenous bovine cerebrosides are optimal for enzyme activity. The pH optimum of the reaction is at pH 6.5 using 0.1 M imidazole buffer. The enzyme reaction is stimulated by NaCl, KCl, MgCl2, CaCl2, MnCl2, ATP and inhibited by ADP. The developmental enzyme activity pattern of mouse brain is the same, if derived from homogenates and microsomes, respectively, under our assay conditions.     

Quantitative recovery, selective removal and one-step purification of human parotid and leukemic lysozymes by immunoadsorption

Immunoadsorption affinity chromatography was used to isolate and purify human lysozyme. The immunoadsorbent was prepared by coupling sheep anti-(human leukemic lysozyme) IgG to epoxy-activated Sepharose 6B. Lyophilized parotid saliva (21) was resuspended in distilled water (325 ml, 50 mg/ml, w/v) and applied to a column which had a capacity to bind 4.25 mg human enzyme. Non-adsorbed material did not contain lysozyme, as determined by enzymatic and immunological analyses. All lysozyme activity present in the applied sample (1.97 mg) bound to and was desorbed from the column by elution with 0.2 M sodium acetate HCl buffer, pH 1.8. The isolated material was homogeneous as determined by cationic and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, ultracentrifugation, amino acid and amino-terminal analyses, and immunoelectrophoretic analysis. The one-step purification procedure yielded a 1370-fold increase in specific activity. Human lysozyme was also selectively purified by this method from an ammonium sulfate precipitate of the urine of a patient with chronic monocytic leukemia. Amino acid and polyacrylamide gel electrophoretic analyses indicated that the purified enzyme was identical to human lysozyme isolated from leukemic urine by classical biochemical techniques.     

Studies on a partially purified bovine milk lysozyme

Bovine milk lysozyme has been partially purified by a method developed in this laboratory. We have shown, by preliminary sequential analysis, and by gel filtration on HPLC, that the product is a mixture of two components. One of these, the enzymically active one, differs in its N-terminal sequence from that of "lysozyme 2", a bovine stomach mucosal enzyme, by 7 residues within the first 39 residues. However, some of its properties differ markedly from those of lysozyme 2. The other component, comprising 70% by weight of the total mixture, bears no sequential resemblance to any protein known to us. Our two component system appears to be the same as the preparation of Chandan et al. (Biochim. Biophys. Acta 110, 289 (1965], which they concluded was an homogeneous preparation of lysozyme.     

Fluorometric determination of isocitrate dehydrogenase (EC 1.1.1.42; 1; NADP+ dependent) in ruminant milk

The enzyme isocitrate dehydrogenase (EC 1.1.1.42; 1; NADP⁺ dependent) located in the mammary cell cytosol mediates the synthesis of the majority of reducing equivalents for the energetically demanding milk fat and cholesterol synthesis in mammary cell cytosol. The present article presents a novel fluorometric method for quantification of the activity of this enzyme (IDH) in ruminant milk without pretreatment of the sample. Further, 493 goat milk samples – harvested before, during and after a nutritional restriction – were analysed for IDH activity i) with addition of extra substrate (isocitrate), and ii) with the intrinsic isocitrate solely. The IDH activity ranged from 0.22 to 15.4 units [nano moles product/(ml * min)] (un-supplemented) and from 0.22 to 45.6 units (isocitrate supplemented). The IDH activity increased considerably in milk during the nutritional restriction period concomitant with the increase in the metabolite isocitrate concentration and somatic cell count and returned to the initial level shortly after restriction period. The present ‘high through-put’ analytical method may be beneficial in future studies to phenotype modifications in mammary energy metabolism and milk fat synthesis, for which IDH activity may be a biomarker.     

A kinetic study of the competition between renaturation and aggregation during the refolding of denatured-reduced egg white lysozyme

The recovery of proteins following denaturation is optimal at low protein concentrations. The decrease in yield at high concentrations has been explained by the kinetic competition of folding and "wrong aggregation". In the present study, the renaturation-reoxidation of hen and turkey egg white lysozyme was used as a model system to analyze the committed step in aggregate formation. The yield of renatured protein for both enzymes decreased with increasing concentration in the folding process. In addition, the yield decreased with increasing concentrations of the enzyme in the denatured state (i.e., prior to its dilution in the renaturation buffer). The kinetics of renaturation of turkey lysozyme were shown to be very similar to those of hen lysozyme, with a half-time of about 4.5 min at 20 degrees C. The rate of formation of molecular species that lead to formation of aggregates (and therefore fail to renature) was shown to be rapid. Most of the reaction occurred in less than 5 s after the transfer to renaturation buffer, and after 1 min, the reaction was essentially completed. Yet, by observing the effects of the delayed addition of denatured hen lysozyme to refolding turkey lysozyme, it was shown that folding intermediates become resistant to aggregation only much more slowly, with kinetics indistinguishable from those observed for the appearance of native molecules. The interactions leading to the formation of aggregates were nonspecific and do not involve disulfide bonds. These observations are discussed in terms of possible kinetic and structural aspects of the folding pathway.     

Enzyme-Linked Immunosorbent Assay of Ampicillin in Milk

An indirect immunoassay for quantitative determination of ampicillin (range, 10–1000 ng/ml) in buffer or milk has been developed. Polyclonal antibodies were obtained against ampicillin conjugated with bovine serum albumin; the conjugate was synthesized by direct condensation using carbodiimide. The antibodies were specific for ampicillin and exhibited low cross-reactivity to other penicillins (azlocillin, 17%; penicillin G, 10%; piperacillin, 5%; and carbenicillin, 4%). Matrix effects were minimized by combining the use of a casein-supplemented buffer (content of casein, 1%) with sample dilution. Limit of detection for ampicillin in milk (diluted tenfold) was equal to 5.0 ng/ml (which corresponded to 50 ng/ml of the original sample).     

Enzyme immunoassay of ampicillin in milk

An indirect immunoassay for quantitative determination of ampicillin (range, 10-1000 ng/ml) in buffer or milk has been developed. Polyclonal antibodies were obtained against ampicillin conjugated with bovine serum albumin; the conjugate was synthesized by direct condensation using carbodiimide. The antibodies were specific for ampicillin and exhibited low cross-reactivity to other penicillins (azlocillin, 17%; penicillin G, 10%; piperacillin, 5%; and carbenicillin, 4%). Matrix effects were minimized by combining the use of a casein-supplemented buffer (content of casein, 1%) with sample dilution. The threshold of ampicillin detection in milk (diluted tenfold) was equal to 5.0 ng/ml (which corresponded to 50 ng/ml of the original sample).     

Effect of beta-lactoglobulin on the activity of pregastric lipase. A possible role for this protein in ruminant milk.

The interaction of bovine beta-lactoglobulin with palmitic and oleic acids has been studied by a partition equilibrium method. Bovine beta-lactoglobulin displays only one high affinity binding site for fatty acids whose association constants for palmitic and oleic acids are 4.2 x 10(6) and 2.3 x 10(6) M-1, respectively. However, other binding sites with low affinity are also present. The existence of one high affinity binding site is in accordance with the amount of fatty acids naturally bound to beta-lactoglobulin isolated from milk. The effect of beta-lactoglobulin on ruminant pregastric lipases from a pharyngeal extract has been assayed. The activity of pharyngeal lipase on a triglyceride emulsion is increased about 200%, 250% and 190% in the presence of 10 mg/ml, 20 mg/ml and 40 mg/ml of beta-lactoglobulin, respectively, the last concentration representing that found physiologically in colostrum. Albumin, another ligand-binding protein, increases the activity of this enzyme to a lesser extent and high levels tend to inhibit enzyme action. These results indicate that beta-lactoglobulin could participate in the digestion of milk lipids during the neonatal period by enhancing the activity of pregastric lipase through removal of the fatty acids that inhibit this enzyme.     

Rabbit muscle phosphofructokinase: the effect of the state of the enzyme and assay procedure on the kinetic properties.

The specific activity and/or the allosteric behavior of rabbit muscle phosphofructokinase is dependent on several factors including (1) the method of assay, and (2) the concentration, pH, and temperature of the enzyme solution from which dilution is made into the assay mixture. These observations suggest that quantitative interpretation of the allosteric characteristics of this enzyme may be in error because of lack of control of any or all of these factors. In some buffer systems, such as imidazole at pH 7, instability of the enzyme in the assay leads to further complications in the interpretation of previous studies.The results of the present paper show that under specific conditions it is possible to obtain allosteric kinetic data from which quantitative interpretations can be made. This is best accomplished by performing experiments in phosphate buffer and coupling the reaction through pyruvate kinase and lactic dehydrogenase. The experiments must be carried out either in the presence of excess fructose 1,6-bisphosphate or fructose diphosphatase in order to control the level of the activator fructose 1,6-bisphosphate. Under such conditions, the allosteric kinetic behavior observed at pH 6.5 does not appear to be a consequence of polymerization between an active (four subunit) and inactive (two subunit) form of the enzyme, but is inherent in the active form.     

Sensitization of heat-treated Listeria monocytogenes to added lysozyme in milk.

Listeria monocytogenes was highly resistant to hen egg white lysozyme in whole milk but was sensitive in media and in phosphate buffer. Methods to sensitize the pathogen to lysozyme in milk were investigated. Treatment of whole milk by cation exchange to remove minerals, particularly Ca2+ and Mg2+, slightly promoted inactivation of L. monocytogenes by lysozyme at 4 degrees C over a period of 6 days. Heat treatment (62.5 degrees C for 15 s) strongly sensitized L. monocytogenes to lysozyme in demineralized milk and in MES [2-(N-morpholino)ethanesulfonic acid] buffer. Addition of Ca2+ or Mg2+ to the demineralized milk restored resistance to lysozyme. Cells were more rapidly heat inactivated at 55 degrees C in demineralized milk containing lysozyme, and addition of Ca2+ to the demineralized milk restored the resistance to heat. The results indicate that minerals or mineral-associated components protect L. monocytogenes from inactivation by lysozyme and heat in milk, probably by increasing cell surface stability. The heat treatment of foods containing added lysozyme can probably play a significant role in producing microbiologically safe foods.     

Kinetic characterization of rat liver nuclear lysozyme

The kinetic properties of rat liver nuclear lysozyme, earlier purified to homogeneity in our laboratory, have been studied. The enzyme was found to be maximally active in the pH range 4.2 to 5.4 in 0.02 M buffer. Its Km was found to be 333 mg/litre. It was heat sensitive even in the acidic pH range. The enzyme exhibited tissue specific differences when compared with the rat kidney nuclear lysozyme.     

A simple and ultrasensitive enzymatic assay for the quantitative determination of lysozyme in the picogram range

A simple method for the ultrasensitive quantitation of lysozyme has been developed. The enzyme's lytic activity against Micrococcus lysodeikticus is measured spectrophotometrically after an 18-h incubation period. The method is capable of quantitating lysozyme at concentrations as low as 5 pg/ml, and is applicable to determinations of the enzyme in complex biological mixtures.     

High-performance liquid chromatography method for the simultaneous determination of sulfamethoxazole and trimethoprim in bovine milk using an on-line clean-up column

A bidimensional HPLC method for the simultaneous determination of sulfamethoxazole (SMX) and trimethoprim (TMP) in bovine milk has been developed and validated. After centrifugation, aliquots (150 microl) of milk samples were directly injected to a column-switching HPLC system. At the first step a RAM octyl-BSA column was employed to automatically remove proteins that otherwise would interfere with milk analysis. The mobile phase 0.01 M phosphate buffer pH 6.0:acetonitrile (95:5, v/v) was used in the first 5 min for the elution of milk proteins and then 0.01 M phosphate buffer pH 6.0:acetonitrile (83:17, v/v) for transfer SMX and TMP to the analytical column. The separation of SMX and TMP from one another and from other remaining milk components was performed on an octyl column using the mobile phase 0.01 M phosphate buffer pH 5.0:acetonitrile (82:18, v/v), which were detected by UV at 265 nm. The calibration graphs were linear in the concentration ranges of 25-800 ng/ml and 50-400 ng/ml for SMX and TMP, respectively. The intra- and inter-assay coefficients of variation were less than 15% for both drugs. The validated method was applied to the analysis of milk samples of twelve (two groups of six) cows after administration (intramuscular or subcutaneous) of a single recommended therapeutic dose of the SMX-TMP combination.     

In vitro formation of retinoic acid from retinal in rat liver.

Enzymatic conversion of retinal to retinoic acid in rat liver cytosol was detected using a rapid and sensitive assay based on high pressure liquid chromatography (HPLC). This retinal oxidase assay system did not require extraction steps or any other manipulation of the sample mixture once the sample vial was sealed for incubation. The product (retinoic acid) and the reactant (retinal) were separated by HPLC in 14.0 min with a sensitivity of 15 and 40 pmol per injection for retinoic acid and retinal, respectively. Enzymatic activity was observed to be linear with protein concentration (0-2.4 mg/mL) and time (0-30 min) and displayed a broad pH maximum of 7.7-9.7. The enzyme exhibited Michaelis-Menten single-substrate kinetics with an apparent Km of 0.25 mM. The average specific activity in nine normal rats was 35.6 +/- 3.3 nmol retinoic acid formed/h per mg protein. Incubation of the enzyme with zinc did not affect the rate of retinoic acid synthesis. Dithiothreitol inhibited the reaction. Both NAD and NADH stimulated retinoic acid formation. Formation of retinol was also observed when these pyridine nucleotides were added to the reaction mixture, indicating the presence of retinal reductase activity. The results of kinetic studies suggest that NADH may act indirectly to stimulate retinoic acid formation.     

The isolation and amino acid sequences of echidna (Tachyglossus aculeatus) milk lysozyme I and II.

Comparative studies of monotreme proteins are of particular value in gaining an understanding of the origin of mammals and their interrelationships. The presence of two lysozyme variants, echidna lysozyme I and II, has been confirmed in mature milk samples of Tachyglossus aculeatus multiaculeatus and Tachyglossus aculeatus aculeatus respectively. A simplified procedure is described for their isolation. Their amino acid sequences, the first determined for a monotreme secretory protein, are unusual. They are shown to be c-type lysozymes, each consisting of a single chain of 125 residues (terminating at Cys 125). The only other known c-type lysozyme with this termination is that of pigeon eggwhite. Echidna lysozyme is unique in having no Cys at position 6, but at position 9. It has precisely the residues relevant to the binding of Ca(II), and most of the residues implicated in the galactosyl transferase modifier action of alpha-lactalbumin. However, the weak modifier action previously observed for variant I, prepared by a different method, was not found for the present preparation. The evolutionary significance of the results is discussed.     

Kinetic modeling of lactose hydrolysis with an immobilized beta-galactosidase from Kluyveromyces fragilis

The kinetic model of the hydrolysis of lactose with a beta-galactosidase from Kluyveromyces fragilis immobilized on a commercial silica-alumina (KA-3, from Südchemie) has been determined. A wide experimental range of the main variables has been employed: temperature, concentrations of substrate, and products and concentration of enzyme. The runs were performed in a complex buffer with the salt composition of milk. The effect of pH and temperature on the stability and the activity of the enzyme have been studied. The optimum pH for the enzyme activity was, approximately, seven. The immobilized enzyme was more stable than the free one at acidic pH, but more instable at basic pH. The maximum temperature used for the hydrolysis runs performed to select the kinetic model was 40 degrees C, so inactivation of the enzyme during the kinetic runs has been avoided. Agitation, concentration of enzyme in the solid and particle size were selected to ensure that the overall rate was that of the chemical reaction. Eleven kinetic models were proposed to fit experimental data, from first order to more complex ones, such as those taking into account inhibition by one of the compounds involved in the hydrolysis reaction. Applying statistical and physical criteria, a Michaelis-Menten model with a competitive inhibition by galactose has been selected. The model is able to fit the experimental data correctly in the wide experimental range studied. Finally, the model obtained is compared to the one selected in a previous work for the hydrolysis of lactose with the free enzyme.