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Biomedical subjects

P L McSweeney

Publications and source records attributed to P L McSweeney.

10 recordsLinked to original sources
PubMed2001

Thermal inactivation kinetics of bovine cathepsin D.

Cathepsin D, the principal indigenous acid proteinase in bovine milk, is a lysosomal proteinase, which exists in milk in four forms, including the inactive zymogen procathepsin D. The thermal inactivation kinetics of bovine cathepsin D, isolated from spleen and milk, were studied under isothermal conditions, using a specific HPLC assay to determine residual activity. Inactivation of the blood enzyme preparation followed first order kinetics, with z-values in phosphate buffer (pH 6.7) and skimmed milk of 6.5 and 7.6 degrees C, respectively, the enzyme being far more stable in the latter environment. Inactivation kinetics of the enzyme purified from milk were more complex, and could be best approximated by a double exponential model. Again, stability was higher in milk than in buffer. The double exponential model may indicate differing heat stabilities of isoforms of the enzyme, or stabilization of the enzyme by some milk constituent. It is clear that the enzyme can survive, at least partially, processes such as heating at 55 degrees C for 30 min during manufacture of high-cook cheese varieties (45% survival), and HTST pasteurization (8% survival), and thus may contribute to proteolysis in a range of dairy products.

Animals↗
PubMed2001

Solution structures of casein peptides: NMR, FTIR, CD, and molecular modeling studies of alphas1-casein, 1-23.

To determine its potential for interacting with other components of the casein micelle, the N-terminal section of bovine alphas1-casein-B, residues 1-23, was investigated with nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies, and molecular modeling. NMR data were not consistent with conventional alpha-helical or beta-sheet structures, but changes in N-H proton chemical shifts suggested thermostable structures. Both CD and FTIR predicted a range of secondary structures for the peptide (30-40% turns, 25-30% extended) that were highly stable from 5 degrees C to 25 degrees C. Other conformational elements, such as loops and polyproline II helix, were indicated by FTIR only. Molecular dynamics simulation of the peptide predicted 32% turns and 27% extended, in agreement with FTIR and CD predictions and consistent with NMR data. This information is interpreted in accord with recent spectroscopic evidence regarding the nature of unordered conformations, leading to a possible role of alphas1-casein (1-23) in facilitating casein-casein interactions.

Caseins↗
PubMed2001

Amino acid catabolism in cheese-related bacteria: selection and study of the effects of pH, temperature and NaCl by quadratic response surface methodology.

AIMS: To screen the cystathionine lyase and L-methionine aminotransferase activities of cheese-related bacteria (lactococci, non-starter lactobacilli and smear bacteria) and to determine the individual and interactive effects of temperature, pH and NaCl concentration on selected enzyme activities. METHODS AND RESULTS: A subcellular fractionation protocol and specific enzyme assays were used, and a quadratic response surface methodology was applied. The majority of the strains, 21 of 33, had detectable cystathionine lyase activity which differed in the specificity. Aminotransferase activity on L-methionine was observed in only three strains. The cystathionine lyase activities of Lactobacillus reuteri DSM20016, Lactococcus lactis subsp. cremoris MG1363, Brevibacterium linens 10 and Corynebacterium ammoniagenes 8 and the L-methionine aminotransferase activity of Lact. reuteri DSM20016 had temperature and pH optima of 30-45 degrees C, and 7.5-8.0, respectively. As shown by the quadratic response surface methodology these enzymes retained activities in the range of temperature, pH and NaCl concentration which characterized the cheeses from which the bacteria originated. CONCLUSION: The enzyme activities may have a role in flavour development during cheese ripening. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work contribute to the knowledge about the amino acid catabolic enzymes in order to improve cheese ripening.

Amino Acids↗
PubMed1994

Proteolysis of bovine alpha s2-casein by chymosin.

Proteolysis of bovine alpha s2-casein by chymosin (E. C. 3.4.23.4) in solution in 100 mM Na phosphate buffer, pH 6.5, at 30 degrees C was studied by reversed-phase (RP)-HPLC and urea-polyacrylamide gel electrophoresis (PAGE). Chymosin hydrolyzed alpha s2-casein in solution to eight peptides detectable by urea-PAGE. Peptides soluble in acetate buffer, pH 4.6, were isolated by RP-HPLC on a C18 column using an acetonitrile/water gradient and identified from their N-terminal amino acid sequence. The chymosin cleavage sites were at the bonds Phe88-Tyr89, Tyr95-Leu96, Gln97-Tyr98, Tyr98-Leu99, Phe163-Leu164, Phe174-Ala175 and Tyr179-Leu180. Chymosin cleavage sites were restricted to the hydrophobic regions of the molecule. The bond-type in alpha s2-casein cleaved by chymosin was in agreement with that found to be susceptible to chymosin in other caseins. The primary site of chymosin action on alpha s2-casein appeared to be at Phe88-Tyr89.

Amino Acid Sequence↗
PubMed1993

Proteolytic specificity of chymosin on bovine alpha s1-casein.

The proteolytic specificity of chymosin (EC 3.4.23.4) on bovine alpha s1-casein at 30 degrees C in phosphate buffer, pH 6.5 and at pH 5.2 in the presence of 5% (w/v) NaCl was investigated. Peptides (pH 4.6-soluble) were isolated by reversed-phase HPLC and identified from their amino acid sequence; the identity of some peptides was confirmed by mass spectrometry and/or amino acid composition. The small peptides produced at pH 6.5 were Arg1-Phe23, Phe24-Phe28, Phe24-Leu40(?), Phe150-Phe153, Phe150-Leu156, Tyr154-Tyr159, Tyr154-Trp164, Asp157-Trp164 and Tyr165-Trp199. The same peptides, except Tyr154-Trp164, were produced at pH 5.2 in the presence of NaCl and, in addition, the peptides Arg1-Leu11, Phe24-Phe32, Lys102-Leu142, Ala143-Leu149 and Tyr165-Phe179. The rates of production of individual peptides differed under the two conditions studied but Arg1-Phe23 and Tyr165-Trp199 were the first and second peptides produced under both conditions. Pathways are proposed to interpret the proteolysis of alpha s1-casein in solution under the conditions of this study.

Amino Acid Sequence↗