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H M Farrell

Publications and source records attributed to H M Farrell.

At least 37 records · Page 2Linked to original sources
PubMed1997

Arginase in lactating bovine mammary glands: implications in proline synthesis.

The occurrence and subcellular distribution of arginase have been studied in mammary glands from lactating dairy cattle. The enzyme appears to be localized in the mitochondrial fraction, although a significant amount has been found to be associated with the cytosolic fraction. Both mitochondrial and cytosolic arginase are activated by heating with Mn2+. The Michaelis constants for the two fractions, however, are different: 49.5 and 18.5 mM for the mitochondrial fraction and cytosolic fraction, respectively. Overall the total enzyme concentration in the gland suggests that these enzymes contribute to the conversion of arginine to ornithine. Ornithine, in turn, may be converted by ornithine aminotransferase into an intermediate, L-delta 1-pyrroline-5-carboxylate; concurrently. alpha-keto-glutarate is transformed into glutamic acid. Finally, pyrroline-5-carboxylate reductase yields proline, an important amino acid that is needed for casein synthesis. Because pyrroline-5-carboxylate reductase requires NADPH, and because ornithine aminotransferase uses alpha-ketoglutarate, this new pathway is linked to the Krebs cycle through the cytosolic isocitrate dehydrogenase, which is the source of both of these intermediates.

Animals↗
PubMed1996

Particle sizes of purified kappa-casein: metal effect and correspondence with predicted three-dimensional molecular models.

kappa-Casein as purified from bovine milk exhibits a rather unique disulfide bonding pattern as revealed by SDS-PAGE. The disulfide-bonded caseins present range from dimer to octamer and above and preparations contain about 10% monomer. All of these heterogenous polymers, however, self-associated into nearly spherical uniform particles with an average radius of 8.9 nm as revealed by negatively stained transmission electron micrographs. Evidence is presented that multivalent cations play a role in the stabilization of these spherical particles. Treatment with EDTA causes disruption of the kappa-casein particles and leads to a broder size distribution as judged by electron microscopy and dynamic light scattering. The size and shape of the particles are in accord with earlier proposed 3D models for kappa-casein that actually predicted participation of divalent cations in the structure.

Amino Acids↗
PubMed1996

Pyrroline-5-carboxylate reductase in lactating bovine mammary glands.

The occurrence and subcellular distribution of pyrroline-5-carboxylate reductase have been studied in lactating bovine mammary glands. The enzyme appears to have only a cursory association with the mitochondrial fraction, because significant amounts of the enzyme are found in other membrane-containing fractions and in the cytosol. Polyamines stimulate the enzyme in vitro, supporting the mediation of cursory attachment to membrane fractions by these compounds. The enzyme is selective for NADPH but can utilize NADH as well. Long-chain coenzyme A derivatives, which are generated during lipid metabolism, almost completely inhibit this enzyme, which is responsible for the synthesis of a portion of the proline needed for casein production. Overall, the enzyme concentration in the gland correlates well with a role in the conversion of an intermediate, L-delta 1-pyrroline-5-carboxylate, into proline, an important amino acid for the mammary secretory process, especially casein synthesis.

Animals↗
PubMed1995

Effects of long-chain acyl-coenzyme A's on the activity of the soluble form of nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase from lactating bovine mammary gland.

The cytosolic form of NADP+:isocitrate dehydrogenase, a primary source of the NADPH required for de novo fatty acid synthesis in lactating bovine mammary gland, was studied to determine possible mechanisms of regulation by fatty acyl-coenzyme A (CoA). The reduction of NADP+ by the enzyme is inhibited by palmitoyl-CoA. In steady-state experiments, when added enzyme is used to start the reaction, analyses of velocity versus palmitoyl-CoA concentration as a binding isotherm, following the assumptions of Wyman's theory of thermodynamic linkage, suggested that binding of palmitoyl-CoA produced two different inhibitory effects on the enzyme. This analysis suggested inhibition first through binding to sites with an average dissociation constant of 3.3 microM, then by binding to sites with an average dissociation constant of 294 microM. When the enzyme is preincubated with palmitoyl-CoA there is an induction of a significant lag-burst reaction rate (hysteretic kinetics). Preincubation of the enzyme with its substrate, metal-isocitrate complex, nearly abolished the lag time and decreased the degree of inhibition. Changes in lag time and percentage inhibition as a function of concentration of palmitoyl-CoA followed patterns, similar to those observed in steady-state reactions, where the enzyme is not preincubated. Examination of the effect of acyl chain length at 300 microM demonstrated that only long-chain CoA's with carbon numbers > 14 have pronounced effects on kinetics. CoA alone has little or no effect, while stearoyl-CoA completely inhibited the enzyme. Other C18 acyl groups produced varying effects depending on the degree of unsaturation and cis-trans isomerism. NADP+:Isocitrate dehydrogenases, from other sources including that from Escherichia coli, do not show such sensitivity to acyl chain character under these conditions. Concentration ranges observed for these transitions are compatible with physiological conditions. This suggests that complexes of acyl-CoA's and NADP+:isocitrate dehydrogenase, in tissue rich in the cytoplasmic form of the enzyme, could be related to cytoplasmic events in the synthesis and secretion of lipid and possibly protein, since palmitoyl-CoA is known to promote secretory processes through acylation reactions which lead to vesicle fusion.

Acyl Coenzyme A↗
PubMed1995

Ornithine-delta-aminotransferase in lactating bovine mammary glands.

The occurrence and subcellular distribution of ornithine-delta-aminotransferase have been studied in lactating bovine mammary glands. The enzyme is localized in the mitochondria and has a unique thermal reaction profile that distinguishes it from putative liver and kidney isozymes. The enzyme concentration in the gland correlates well with a role in the conversion of ornithine into the proline precursor, L-delta 1-pyrroline-5-carboxylate. However, an unusually high Michaelis constant for the mitochondrial enzyme (8.4 mM) raises the question of enzyme efficiency in vivo such that this pathway needs to be considered in estimating barriers to protein secretion into milk.

Animals↗
PubMed1994

An energy-minimized casein submicelle working model.

To develop a molecular basis for structure-function relationships of the complex milk protein system, an energy-minimized, three-dimensional model of a casein submicelle was constructed consisting of kappa-casein, four alpha s1-casein, and four beta-casein molecules. The models for the individual caseins were from previously reported energy-minimized, three-dimensional structures. Docking of one kappa-casein and four alpha s1-casein molecules produced a framework structure through the interaction of two hydrophobic antiparallel sheets of kappa-casein with two small hydrophobic antiparallel sheets (residue 163-174) of two preformed alpha s1-casein dimers. The resulting structure is approximately spherically symmetric, with a loose packing density; its external portion is composed of the hydrophilic domains of the four alpha s1-caseins, while the central portion contains two hydrophbic cavities on either side of the kappa-casein central structure. Symmetric and asymmetric preformed dimers of beta-casein formed from the interactions of C-terminal beta-spiral regions as a hinge point could easily be docked into each of the two central cavities of the alpha-kappa framework. This yielded two plausible energy-minimized, three-dimensional structures for submicellar casein, one with two symmetric beta-casein dimers and one with two asymmetric dimers. These refined submicellar structures are in good agreement with biochemical, chemical, and solution structural information available for submicellar casein.

Animals↗
PubMed1994

Comparison of the three-dimensional molecular models of bovine submicellar caseins with small-angle X-ray scattering. Influence of protein hydration.

To test the applicability of two energy-minimized, three-dimensional structures of the bovine casein submicelle, theoretical small-angle X-ray scattering curves in the presence and absence of water were compared to experimental data. The published method simulates molecular dynamics of proteins in solution by employing adjustable Debye-Waller temperature factors (B factors) for the protein, for the solvent, and for protein-bound water. The programs were first tested upon bovine pancreatic trypsin inhibitor beginning with its known X-ray crystal structure. To approximate the degree of protein hydration previously determined by NMR relaxation experiments (0.01 g water/g protein), 120 water molecules were docked into the large void of the kappa-casein portion of the structure for both the symmetric and asymmetric casein submicelle models. To approximate hydrodynamic hydration (0.244 g water/g protein), 2703 water molecules were added to each of the above structures using the "droplet" algorithm in the Sybyl molecular modeling package. All structures were then energy-minimized and their solvation energies calculated. Theoretical small-angle X-ray scattering curves were calculated for all unhydrated and hydrated structures and compared with experimentally determined scattering profiles for submicellar casein. Best results were achieved with the 120-bound-water structure for both the symmetric and asymmetric submicelle models. Comparison of results for the protein submicelle models with those for the theoretical and literature values of bovine pancreatic trypsin inhibitor demonstrates the applicability of the methodology.

Animals↗
PubMed1994

In vitro DNA synthesis as indicator of mammary epithelial cell division: [14C]thymidine uptake versus flow cytometry cell cycle analysis.

Mammary and adipose explants from eight mid-lactation Holstein cows were co-cultured for 24 h in the presence or absence of liver explants, 1 microgram/ml pituitary bovine somatotrophin, or 100 ng/ml insulinlike growth factor-I. Liver explants in the media significantly depressed DNA and protein synthesis by mammary tissue as measured by [14C]-thymidine and amino acid incorporation. As measured by flow cytometry, the concentration of DNA in the G0G1 and G2M cells and the percentage of cells in the G0G1 population of mammary tissue was also significantly depressed by liver tissue. Changes in the percentage of cells in the S and G2M phases were not significant. Insulinlike growth factor-I in the presence of liver explants depressed protein synthesis, thymidine incorporation, and the concentration of DNA in the G0G1 and G2M cells compared to control but did not affect the percentage of cells in the G0G1, S, or G2M phases. Previously it was assumed that changes in [14C]thymidine incorporation indicated that changes in cell division were occurring. Flow cytometry revealed that changes in DNA content of mammary cells as a result of liver or hormonal stimulation were not due to changes in cell division. Indications are that differences in cellular DNA content result from changes in the rate of amplification of individual genes responsible for milk protein synthesis.

Adipose Tissue↗
PubMed1993

Properties of [Ca2+ + Mg2+]-adenosine triphosphatases in the Golgi apparatus and microsomes of the lactating mammary glands of cows.

The [Ca2+ + Mg2+]-ATPase activity of bovine lactating mammary gland is associated with membranes. This study compares the ATPase activity in microsomal membranes to that of the Golgi apparatus. The enzyme activity in both fractions hydrolyzed Ca(2+)-ATP and Mg(2+)-ATP. The ATPase activities were inhibited by p-chloromercuribenzoate, indicating the involvement of a sulfhydryl group for activity. Although calmodulin had no effect on the ATPase activities of the two fractions, calmodulin antagonists (chlorpromazine, fluphenazine, and trifluoperazine) were inhibitory. Strong inhibitors of the ATPase activities were vanadate, dicyclohexyl-carbodiimide, La3+, and Zn2+. There were some differences in the activities from two membrane fractions. Although both fractions could hydrolyze all of the triphosphonucleotides, cytidine-5'-triphosphate and uridine-5'-triphosphate were poor substrates for the Golgi enzyme. Detergents diminished the activity of the microsomal enzyme to a much greater extent than the ATPase of the Golgi apparatus. Thus, the intact membrane may be more critical to microsomal activity. The role of these enzymes in Ca2+ accumulation in milk is discussed.

Animals↗
PubMed1993

Three-dimensional molecular modeling of bovine caseins: an energy-minimized beta-casein structure.

To obtain a molecular basis for the similarities and dissimilarities in the functional, chemical, and biochemical properties between beta-casein and the other caseins, a predicted three-dimensional model is presented. The predicted structure was assembled using molecular modeling techniques, as well as secondary structural prediction algorithms, in conjunction with global secondary structural information from Raman spectroscopy. To add validity to this model, the structure was refined using energy minimization techniques to diminish the likelihood of structural overlaps and energetically unfavorable van der Waals contacts arising from the large number of proline residues present in the beta-casein sequence. The refined model overall showed a loosely packed, asymmetrical structure with an axial ratio of 2:1. Hydrophobic side chains were uniformly dispersed over one end (C terminal) and the center surface of the structure; the other end (N terminal) was hydrophilic. The hydrophobic section also possessed a large loop through which water could easily travel. Such a suprasurfactant structure could account for the micellar type of hydrophobically driven self-association exhibited by beta-casein. Other chemical and biochemical data are in good agreement with the refined structure.

Amino Acid Sequence↗
PubMed1993

Three-dimensional molecular modeling of bovine caseins: a refined, energy-minimized kappa-casein structure.

A refined three-dimensional molecular model of kappa-casein has been produced using energy minimization techniques and a Kollman force field on a previously reported predicted three-dimensional structure. This initial model was constructed via molecular modeling techniques from sequence-based secondary structural prediction algorithms. Both the initial and refined structures agreed with global secondary structure analysis from vibration spectroscopy. The refined structure contained many of the features of the initial model, including two sets of antiparallel beta-sheet structures containing predominantly hydrophobic side chains, which could form interaction sites with alpha s1-casein. Two types of energy-minimized dimer and tetramer models are presented: 1) using Cys as potential intermolecular disulfide binding sites and 2) using the two sheets as possible hydrophobic self-association sites, without Cys interactions. All structures yielded good stabilization energies and are in agreement with chemical, biochemical, and physical chemical results obtained for kappa-casein.

Amino Acid Sequence↗
PubMed1993

Regulation of the soluble form of nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase from lactating bovine mammary gland: effects of metabolites on activity and structure.

The cytosolic form of NADP+: isocitrate dehydrogenase, a primary source of the NADPH required for de novo fatty acid synthesis in lactating bovine mammary gland, was studied to determine possible mechanisms of regulation by metabolites. The enzymatic reduction of NADP+ exhibits lag-burst (hysteretic) kinetics that are eliminated by the noncatalytic binding of the substrate, a complex (1:1) of a metal ion (Mn2+ or Mg2+) and isocitrate. Preincubation of the enzyme with metal-citrate complex also nearly abolished the lag or activation time. In steady-state experiments, analyses of velocity versus metal-citrate complex as a binding isotherm, following the assumptions of Wyman's theory of thermodynamic linkage, showed that binding of metal-citrate complex could both stimulate and inhibit the enzyme. This analysis suggested hyperactivation by binding to sites with an average dissociation constant of .25 mM, inhibition by binding to sites with an average dissociation constant of 3.83 mM, and modulation (reactivation) by binding to sites with an average dissociation constant of 1.54 mM. Conformational changes induced by the binding of ligands were assessed using circular dichroism. The results suggest that binding of metal-isocitrate induces a conformational transition involving tyrosyl residues that is related to the altered kinetic processes. Reexamination of Michaelis-Menten kinetics using non-linear regression analysis also demonstrated hyperactivation of enzyme activity by metal-isocitrate with a dissociation constant equal to 21 microM (which is nearly seven times greater than the Michaelis constant). Concentration ranges observed for these transitions are compatible with physiological conditions, suggesting that complexes of metal-citrate and metal-isocitrate serve to modulate the activity of NADP+: isocitrate dehydrogenase.

Animals↗
PubMed1992

Distribution of ADPase activity in the lactating rat mammary gland and its possible role in an ATP cycle in the Golgi apparatus.

A Ca2+/Mg(2+)-stimulated ADPase has been found to occur in the lactating rat mammary gland. The enzyme is membrane associated and occurs in mitochondrial, microsomal, and Golgi apparatus fractions. The pH activity curves for the Golgi apparatus and microsomal fractions display two distinct maxima, one at pH 6.3 and one at pH 7.4. Studies with inhibitors and activators indicate that the enzyme is similar to ADPases found in other tissues and is distinct from the uridine nucleoside diphosphatase previously reported in the mammary Golgi apparatus. The occurrence of ADPase in the Golgi apparatus indicates a possible role for this enzyme in the milk secretory process, while the microsomal enzyme could be involved in extracellular activities.

Adenosine Triphosphate↗
PubMed1992

Reexamination of the polymeric distributions of kappa-casein isolated from bovine milk.

kappa-Casein the stabilizing protein of the colloidal milk protein complex was purified from bovine skim milk by the method of McKenzie and Wake (Biochim, Biophys. Acta. 47, 240, 1961). The preparations were examined by sodium dodecyl sulfate gel electrophoresis in the presence and absence of a reducing agent. In the presence of a reducing agent, the kappa-casein migrates as a single low molecular weight band. However, in the absence of a reducing agent, a characteristic pattern of aggregates of varying molecular weight was observed with components ranging from monomer to octamer in integer steps. Densitometry of the Coomassie blue stained gels showed an almost equal distribution of components in each band; carbohydrate staining showed preferential location of sugar residues in lower molecular weight components. Treatment with chymosin (rennin) caused a downward shift in apparent molecular weight for each band with no change in the relative intensity of the Coomassie blue stained bands. Similar gel patterns were observed in whole caseins and partially purified kappa-caseins, indicating that this size distribution is a natural disulfide-linked reporter for the distribution of kappa-casein in casein colloids (micelles).

Animals↗
PubMed1992

Distribution of adenosine-5'-diphosphatase activity in the lactating bovine mammary gland.

A Ca2+- and Mg(2+)-stimulated adenosine-5'-diphosphatase has been found in lactating bovine mammary glands. The enzyme is associated with membranes of mitochondrial, microsomal, and Golgi apparatus fractions. The pH activity curves for the Golgi apparatus and microsomal fractions display two distinct maxima, one at pH 5.8 and the other between pH 7.4 and 8.4. Studies with activators and inhibitors indicate the enzyme is similar to adenosine-5'-diphosphatase found in other tissues. Its occurrence in the Golgi apparatus fraction indicates a possible role for this enzyme in the milk secretory process, particularly in ATP cycling in vesicles. Its occurrence in the microsomal fraction suggests a role in plasma membrane functioning.

Animals↗
PubMed1991

Protein-water interactions from 2H NMR relaxation studies: influence of hydrophilic, hydrophobic, and electrostatic interactions.

The importance of water interactions with proteins in food systems is well documented. A controversy exists, however, as to the nature of these interactions and the effect of protein structural changes on them. To clarify these questions, a method has been developed for determining hydration from the protein concentration-dependence of deuteron resonance relaxation rates. Measurements were made in D2O on beta-lactoglobulin A to study effects of hydrophilic interactions, and on both casein micelles and submicelles to study hydrophobic and electrostatic effects. From the protein concentration-dependent relaxation rates, the second viral coefficients of the proteins were obtained by nonlinear regression analysis. Using either an isotropic tumbling or an intermediate asymmetry model, hydrations, upsilon, and correlation times, tau c, were calculated for the protein-associated water; from tau c, the Stokes radius, R, was obtained. Variations in upsilon and R were in accord with known structural changes in molecular states of the proteins. The NMR results are compared with hydrations and structural information derived independently from small-angle X-ray scattering.

Chemical Phenomena↗
PubMed1991

Calcium-induced associations of the caseins: thermodynamic linkage of calcium binding to colloidal stability of casein micelles.

The caseins occur in milk as colloidal complexes of protein aggregates, calcium, and inorganic phosphate. As determined by electron microscopy, these particles are spherical and have approximately a 650 A radius (casein micelles). In the absence of calcium, the protein aggregates themselves (submicelles) have been shown to result from mainly hydrophobic interactions. The fractional concentration of stable colloidal casein micelles can be obtained in a calcium caseinate solution by centrifugation at 1500 g. Thus, the amount of stable colloid present with varying Ca2+ concentrations can be determined and then analyzed by application of equations derived from Wyman's Thermodynamic Linkage Theory. Ca(2+)-induced colloid stability profiles were obtained experimentally for model micelles consisting of only alpha s1- (a calcium insoluble casein) and the stabilizing protein kappa-casein, eliminating the complications arising from beta- and minor casein forms. Two distinct genetic variants alpha s1-A and B were used. Analysis of alpha s1-A colloid stability profiles yielded a precipitation (salting-out) constant k1, as well as colloid stability (salting-in) parameter k2. No variations of k1 or k2 were found with increasing amounts of kappa-casein. From the variation of the amount of colloidal casein capable of being stabilized vs. amount of added kappa-casein an association constant of 4 L/g could be calculated for the complexation of alpha s1-A and kappa-casein. For the alpha s1-B and kappa-casein micelles, an additional Ca(2+)-dependent colloidal destabilization parameter, k3, was added to the existing k1 and k2 parameters in order to fully describe this more complex system. Furthermore, the value of k3 decreased with increasing concentration of kappa-casein. These results were analyzed with respect to the specific deletion which occurs in alpha s1-casein A in order to determine the sites responsible for these Ca(2+)-induced quaternary structural effects.

Amino Acid Sequence↗