Phenylalanine stable isotope tracer labeling of cow milk and meat and human experimental applications to study dietary protein-derived amino acid availability.

BACKGROUND & AIMS: Availability of dietary protein-derived amino acids (AA) is an important determinant for their utilization in metabolism and for protein synthesis. Intrinsic labeling of protein is the only method to directly trace availability and utilization. The purpose of the present study was to produce labeled milk and meat proteins and investigate how dietary protein-derived AA availability is affected by the protein-meal matrix. METHODS: Four lactating cows were infused with L-[ring-d5]phenylalanine and one with L-[15N]phenylalanine for 72 h. Milk was collected, and three of the [d5]phenylalanine cows were subsequently slaughtered. Two human studies were performed to explore plasma AA availability properties utilizing the labeled proteins. One study compared the intake of whey protein either alone or together with carbohydrates-fat food-matrix. The other study compared the intake of meat hydrolysate with minced beef. Cow blood, milk, meat and human blood samples were collected and analyzed by mass spectrometry. RESULTS: Whey and caseinate acquired label to 15-20 mol percent excess (MPE), and the meat proteins reached 0.41-0.73 MPE. The [d5]phenylalanine appeared fast in plasma and peaked 30 min after whey protein alone and meat hydrolysate intake, whereas whey protein with a food-matrix and the meat minced beef postponed the [d5]phenylalanine peak until 2 and 1 h, respectively. CONCLUSIONS: Phenylalanine stable isotope-labeled milk and meat were produced and proved a valuable tool to investigate AA absorption characteristics. Dietary protein in food-matrices showed delayed postprandial plasma AA availability as compared to whey protein alone and meat hydrolysate.

The stress response protein Gls24 is induced by copper and interacts with the CopZ copper chaperone of Enterococcus hirae.

Intracellular copper routing in Enterococcus hirae is accomplished by the CopZ copper chaperone. Under copper stress, CopZ donates Cu(+) to the CopY repressor, thereby releasing its bound zinc and abolishing repressor-DNA interaction. This in turn induces the expression of the cop operon, which encodes CopY and CopZ, in addition to two copper ATPases, CopA and CopB. To gain further insight into the function of CopZ, the yeast two-hybrid system was used to screen for proteins interacting with the copper chaperone. This led to the identification of Gls24, a member of a family of stress response proteins. Gls24 is part of an operon containing eight genes. The operon was induced by a range of stress conditions, but most notably by copper. Gls24 was overexpressed and purified, and was shown by surface plasmon resonance analysis to also interact with CopZ in vitro. Circular dichroism measurements revealed that Gls24 is partially unstructured. The current findings establish a novel link between Gls24 and copper homeostasis.

The interaction of Fusarium solani pisi cutinase with long chain spin label esters.

We here present a study of the interaction between the Fusarium solani pisi cutinase mutant S120A and spin-labeled 4,4-dimethyloxazoline-N-oxyl-(DOXYL)-stearoyl-glycerol substrates in a micellar system. The interaction is detected by NMR measuring changes in chemical shift for 1H and 15N as well as relaxation parameters for backbone 1H (T1) and 15N (T1, T2) atoms as well as for side chain methyl groups 1H (T1). The detected interaction shows a weak binding of cutinase to the lipid micelles. Structural and mobility changes are located inside and around the active site, its flanking loops, and the oxyanion hole, respectively. Relaxation changes in the amino acid pairs Ser 92, Ala 93 and Thr 173, Gly 174 positioned at the edge of each of the active site flanking loops make these residues prime candidates for hinges, allowing for structural rearrangement during substrate binding. The cutinase mutant S120A used carries a 15 amino acid pro-peptide; the significance of this pro-peptide was so far undetermined. We show here that the pro-peptide is affected by the presence of the micellar substrate. Relaxation enhancements indicative of spatial proximity between the DOXYL group in the lipid chain and some hydrophobic residues surrounding the active site could be found.

CopY-like copper inducible repressors are putative 'winged helix' proteins.

CopY of Enterococcus hirae is a well characterized copper-responsive repressor involved in copper homeostasis. In the absence of copper, it binds to the promoter. In high copper, the CopZ copper chaperone donates copper to CopY, thereby releasing it from the promoter and allowing transcription of the downstream copper homeostatic genes of the cop operon. We here show that the CopY-like repressors from E. hirae, Lactococcus lactis, and Streptococcus mutans have similar affinities not only for their native promoters, but also for heterologous cop promoters. CopZ of L. lactis accelerated the release of CopY from the promoter, suggesting that CopZ of L. lactis acts as copper chaperone, similar to CopZ in E. hirae. The consensus binding motif of the CopY-like repressors was shown to be TACAxxTGTA. The same binding motif is present in promoters controlled by BlaI of Bacillus licheniformis, MecI of Staphylococcus aureus and related repressors. BlaI and MecI have known structures and belong to the family of 'winged helix' proteins. In the N- terminal domain, they share significant sequence similarity with CopY of E. hirae. Moreover, they bind to the same TACAxxTGTA motif. NMR analysis of the N-terminal DNA binding domain of CopY of L. lactis showed that it contained the same alpha-helical content like the same regions of BlaI and MecI. These findings suggest that the DNA binding domains of CopY-like repressors are also of the 'winged helix' type.

Quantization of pH: evidence for acidic activity of triglyceride lipases.

Here we present a study of lipolytic activity of lipases from Fusarium solani pisi (cutinase), Rhizomucor miehei, Pseudomonas cepacia, and Humicola lanuginosa. Their activities toward triolein provide clear evidence for considerable enzymatic activity under acidic conditions. The activity was followed using Fourier transform infrared attenuated total reflection (FTIR-ATR) and nuclear magnetic resonance (NMR). Using these approaches, all the lipases that were studied exhibited lipolytic activity down to pH 4. The common model for the catalytic activity of the F. solani pisi cutinase, and lipases in general, requires the deprotonation of the active site histidine. Measurements using (13)C NMR spectroscopy showed a pK(a) value in the absence of substrate that is not consistent with the detected acid activity. We propose a novel model for the electrostatics in the active site of cutinase that could explain the observed acidic activity. The active site is essentially covered with the lipid surface during catalysis, thus preventing chemical communication between the active site and the bulk solvent. We propose that the classical definition of pH in bulk solution is not applicable to the active site environment of a lipase when the active site is inaccessible to solvent. In small restricted volumes, the pH must be quantized, and since much of the biological world is dependent on compartmentalization of processes in small volumes, it becomes relevant to investigate when this mechanism comes into play. We have made a quantitative assessment of how large the restricted volume can be and still lead to quantization of pH.