Kinetics of the inhibition of renin and angiotensin I-converting enzyme by cod (Gadus morhua) protein hydrolysates and their antihypertensive effects in spontaneously hypertensive rats.

BACKGROUND: Cod muscle has a balanced protein profile that contains potentially bioactive amino acid sequences. However, there is limited information on release of these peptides from the parent proteins and their ability to modulate mammalian blood pressure. OBJECTIVE: The aim of this study was to generate cod antihypertensive peptides with potent in vitro inhibitory effects against angiotensin-converting enzyme (ACE) and renin. The most active peptides were then tested for systolic blood pressure (SBP)-reducing ability in spontaneously hypertensive rats (SHRs). DESIGN: Cod protein hydrolysate (CPH) was produced by subjecting the muscle proteins to proteolysis first by pepsin and followed by trypsin+chymotrypsin combination. In order to enhance peptide activity, the CPH was subjected to reverse-phase (RP)-HPLC separation to yield four fractions (CF1, CF2, CF3, and CF4). The CPH and RP-HPLC fractions were each tested at 1 mg/mL for ability to inhibit in vitro ACE and renin activities. CPH and the most active RP-HPLC fraction (CF3) were then used for enzyme inhibition kinetics assays followed by oral administration (200 and 30 mg/kg body weight for CPH and CF3, respectively) to SHRs and SBP measurements within 24 h. RESULTS: The CPH, CF3, and CF4 had similar ACE-inhibitory activities of 84, 85, and 87%, which were significantly (p<0.05) higher than the values for CF1 (69%) and CF2 (79%). Conversely, the CF3 had the highest (63%) renin-inhibitory activity (p<0.05) when compared to CPH (43%), CF1 (15%), and CF4 (44%). CPH and CF3 exhibited uncompetitive mode of ACE inhibition, whereas renin inhibition was non-competitive. Even at a 6.7-fold lower dosage, the CF3 significantly (p<0.05) reduced SBP (maximum -40.0 mmHg) better than CPH (maximum -19.1 mmHg). CONCLUSIONS: RP-HPLC fractionation led to enhanced antihypertensive effects of cod peptides, which may be due to a stronger renin-inhibitory activity.

Low-Molecular-Weight Peptides from Salmon Protein Prevent Obesity-Linked Glucose Intolerance, Inflammation, and Dyslipidemia in LDLR-/-/ApoB100/100 Mice.

BACKGROUND: We previously reported that fish proteins can alleviate metabolic syndrome (MetS) in obese animals and human subjects. OBJECTIVES: We tested whether a salmon peptide fraction (SPF) could improve MetS in mice and explored potential mechanisms of action. METHODS: ApoB(100) only, LDL receptor knockout male mice (LDLR(-/-)/ApoB(100/100)) were fed a high-fat and -sucrose (HFS) diet (25 g/kg sucrose). Two groups were fed 10 g/kg casein hydrolysate (HFS), and 1 group was additionally fed 4.35 g/kg fish oil (FO; HFS+FO). Two other groups were fed 10 g SPF/kg (HFS+SPF), and 1 group was additionally fed 4.35 g FO/kg (HFS+SPF+FO). A fifth (reference) group was fed a standard feed pellet diet. We assessed the impact of dietary treatments on glucose tolerance, adipose tissue inflammation, lipid homeostasis, and hepatic insulin signaling. The effects of SPF on glucose uptake, hepatic glucose production, and inducible nitric oxide synthase activity were further studied in vitro with the use of L6 myocytes, FAO hepatocytes, and J774 macrophages. RESULTS: Mice fed HFS+SPF or HFS+SPF+FO diets had lower body weight (protein effect, P = 0.024), feed efficiency (protein effect, P = 0.018), and liver weight (protein effect, P = 0.003) as well as lower concentrations of adipose tissue cytokines and chemokines (protein effect, P ≤ 0.003) compared with HFS and HFS+FO groups. They also had greater glucose tolerance (protein effect, P < 0.001), lower activation of the mammalian target of rapamycin complex 1/S6 kinase 1/insulin receptor substrate 1 (mTORC1/S6K1/IRS1) pathway, and increased insulin signaling in liver compared with the HFS and HFS+FO groups. The HFS+FO, HFS+SPF, and HFS+SPF+FO groups had lower plasma triglycerides (protein effect, P = 0.003; lipid effect, P = 0.002) than did the HFS group. SPF increased glucose uptake and decreased HGP and iNOS activation in vitro. CONCLUSIONS: SPF reduces obesity-linked MetS features in LDLR(-/-)/ApoB(100/100) mice. The anti-inflammatory and glucoregulatory properties of SPF were confirmed in L6 myocytes, FAO hepatocytes, and J774 macrophages.

Evaluation of the in vitro antioxidant properties of a cod (Gadus morhua) protein hydrolysate and peptide fractions.

Mechanically-deboned cod muscle proteins were sequentially hydrolysed using pepsin and a trypsin+chymotrypsin combination, which was followed by passing the digest through a 1 kDa equipped tangential flow filtration system; the permeate (<1 kDa peptides) was collected as the cod protein hydrolysate (CPH). Reversed-phase high performance liquid chromatography (RP-HPLC) was used to separate the CPH into four peptide fractions (CF1-CF4) and their in vitro antioxidant properties investigated. Results showed that most of the peptide fractions (CF2-CF4) displayed significantly higher (p<0.05) oxygen radical absorbance capacity values (698-942 µM Trolox equivalents, TE/g) and 2,2-diphenyl-1-picrylhydrazyl scavenging activities (17-32%) than those of CPH (613 µM TE/g and 19%, respectively). However, the unfractionated CPH displayed improved capability to scavenge superoxide and hydroxyl radicals as well as significantly higher (p<0.05) ferric iron reduction and chelation of iron than the RP-HPLC peptides. The CPH and peptide fractions displayed a dose-dependent inhibition of linoleic acid oxidation.

Interaction of protamine with gram-negative bacteria membranes: possible alternative mechanisms of internalization in Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa.

This study was concerned with the interaction between the cationic antimicrobial peptide, protamine (Ptm) and the cytoplasmic membranes of the gram-negative bacteria Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa. The objective of the study was to explain the observed paradox of internalization without permanent disruption of the cell envelope. We carried out Monte Carlo computer simulation of Ptm in an aqueous environment in the presence of ~100 mM NaCl and model membranes consisting of either (65:35) or (75:25) PE:PG molar ratios. The (75:25) model, representative of the gram-negative cytoplasmic membrane, showed that the Ptm center of mass remained at least 7 nm from the membrane surface leading to the prediction that Ptm would not internalize via disruption of the inner membrane. By using immunoelectron microscopy of Ptm-treated cells, we showed that Ptm internalization to the cytoplasm took place rapidly in the presence or absence of the outer envelope. Ultrastructural examination revealed no obvious morphological changes to cells that were treated with subinhibitory or bactericidal levels of Ptm. Reconstituted phospholipid bilayers were constructed and were unperturbed by Ptm treatment over a wide range of concentrations and applied transmembrane voltages. We conclude that in the cases of the cell envelopes of E. coli, S. typhimurium and P. aeruginosa, Ptm internalized by means independent of the phospholipid bilayer, most likely mediated by one or more membrane proteins such as cation-selective barrel-like proteins. Work is currently underway to test this hypothesis.

Screening of ACE-inhibitory peptides from a random peptide-displayed phage library using ACE-coupled liposomes.

Angiotensin I converting enzyme (ACE)-inhibitory peptides were screened from a random peptide-displayed phage library using ACE-coupled liposomes. Among four kinds of inhibitory peptides selected by biopanning with two different elution strategies, a peptide (LSTLRSFCA) showed the highest inhibitory activity with an IC(50) value of 3microM. By measuring inhibitory activities of fragments of the peptide, it was found that the RSFCA region was a functional site to inhibit strongly the ACE catalytic activity, and particularly both Arg and Cys residues were essential for the strong inhibitory activity. The inhibitory activity of RRFCA was slightly increased, while that of the RSFRA, in which the Cys residue was replaced by Arg, was decreased to greater extent in comparison with the inhibitory activity of RSFCA. Taking into account the results obtained from the SPOT analysis, it was suggested that the Arg and Phe residues in RSFCA were important for a specific interaction with ACE, and the Cys residue inhibited the ACE activity. The cystein-based ACE-inhibitory peptides have not been isolated from processed food materials. These findings suggested that the biopanning method utilizing protein-coupled liposomes and random peptide libraries might have a possibility to screen new functional peptides that are not found in processed food materials.