The role of free amino groups of peptides and proteins in the Folin-Lowry and biuret methods

On an equal weight basis polymyxin B and EM 49 which do not contain tyrosine or tryptophan yielded the same colour intensity as proteins in the Folin-Lowry and biuret methods. But, in the absence of reagent C (alkaline copper reagent) polymyxin B and EM 49 yielded no colour in the Folin-Lowry method. Mono-, di- and tri-formyl polymyxins B formed identical amounts of coloured complexes as polymyxin B in the two methods. However, the tetra- and penta-formyl polymyxins B yielded only one-fifth and one-sixth, respectively, of the expected colour in the Folin-Lowry method. Similarly, 40% and 30%, respectively, of the anticipated amount of colour is formed in the biuret method. Formylated and methylated lysozyme and bovine serum albumins form only 70–75% of the expected colour in the Folin-Lowry method. Since formation of colour by reduction of Folin reagent, in the Folin-Lowry method, is at least partly due to complexes of copper, it was inferred that polymyxin B as well as its mono-, di- and tri-formyl derivatives on the one hand and the tetra- and penta-formyl derivatives on the other differ in their ability to complex Cu(II) The former group of compounds was indeed found to complex as many as three Cu(II) ions whereas the tetra- and penta-formyl polymyxins B complexed only one equivalent, under conditions of excess Cu(II). Under conditions of low Cu(II), polymyxin B and all its derivatives complexed only one Cu(II). In proteins, sites other than amino groups which complex Cu(II) probably play a major role in the reduction of the Folin reagent, since methylated lysozyme and bovine serum albumin yield 70–75% of the colour formed by the unmodified proteins in the Folin-Lowry reaction.     

Insulin and IGF-1 receptors contain covalently bound palmitic acid

We have studied the biosynthesis of the insulin receptor in a human hepatoma cell line, HepG2. As previously reported, these cells synthesize a disulphide-bonded alpha 2 beta 2 tetrameric insulin receptor. Labelling of HepG2 cells with [3H]palmitate or [3H]myristate followed by immunoprecipitation with a polyclonal antireceptor antibody revealed the incorporation of palmitate, but not myristate, into the beta-subunit and alpha beta-precursor of the receptor in a hydroxylamine-sensitive linkage. The extracellular alpha-subunit was not labelled, demonstrating the specificity of incorporation. Acylation of the insulin receptor was an early event as judged by fatty acid incorporation into the alpha beta-precursor and prevention by protein synthesis inhibitors. Pulse-chase studies demonstrated the expected processing of the alpha beta-precursor to mature alpha- and beta-subunits, but no evidence for preferential turnover of the fatty acid moiety was found. The site of acylation appears to be in the transmembrane or cytoplasmic domain since proteolytic treatment of intact cells produced a truncated beta-subunit still containing label. Binding studies showed that HepG2 cells contain approximately half as many insulin-like growth factor-1 receptors as insulin receptors, raising the possibility that this receptor may also be acylated. Indeed, immunoprecipitation with the antiinsulin receptor serum of MDCK cells expressing IGF-1 receptors, but not insulin receptors, revealed bands corresponding to the alpha beta-precursor, alpha- and beta-subunits, of which the alpha beta-precursor and beta-subunits incorporated [3H]palmitate but the alpha-subunit did not.     

Fatty acid acylated antibodies against virus suppress its reproduction in cells

A method for suppression of virus reproduction in cells using fatty acylated antiviral antibodies, which in contrast to non-modified antibodies are capable of intracellular penetration, has been suggested. The addition of stearoylated antiviral antibodies to influenza A/Chili virus-infected cells causes a 100-fold suppression of virus reproduction. Non-modified antibodies do not produce any effect on virus reproduction.     

Antimicrobial activity of human α-defensin 5 and its linear analogs: N-terminal fatty acylation results in enhanced antimicrobial activity of the linear analogs

Human α-defensin 5 (HD5) exhibits broad spectrum antimicrobial activity and plays an important role in mucosal immunity of the small intestine. Although there have been several studies, the structural requirements for activity and mechanism of bacterial killing is yet to be established unequivocally. In this study, we have investigated the antimicrobial activity of HD5 and linear analogs. Cysteine deletions attenuated the antibacterial activity considerably. Candidacidal activity was affected to a lesser extent. Fatty acid conjugated linear analogs showed antimicrobial activity comparable activity to HD5. Effective surface charge neutralization of bacteria was observed for HD5 as compared to the non-fatty acylated linear analogs. Our results show that HD5 and non-fatty acylated linear analogs enter the bacterial cytoplasm without causing damage to the bacterial inner membrane. Although fatty acylated peptides exhibited antimicrobial activity comparable to HD5, their mechanism of action involved permeabilization of the Escherichia coli inner membrane. HD5 and analogs had the ability to bind plasmid DNA. HD5 had greater binding affinity to plasmid DNA as compared to the analogs. The three dimensional structure of HD5 favors greater interaction with the bacterial cell surface and also with DNA. Antibacterial activity of HD5 involves entry into bacterial cytoplasm and binding to DNA which would result in shut down of the bacterial metabolism leading to cell death. We show how a moderately active linear peptide derived from the α-defensin HD5 can be engineered to enhance antimicrobial activity almost comparable to the native peptide.     

Chemoenzymatic synthesis of hydroxytyrosol monoesters and their suppression effect on nitric oxide production stimulated by lipopolysaccharides

Fatty acid monoesters of hydroxytyrosol [2-(3,4-dihydroxyphenyl)ethanol] were synthesized in two steps from tyrosol (4-hydroxyphenylethanol) by successive Candida antarctica lipase B-catalyzed chemoselective acylation on the primary aliphatic hydroxy group over phenolic hydroxy group in tyrosol, and 2-iodoxybenzoic acid (IBX)-mediated hydroxylation adjacent to the remaining free phenolic hydroxy group. Examination of their suppression effects on nitric oxide production stimulated by lipopolysaccharides in RAW264.7 cells showed that hydroxytyrosol butyrate exhibited the highest inhibition (IC₅₀ 7.0 μM) among the tested compounds.     

Formation of acylated growth hormone-releasing peptide-6 by poly(lactide-co-glycolide) and its biological activity

The purpose of this study was to investigate the formation of acylated impurity resulting from a chemical reaction between the growth hormone-releasing peptide-6 (GHRP-6) and poly(lactide-co-glycolide) (PLGA) and the effect of peptide acylation on the in vivo biological activity of GHRP-6. The peptide acylation pattern of GHRP-6 by hydrophilic PLGA polymers with different molecular weights was characterized by reversed-phase high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Higher levels of acylated GHRP-6 were produced with the higher molecular weight PLGA, which might be due to the slower degradation rate of the polymer. The evaluation of the biological activity in rats showed that the acylated GHRP-6 had a much lower activity than the intact GHRP-6. This finding suggests that the acylation reaction would decrease the effectiveness of the GHRP-6 formulation such as PLGA microspheres. There-fore, a strategy for stabilizing the GHRP-6 will be necessary for the development of a successful formulation of PLGA microspheres. Published: June 8, 2007     

Increased plasma acylation-stimulating protein correlates with hyperlipidemia at late gestation

Objectives: Obesity is often associated with negative consequences, including hyperlipidemia and insulin resistance. Weight gain during pregnancy is also associated with major lipid alterations. Fat storage is enhanced in early pregnancy. At late gestation, hyperlipidemia becomes a major manifestation. The acylation‐stimulating protein (ASP) is a potent lipogenic adipocytokine that correlates with postprandial triglyceride (TG) clearance in vivo and has been linked to hyperlipidemic disorders. The role of ASP during a normal pregnancy is unknown. The objective of this study was to investigate plasma ASP levels in correlation with the lipid profile during late gestation. Research Methods and Procedures: Seventy healthy women at late gestation and 60 non‐pregnant controls of similar age and prepregnancy BMI were included in a cross‐sectional study. Fasting plasma ASP levels and the lipid profile of all of the women were measured. Results: ASP levels were markedly elevated in the pregnant women (66%, p < 0.001). ASP levels correlated strongly with the elevated levels of TGs (r = 0.608, p < 0.000), apolipoprotein B (0.519, p < 0.000), and low‐density lipoprotein‐cholesterol (r = 0.405, p < 0.000). Multivariate analysis adjusting for BMI and age showed that changes in ASP levels at late gestation were best predicted by TG and apoB levels, accounting for 53.8% of plasma ASP variation. For the controls, ASP strongly correlated with BMI, which was the only significant predictor of ASP levels. Discussion: Gestational hormone alterations during pregnancy may affect ASP function as a lipogenic factor. Increased plasma ASP levels at late gestation and their strong correlation with parameters reflecting very low‐density lipoprotein accumulation are suggestive of ASP resistance, which may further contribute to the hyperlipidemic state, shifting energy in the form of TGs to the rapidly growing fetus.     

Screening of lipases for regioselective hydrolysis of peracetylated β-monosaccharides

The monodeacetylation of peracetylated-β-d-galactose (1) and peracetylated N-acetyl-β-d-glucosamine (2) by different lipases is here described. Lipases from different sources in an immobilized form were evaluated to find those that offer the higher activity and regioselectivity in the reactions. In the hydrolysis of 1, the lipase from Aspergillus niger was the most active one, although it hydrolyzed the anomeric position. Using the lipase from Candida rugosa, 30% yield of the corresponding 6-OH isomer was achieved. On the other hand, in the hydrolysis of 2, the lipase from A. niger was the most active and regioselective catalyst, producing more than 75% of the 6-OH derivative product.     

Alkylalanes and methyl furanosides: regioselective O-debenzylation or acetal cleavage

Perbenzylated methyl pentofuranosides were submitted to the action of three alkylalanes and regioselective debenzylation at O-2 of the four pentoses was observed when choosing the right match between anomeric configuration and aluminium reagent. Diisobutylalane (DIBAL-H) allowed an easy access to reduced open-chain compounds, whereas trimethylalane (TMAL) stereoselectively produced methylated open chain derivatives.