Sequential Gel Permeation Research Articles

Purification and characterization of two novel angiotensin I-converting enzyme inhibitory peptides derived from R-phycoerythrin of red algae (Bangia fusco-purpurea)

R-phycoerythrin prepared from red algae (Bangia fusco-purpurea) was hydrolyzed by pepsin followed by trypsin to produce angiotensin I-converting enzyme (ACE) inhibitory peptides. The IC50 of the hydrolysate of R-phycoerythrin (HRPE) was 191.1 ± 4.1 μg/mL, and the molecular weight of most products (89.9 %) was below 2 kDa. After sequential gel permeation and reversed-phase chromatography steps to purify the hydrolysate, two peptides with the sequences of ALLAGDPSVLEDR and VVGGTGPVDEWGIAGAR were obtained and their IC50 values were 57.2 ± 5.0 and 66.2 ± 4.2 μg/mL, respectively. The ALLAGDPSVLEDR and VVGGTGPVDEWGIAGAR peptides were derived from the β- and α-subunit of R-phycoerythrin from Polysiphonia urceolata, with a 92.3 % (12/13) and 94.1 % (16/17) match, respectively. Both peptides were resistant to digestion by proteinases common in the gastrointestinal tract. Therefore, the identified novel peptides derived from R-phycoerythrin may be used as potential nutraceuticals for development of functional foods.

Alternative Luminal Activation Mechanisms for Paneth Cell α-Defensins

Paneth cell α-defensins mediate host defense and homeostasis at the intestinal mucosal surface. In mice, matrix metalloproteinase-7 (MMP7) converts inactive pro-α-defensins (proCrps) to bactericidal forms by proteolysis at specific proregion cleavage sites. MMP7(-/-) mice lack mature α-defensins in Paneth cells, accumulating unprocessed precursors for secretion. To test for activation of secreted pro-α-defensins by host and microbial proteinases in the absence of MMP7, we characterized colonic luminal α-defensins. Protein extracts of complete (organ plus luminal contents) ileum, cecum, and colon of MMP7-null and wild-type mice were analyzed by sequential gel permeation chromatography/acid-urea polyacrylamide gel analyses. Mature α-defensins were identified by N-terminal sequencing and mass spectrometry and characterized in bactericidal assays. Abundance of specific bacterial groups was measured by qPCR using group specific 16 S rDNA primers. Intact, native α-defensins, N-terminally truncated α-defensins, and α-defensin variants with novel N termini due to alternative processing were identified in MMP7(-/-) cecum and colon, and proteinases of host and microbial origin catalyzed proCrp4 activation in vitro. Although Paneth cell α-defensin deficiency is associated with ileal microbiota alterations, the cecal and colonic microbiota of MMP7(-/-) and wild-type mice were not significantly different. Thus, despite the absence of MMP7, mature α-defensins are abundant in MMP7(-/-) cecum and colon due to luminal proteolytic activation by alternative host and microbial proteinases. MMP7(-/-) mice only lack processed α-defensins in the small intestine, and the model is not appropriate for studying effects of α-defensin deficiency in cecal or colonic infection or disease.

Involvement of xylem sap zeatin- O-glucoside in cucumber shoot greening

A fraction of xylem sap collected from squash ( Cucurbita maxima) root and separated with gel filtration showed strong promoting activity in a greening bioassay using etiolated cucumber cotyledons. This activity decreased with waterlogging of the root. The promoting factor was purified with sequential gel-permeation, and reverse-phase and normal-phase column chromatographies. Zeatin- O-glucoside (ZOG) was identified as the promoting factor by monitoring liquid chromatography tandem mass spectrometry. Although the glycosyl conjugate of zeatin was thought to be inactive, ZOG had a promoting activity that was 100 times higher than those of zeatin and zeatin riboside between concentrations of 10 –9 to 10 –4 M in the greening bioassay. These results suggest that in some developmental stages, the conjugation of cytokinins with a sugar moiety, such as glucose, might be a key factor in the control of shoot greening by roots.

Purification to homogeneity and characterization of major fatty acid ethyl ester synthase from human myocardium

Non-oxidative metabolism of ethanol via fatty acid ethyl ester synthase is present in those extrahepatic organs most commonly damaged by alcohol abuse. DEAE-cellulose chromatography of human myocardial cytosol at pH 8.0 separated synthase I, minor and major activities, eluting at conductivities of 5,7 and 11 mS, respectively. The major synthase was purified 8900-fold to homogeneity by sequential gel permeation, hydrophobic interaction, and anti-human albumin affinity-chromatographies with an overall yield of 2596. SDS-PAOE showed a single polypeptide with a molecular mass of 26 kDa and gel permeation chromatography under nondenaturing conditions indicated a molecular mass of 54 kDa for the active enzyme. The purified enzyme catalyzed ethyl ester synthesis at the highest rates with unsaturated octadecanoic fatty acid substrates ( V max = 100 and 65 nmol/mg h for oleate and linoleate, respectively). K m values for oleate, linoleate, arachidonate, palmitate and stéarate were 0.22 mM, 0.20 mM, 0.13 mM, 0.18 mM and 0.12 mM, respectively. Thus, human heart fatty acid ethyl ester synthase (major form) is a soluble dimeric enzyme comprised of two identical, or nearly identical, subunits ( M r = 26000).

Nonoxidative ethanol metabolism in rabbit myocardium: purification to homogeneity of fatty acyl ethyl ester synthase.

Fatty acyl ethyl esters, previously identified in our laboratory as metabolites of ethanol in human and rabbit myocardium, arise from an esterification of free fatty acids with ethanol in the absence of ATP and coenzyme A. This study was designed to isolate and purify the enzyme(s) in rabbit myocardium that catalyze(s) this reaction. Enzyme activity in homogenates of rabbit myocardium, as assayed by the rate of synthesis of ethyl [14C]oleate from 0.4 mM [14C]oleic acid and 0.2 M ethanol, was 31 nmol/(g.h), and all of it was recovered in the 48400g supernatant. This soluble ethyl ester synthase activity bound to DEAE-cellulose at pH 8, and elution with a NaCl gradient (0-0.25 M) separated two enzyme activities accounting for 13 and 87% of recovered synthase activity. The major enzyme activity was then purified over 5000-fold to homogeneity by sequential gel permeation, hydrophobic interaction, and anti-albumin affinity chromatographies with an overall yield of 40%. Up to 45 micrograms of enzyme was present per g of myocardium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single polypeptide with Mr 26 000, and gel permeation chromatography under nondenaturing conditions indicated a Mr of 50 000 for the active enzyme. Kinetic analyses using the purified enzyme indicated that greatest rates of ethyl ester synthesis were observed with unsaturated octadecanoic fatty acid substrates [Vmax = 1.9 and 1.5 nmol/(mg.s) for linoleate and oleate, respectively], with lesser rates associated with palmitate, stearate, and arachidonate substrates [0.14, 0.03, and 0.35 nmol/(mg.s), respectively].(ABSTRACT TRUNCATED AT 250 WORDS)