Exclusion High Pressure Liquid Chromatography Research Articles

Characterization of the Molecular Weight of Hyaluronan in Eye Products Using a Novel Method of Size Exclusion High-Pressure Liquid Chromatography.

Hyaluronan (HA) exists in two forms, high molecular weight HA (HMWHA) and low molecular weight HA (LMWHA), which have distinct physiological functions. Therefore it is imperative to know the form of HA within pharmaceutical products, including eye products. This study developed an accurate, sensitive, and quantitative method to characterize the form of HA in eye products. Thereafter, the effects of the HA-containing eye products on corneal wound healing were investigated. The MW distributions and concentrations of HA in over the counter eye products were determined by size exclusion chromatography (SEC) high-pressure liquid chromatography (HPLC). The effects of the eye products containing HA on corneal wound healing were characterized both in vitro and in vivo using the scratch assay and the debridement wound model, respectively. The concentrations and MWs of HA were successfully determined within a range of 0.014 to 0.25 mg/mL using SEC HPLC. The concentrations of HA in the ophthalmic products varied from 0.14 to 4.0 mg/mL and the MWs varied from ∼100 kDa to >2500 kDa. All but one HA-containing eye product had an inhibitory effect on corneal wound healing, whereas pure HA promoted corneal wound healing. A novel SEC-HPLC method was developed for quantifying and characterizing the MW of HA in eye products. Although HA promoted corneal wound healing, HA-containing eye products inhibited corneal wound healing, likely caused by preservatives. SEC-HPLC could be implemented as a routine method for determining the form of HA in commercially available ophthalmic products.

Early verification of myocardial ischemia with a novel biomarker of acute tissue damage: C-reactive protein fractional forms

BackgroundWe evaluated the utility of an independent biomarker of early ischemic cellular damage—circulating fractional forms of C-reactive protein (fracCRP), to verify the diagnostic relevance of low Troponin I (TnI) values within the context of a workup for Acute Coronary Syndrome (ACS). MethodsOn a semi-preparative scale, the molecular characteristics of fracCRP were established by electron microscopy and Western Blot, using isolates captured from patient serum on phosphorylcholine beads and purified by size exclusion high-pressure liquid chromatography (SE-HPLC). Captured on an analytical scale, the diagnostic utility of fracCRP was evaluated in first-draw plasma specimens (total CRP not exceeding 6mg/l) recovered from 300 cardiac emergency patients with final discharge diagnoses of ACS ruled out (N=132) or ruled in (N=168). ResultsAt a cutoff value chosen for 97.7% test specificity, the test metric (fracCRP×TnI) identified in the first blood draw 39.9% of all emergency patients ultimately diagnosed with ACS, and 17.9% of ultimately diagnosed patients who arrived with TnI values within the normal reference range (0.01–0.04ng/ml). ConclusionsThese findings suggest that the fracCRP test metric could serve as a rule-in test for ACS in a significant proportion of low to moderate risk emergency patients.

Molecular Control of Phenoloxidase-induced Melanin Synthesis in an Insect

The melanization reaction induced by activated phenoloxidase in arthropods must be tightly controlled because of excessive formation of quinones and excessive systemic melanization damage to the hosts. However, the molecular mechanism by which phenoloxidase-induced melanin synthesis is regulated in vivo is largely unknown. It is known that the Spätzle-processing enzyme is a key enzyme in the production of cleaved Spätzle from pro-Spätzle in the Drosophila Toll pathway. Here, we provide biochemical evidence that the Tenebrio molitor Spätzle-processing enzyme converts both the 79-kDa Tenebrio prophenoloxidase and Tenebrio clip-domain SPH1 zymogen to an active melanization complex. This complex, consisting of the 76-kDa Tenebrio phenoloxidase and an active form of Tenebrio clip-domain SPH1, efficiently produces melanin on the surface of bacteria, and this activity has a strong bactericidal effect. Interestingly, we found the phenoloxidase-induced melanization reaction to be tightly regulated by Tenebrio prophenoloxidase, which functions as a competitive inhibitor of melanization complex formation. These results demonstrate that the Tenebrio Toll pathway and the melanization reaction share a common serine protease for the regulation of these two major innate immune responses.

A field survey of metal binding to metallothionein and other cytosolic ligands in liver of eels using an on-line isotope dilution method in combination with size exclusion (SE) high pressure liquid chromatography (HPLC) coupled to Inductively Coupled Plasma time-of-flight Mass Spectrometry (ICP-TOFMS)

The effect of metal exposure on the accumulation and cytosolic speciation of metals in livers of wild populations of European eel with special emphasis on metallothioneins (MT) was studied. Four sampling sites in Flanders showing different degrees of heavy metal contamination were selected for this purpose. An on-line isotope dilution method in combination with size exclusion (SE) high pressure liquid chromatography (HPLC) coupled to Inductively Coupled Plasma time-of-flight Mass Spectrometry (ICP-TOFMS) was used to study the cytosolic speciation of the metals. The distribution of the metals Cd, Cu, Ni, Pb and Zn among cytosolic fractions displayed strong differences. The cytosolic concentration of Cd, Ni and Pb increased proportionally with the total liver levels. However, the cytosolic concentrations of Cu and Zn only increased above a certain liver tissue threshold level. Cd, Cu and Zn, but not Pb and Ni, were largely associated with the MT pool in correspondence with the environmental exposure and liver tissue concentrations. Most of the Pb and Ni and a considerable fraction of Cu and Zn, but not Cd, were associated to High Molecular Weight (HMW) fractions. The relative importance of the Cu and Zn in the HMW fraction decreased with increasing contamination levels while the MT pool became progressively more important. The close relationship between the cytosolic metal load and the total MT levels or the metals bound on the MT pool indicates that the metals, rather than other stress factors, are the major factor determining MT induction.

Effect of 4-Hydroxy-2-nonenal Modification on α-Synuclein Aggregation

Several observations have implicated oxidative stress and aggregation of the presynaptic protein alpha-synuclein in the pathogenesis of Parkinson disease. alpha-Synuclein has been shown to have affinity for unsaturated fatty acids and membranes enriched in polyunsaturated fatty acids, which are especially sensitive to oxidation under conditions of oxidative stress. One of the most important products of lipid oxidation is 4-hydroxy-2-nonenal (HNE), which has been implicated in the pathogenesis of Parkinson disease. Consequently, we investigated the effects of the interaction of HNE with alpha-synuclein. Incubation of HNE with alpha-synuclein at pH 7.4 and 37 degrees C resulted in covalent modification of the protein, with up to six HNE molecules incorporated as Michael addition products. Fourier transform infrared and CD spectra indicated that HNE modification of alpha-synuclein resulted in a major conformational change involving increased beta-sheet. HNE modification of alpha-synuclein led to inhibition of fibrillation in an HNE concentration-dependent manner. This inhibition of fibrillation was shown to be due to the formation of soluble oligomers based on size exclusion high pressure liquid chromatography and atomic force microscope data. Small angle x-ray scattering analysis indicated that the HNE-induced oligomers were compact and tightly packed. Treatment with guanidinium chloride demonstrated that the HNE-induced oligomers were very stable with an extremely slow rate of dissociation. Addition of 5 mum HNE-modified oligomers to primary mesencephalic cultures caused marked neurotoxicity because the integrity of dopaminergic and GABAergic neurons was reduced by 95 and 85%, respectively. Our observations indicate that HNE modification of alpha-synuclein prevents fibrillation but may result in toxic oligomers, which could therefore contribute to the demise of neurons subjected to oxidative damage.

Structural and Sequence Motifs in Dermatan Sulfate for Promoting Fibroblast Growth Factor-2 (FGF-2) and FGF-7 Activity

Glycosaminoglycans have been implicated in the binding and activation of a variety of growth factors, cytokines, and chemokines. In this way, glycosaminoglycans are thought to participate in events such as development and wound repair. In particular, heparin and heparan sulfate have been well studied, and specific aspects of their structure dictate their participation in a variety of activities. In contrast, although dermatan sulfate participates in many of the same biological processes as heparin and heparan sulfate, the interactions of dermatan sulfate have been less well studied. Dermatan sulfate is abundant in the wound environment and binds and activates growth factors such as fibroblast growth factor-2 (FGF-2) and FGF-7, which are present during the wound repair process. To determine the minimum size and sulfation content of active dermatan sulfate oligosaccharides, dermatan sulfate was first digested and then separated by size exclusion high pressure liquid chromatography, and the activity to facilitate FGF-2 and FGF-7 was assayed by the cellular proliferation of cell lines expressing FGFR1 or FGFR2 IIIb. The minimum size required for the activation of FGF-2 was an octasaccharide and for FGF-7 a decasaccharide. Active fractions were rich in monosulfated, primarily 4-O-sulfated, disaccharides and iduronic acid. Increasing the sulfation to primarily 2/4-O-sulfated and 2/6-O-sulfated disaccharides did not increase activity. Cell proliferation decreased or was abolished with higher sulfated dermatan sulfate preparations. This indicated a preference for specific dermatan sulfate oligosaccharides capable of promoting FGF-2- and FGF-7-dependent cell proliferation. These data identify critical oligosaccharides that promote specific members of the FGF family that are important for wound repair and angiogenesis.

Purification of a myotoxin from the toadfish Thalassophryne maculosa (Günter) venom

Venom was milked by gently pressing the base of the opercular and dorsal fin spines. Three fractions were obtained by molecular exclusion high pressure liquid chromatography (HPLC) (Protein Pak™ 125SW, Millipore Corporation) column, but only the last one with 22.7 min retention time (rt) was biological active (TmPP-22.7). This fraction was rechromatographed on reversed phase HPLC chlorobutylsilane columns (C4, Vydac) nine fractions were obtained, but only one (TmC4-47.2) with 47.2 min rt was biologically active. MALD-TOF mass analysis was carried out on two samples of TmC-47.2 and the results were 15,161.36 and 15,154.70 a.m.u., respectively. Raw venom (1040 μg/ml) depolarised frog ( Hyla crepitans) muscle irreversibly from −85 (−88, −81) mV ( n=20, median and its 95% CI) to −18 (−24, −15) mV ( n=24). The biological activity in TmPP-22.7 (38 μg/ml), which depolarised muscle fibres from −79 (−82, −76) mV ( n=20) to −63 (−69 −57) mV ( n=24). The depolarising fraction was TmC4-47.2 (50 μg/ml) which depolarised muscles from −87 (91, −82) mV ( n=33) to −63 (−76 −51) mV ( n=53); the depolarising effect at this concentration was completely reversed on washing with normal saline for 2 h. Muscles treated with 1 μM tetrodotoxin (TTX) were depolarised from −80 (−85, −72) mV ( n=49) to −44 (−56, −31) mV ( n=44) when 100 μg/ml TmC4-47.2 were applied with TTX; washing 130 min with 1 μM TTX repolarised to −59 (−69, −50) mV ( n=25). We also present evidence that TmC4-47.2 induces myonecrosis in mice.

Dextran-binding human plasma antibody recognizes bacterial and yeast antigens and is inhibited by glucose concentrations reached in diabetic sera

Dextran-binding antibody was isolated in high yield from plasma of all 40 blood donors screened in a South Indian population. The antibody was purified by a single step affinity chromatography on Sephadex G100 using 1- O-methyl α- d-glucoside as eluant. Analysis of protein peaks obtained in size exclusion high pressure liquid chromatography (HPLC) revealed dominance of IgG and suggested the presence of polymeric IgA in this antibody. Methyl and para-nitrophenyl α- d-glucosides, in contrast to their β-anomers, were very efficient inhibitors of binding of this antibody to dextran. Galactose and glucose were equally good inhibitors. Among disaccharide inhibitors sucrose was more efficient than maltose or melibiose. Hemoglobin artificially glycosylated to contain covalently-linked glucose or α-anomeric galactose was sugar-specifically recognized by this antibody. Galactose moieties in glycoproteins or polysaccharides were, however, not recognized. The dextran-binding antibody bound sugar-specifically to glycoconjugates from yeast ( Saccharomyces cerevisiae) and to lipopolysaccharides from Klebsiella and group A Streptococci, but not to lipopolysaccharides from E. coli. Inhibition studies suggested glucose moiety with unsubstituted C2 and C4 and α-anomeric C1 as ideal for recognition by the dextran-binding antibody. Concentration of glucose required for 50% inhibition of binding of the purified antibody to polystyrene-coated dextran in phosphate buffered saline was above the glucose concentrations in normal sera, but well below those reached in diabetic sera. Binding of the antibody from dialysed plasma to immobilized dextran was lowered only marginally in presence of glucose at 4.5 mM (which nears normal serum glucose concentrations), but substantially in presence of the sugar at 20 mM and above which are reached in diabetic sera. If verified in vivo, inhibition of this antibody by high serum glucose may possibly be among reasons for the increased susceptibility of diabetics to infection.

Lysozyme: a mediator of myocardial depression and adrenergic dysfunction in septic shock in dogs

The objective of the present study was to identify the nature of a filterable cardiodepressant substance (FCS) that contributes to myocardial dysfunction in a canine model of Escherichia coli septic shock. In a previous study, it was found that FCS increased in plasma after 4 h of bacteremia (Am J Physiol 1993;264:H1402) in which FCS was identified by a bioassay that included a right ventricular trabecular (RVT) preparation. In that study, FCS was only partially identified by pore filtration techniques and was found to be a protein of molecular weight between 10 and 30 K. In the present study, FCS was further purified by size exclusion high-pressure liquid chromatography, until a single band was identified on one-dimensional gel electrophoresis. This band was then subjected to tandem mass spectrometry and protein-sequencing techniques and both techniques identified FCS as lysozyme c (Lzm-S), consistent with that originating from the canine spleen. Confirmatory tests showed that purified Lzm-S produced myocardial depression in the RVT preparation at concentrations achieved during sepsis in the in vivo preparation. In addition, Lzm-S inhibited the adrenergic response induced by field stimulation and the β - agonist isoproterenol in in vitro preparations, these results suggesting that Lzm-S may inhibit the sympathetic response in sepsis. The present findings indicate that Lzm-S originating from disintegrating leukocytes from organs such as the spleen contributes to myocardial dysfunction in this model. The mechanism may relate to its binding or hydrolysis of a cardiac membrane glycoprotein thereby interfering with myocardial excitation–contraction coupling in sepsis.

Quaternary Structure of Rice Nonsymbiotic Hemoglobin

Plant nonsymbiotic hemoglobins are hexacoordinate heme proteins found in all plants. Although expression is linked with hypoxic environmental conditions (Taylor, E. R., Nie, X. Z., Alexander, W. M., and Hill, R. D. (1994) Plant Mol. Biol. 24, 853-862), no discrete physiological function has yet been attributed to this family of proteins. The crystal structure of a nonsymbiotic hemoglobin from rice has recently been determined. The crystalline protein is homodimeric and hexacoordinate with two histidine side chains coordinating the heme iron atom. Despite the fact that the amino acids responsible for the subunit interface are relatively conserved among the nonsymbiotic hemoglobins, previous work suggests that this group of proteins might display variability in quaternary structure (Duff, S. M. G., Wittenberg, J. B., and Hill, R. D. (1997) J. Biol. Chem. 272, 16746-16752; Arredondo-Peter, R., Hargrove, M. S., Sarath, G., Moran, J. F., Lohrman, J., Olson, J. S., and Klucas, R. V. (1997) Plant Physiol. 115, 1259-1266). Analytical ultracentrifugation and size exclusion high pressure liquid chromatography were used to investigate the quaternary structure of rice nonsymbiotic hemoglobin at various states of ligation and oxidation. Additionally, site-directed mutagenesis was used to test the role of several interface amino acids in dimer formation and ligand binding. Results were analyzed in light of possible physiological functions and indicate that the plant nonsymbiotic hemoglobins are not oxygen transport proteins but more closely resemble known oxygen sensors.

Testing and comparing oxidative stability of vegetable oils and fats at frying temperature

A new method has been developed to estimate the stabilising activity of synthetic and natural food additives at frying. Non-refined and refined vegetable fats and oils were heated at a temperature of 170°C after adding water-conditioned silica gel for two hours. The degraded products were measured to assess the oil stability at frying temperature. The determination of polymeric triglycerides by size exclusion high-pressure liquid chromatography (HPLC) was carried out for the estimation of the oxidative heat stability of vegetable fats and oils. Tocopherols, various tocopherol esters and phytosterol fractions, phenolic compounds, like quercetin, oryzanol, ferulic acid, squalene, butyl hydroxytoluol (BHT), butyl hydroxyanisol (BHA), and other compounds, like ascorbic acid 6-palmitate and gallates, are added to refined sunflower and rapeseed oil and their efficacy determined. Both linoleic and oleic rich oils gave comparable results for the activity of the various compounds. α-tocopherol, tocopherol esters, and BHA have low effects at frying temperature. Ascorbic acid 6-palmitate and some phytosterol fractions were found to have the greatest antioxidant activity. Corn oil was more stable than soybean oil and rapeseed oil better than olive oil. It was also observed that non-refined oils proved to have a better stability at elevated temperature than refined oils. The results show that the stability of the vegetable oils at frying temperature is a function of more than just the fatty acid composition. There is evidence which supports a co-relationship between the unsaponifiable matter content and oxidative stability. It is believed that a radical peroxidation mechanism predominates at lower temperatures. When a large volume of oil is heated in a fryer and the oxygen supply is poor, non-radical reactions such as elimination (acid catalysed dehydration) or nucleophilic substitution take place.

Testing and comparing oxidative stability of vegetable oils and fats at frying temperature

A new method has been developed to estimate the stabilising activity of synthetic and natural food additives at frying. Non-refined and refined vegetable fats and oils were heated at a temperature of 170°C after adding water-conditioned silica gel for two hours. The degraded products were measured to assess the oil stability at frying temperature. The determination of polymeric triglycerides by size exclusion high-pressure liquid chromatography (HPLC) was carried out for the estimation of the oxidative heat stability of vegetable fats and oils. Tocopherols, various tocopherol esters and phytosterol fractions, phenolic compounds, like quercetin, oryzanol, ferulic acid, squalene, butyl hydroxytoluol (BHT), butyl hydroxyanisol (BHA), and other compounds, like ascorbic acid 6-palmitate and gallates, are added to refined sunflower and rapeseed oil and their efficacy determined. Both linoleic and oleic rich oils gave comparable results for the activity of the various compounds. α-tocopherol, tocopherol esters, and BHA have low effects at frying temperature. Ascorbic acid 6-palmitate and some phytosterol fractions were found to have the greatest antioxidant activity. Corn oil was more stable than soybean oil and rapeseed oil better than olive oil. It was also observed that non-refined oils proved to have a better stability at elevated temperature than refined oils. The results show that the stability of the vegetable oils at frying temperature is a function of more than just the fatty acid composition. There is evidence which supports a co-relationship between the unsaponifiable matter content and oxidative stability. It is believed that a radical peroxidation mechanism predominates at lower temperatures. When a large volume of oil is heated in a fryer and the oxygen supply is poor, non-radical reactions such as elimination (acid catalysed dehydration) or nucleophilic substitution take place.

A Curve-Fitting Approach to the near Infrared Reflectance Measurement of Wheat Flour Proteins Which Influence Dough Quality

A curve-fitting approach to the problem of measuring the gliadin and glutenin content of flour is presented. Theoretical spectra for 100% pure gliadin, glutenin and starch are derived based on the analysis of partially purified materials and are shown to have unique characteristics. Spectral differences at 2306 nm and 2350 nm were attributed to the presence of lipid in the glutenin component and a least squares curve-fitting procedure over the range 2050–2220 nm has been used to calculate the proportion of each component present in the spectrum of a flour. High r2 values were achieved when the results were compared with the actual values as measured by size exclusion high pressure liquid chromatography. The results for gliadin ( r2 = 0.73) and glutenin ( r2 = 0.76) suggest that the curve-fitting methodology offers a simple alternative to partial least squares (PLS) and multiple linear regression (MLR) techniques for ranking samples by gliadin and glutenin content. Although there was a large bias between the actual and uncorrected predicted values for gliadin and glutenin content, the results provided a clear justification for assuming that robust calibrations for gliadin and glutenin content can be developed using both curve-fitting and PLS calibration techniques. The least squares methodology can be used as a simple technique for estimating the gliadin and glutenin content of flours where the development and maintenance of a full calibration is either impractical or not desirable.

Inhibition of HLA antibody cytotoxicity by intravenous immunoglobulin G F(ab′) 2 dimers, monomers, and monovalent F(ab)

IVIgG preparations are clinically relevant to sensitized transplant candidates because they inhibit HLA alloantibody in vitro and in vivo. We hypothesized that IVIgG F(ab′) 2 idiotypic-antiidiotypic dimers may possess a greater immunomodulatory capacity when remonomerized than non-dimerizable F(ab′) 2 monomers in IVIgG. We reasoned that when 60–75% of potential antiidiotypic IVIgG monomers fail to bind to IVIgG molecules in a large pool of plasma donors (>10,000), IVIgG monomers may fail to inhibit HLA idiotypic antibodies of sensitized transplant candidates. In the first series of AHG T cell crossmatches, non-fractionated IVIgG F(ab′) 2 was found to inhibit titered HLA antibodies in 13 out of 29 (45%) crossmatch combinations. Crossmatch inhibition was incomplete, i.e., a particular titered HLA antibody specificity was not always inhibited by IVIgG F(ab′) 2 in every HLA antigen-matched target cell crossmatch. Next, the IVIgG F(ab′) 2 product was fractionated into F(ab′) 2 dimers, F(ab′) 2 monomers and Fab monovalent components by size exclusion high pressure liquid chromatography (HPLC) and retested in crossmatches which previously demonstrated inhibition. The percent of crossmatches that were inhibited by HPLC F(ab′) 2 IVIgG fractions in three separate experiments was statistically similar for pH 4.0 remonomerized dimers, 82%; pH 6.0 dimers, 50%; monomers, 64%; and monovalent Fab, 64% ( p = 0.50) . Soluble class I HLA antigen was undetectable in IVIgG F(ab′) 2 by an ELISA assay. In conclusion, IVIgG dimers and monomers appear to have similar immunomodulatory capacities, and separation of whole IVIgG products into dimer and monomer fractions does not appear to be warranted. Further, IVIgG products should be tested for optimal HLA antibody inhibition in vitro prior to in vivo therapy.

Degradation of Low and High Molecular Mass Fractions of Kraft E1 Effluent by Trametes Villosa

A very efficient Trametes villosa strain was selected from a screening in our laboratories, which exhibited a high capability of bleaching effluent (E1) biodegradation. This fungus decolorized 70–80% in 24–48h incubation time and degraded 80% of total phenol acting on E1 effluent. Size exclusion high pressure liquid chromatography (HPLC) showed that 75% of total color can be attributed to the high-molecular mass materials (MM> 15000 Da). The change in the MM distribution during the incubation period showed evidence that both high and low molecular mass fractions were biodegraded by the fungal treatment. The HPLC analyses of the original E1 effluent (MM< 1000 Da) revealed that four compounds were responsible for 99% of the sample composition.

Subunit Composition of Pro-phenol Oxidase from Manduca sexta: Molecular Cloning of Subunit ProPO-p1

Phenol oxidase (PO) is known to play an important role in defense mechanisms in insect immunity. It is present as a zymogen in insect hemolymph, and can be activated by a specific proteolytic reaction that is stimulated by microbial cell wall components. The pro-phenol oxidase (pro-PO) purified from the larval hemolymph of Manduca sexta contains two polypeptides in equal amounts as revealed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). A cDNA for one of the polypeptides, now designated proPO-p2, has been isolated ( Hall et al. (1995) Proc. Natl. Acad. Sci. USA, 92, 7764–7768). We purified pro-PO from plasma of M. sexta and characterized its subunit composition. A cDNA for M. sexta proPO-p1 was isolated from a larval hemocyte cDNA library. M. sexta proPO-p1 is 78% identical in amino acid sequence to Bombyx mori proPO-p1, but only 50% to M. sexta or B. mori proPO-p2. Immunofluorescence labelling and in situ hybridization showed that the pro-PO is synthesized in a single hemocyte type, the oenocytoids. Analysis of pro-PO by size exclusion high-pressure liquid chromatography (HPLC) revealed that pro-PO exists as monomeric, dimeric, trimeric or multimeric structures depending on the ionic strength. All of these isoforms of the protein have phenol oxidase activity upon activation with a detergent, cetylpyridinium chloride. In analysis by non-denaturing PAGE, the majority of the purified pro-PO was present as two dimers of distinct mobility (fast and slow forms). Both forms contain proPO-p1 and proPO-p2, suggesting that they are heterodimers. Individual larvae can contain the slow form, the fast form, or both, which suggests that the slow and fast forms of proPO are allelic variants. These results indicate that there are two pro-PO genes in M. sexta, which are coordinately expressed in oenocytoids, and whose products form predominantly heterodimers in plasma.

Reconstitution of Monomethylamine:Coenzyme M Methyl Transfer with a Corrinoid Protein and Two Methyltransferases Purified fromMethanosarcina barkeri

Methanogenesis from methylamines requires the intermediate methylation of 2-mercaptoethanesulfonate (CoM). In vitro reconstitution of CoM methylation with monomethylamine was achieved with three purified proteins: a monomethylamine corrinoid protein (MMCP), the "A" isozyme of methylcobamide:CoM methyltransferase (MT2-A), and a newly isolated protein termed monomethylamine methyltransferase (MMAMT).MMAMT is a 170-kDa protein with 52-kDa subunits. The MMAMT polypeptide was rate-limiting for methyl transfer until at a 2-fold molar excess over MMCP. MMAMT is a monomethylamine:MMCP methyltransferase, since methylation of MMCP required MMAMT but not MT2-A. MMCP and MMAMT formed a complex detectable by size exclusion high pressure liquid chromatography. Methyl group transfer from methyl-MMCP to CoM was mediated by MT2-A, since methyl iodide:CoM methyl transfer by MMCP and MT2-A did not require MMAMT. MT2-M, an isozyme of MT2-A, was inactive in MMCP-dependent methyl transfer. Immunodepletion of MMCP from the extract inhibited CoM methylation with monomethylamine but not dimethylamine. Purified MMCP reconstituted activity in immunodepleted extracts. These results show that MMCP is the major corrinoid protein for methanogenesis from monomethylamine detectable in extracts and that it interacts with two methyltransferases. MMAMT functions as a MMA:MMCP methyltransferase, while MT2-A functions as a methyl-MMCP:CoM methyltransferase.

The Role of the Circulation in Processing pro-Brain Natriuretic Peptide (proBNP) to Amino-Terminal BNP and BNP-32

Hunt, P. J., E. A. Espiner, G. M. Nicholls, A. M. Richards and T. G. Yandle. The role of the circulation in processing pro-brain natriuretic peptide (proBNP) to amino-terminal BNP and BNP-32. Peptides 18(10) 1475–1481, 1997.—Human proBNP (purified from cardiac tissue) was incubated at 37°C in whole blood, serum and plasma and the products analyzed by size exclusion high pressure liquid chromatography and radioimmunoassay (RIA). In addition to RIAs for BNP-32 and NT-proBNP(1-13), a newly developed RIA for proBNP(62-76) was also used to identify the peptides. Incubation with serum resulted in the formation of a 9 kDa and a 3 kDa peptide, consistent with the N-terminal and the C-terminal peptides of the propeptide. Minimal processing of proBNP was seen in blood or plasma, suggesting that the circulation does not play a major role in the activation of proBNP. Analysis of degradation products of human proBNP using site directed specific antisera indicates that removal of N-terminal amino acids from proBNP occurs in serum. These findings support the view that the “high molecular weight BNP-32” previously identified in human plasma comprises amino-terminal deleted forms, and is unlikely to be intact proBNP(1-108).

Comparison of liquid and gas chromatography for analysis of low molecular weight organic acids in rhizosphere soil

Low molecular weight organic acids from rhizosphere soil of durum wheat (Triticum turgidum var. durum L.) were studied by ion exclusion high pressure liquid chromatography (HPLC) and gas chromatography (GC). Ion exclusion HPLC was found unsatisfactory for acid determination because poor peak resolution hindered acid identification, strongly ionized solutes and oxalic acid co‐eluted in the dead volume of the column, and UV detection was not sensitive for aliphatic acid determination. Excellent resolution was obtained when the same low molecular weight organic acids were analyzed by GC. Non‐volatile low molecular weight organic acids were separated as trimethylsilyl (TMS) derivatives while volatile acids were analyzed without sample derivatization. A combination of these two GC techniques allowed for separation of 16 acids. Ten low molecular weight organic acids were identified and quantified in rhizosphere soil of durum wheat grown in three different soils. Succinic and acetic acids were most abundant among acids extracted from rhizosphere soils followed by butyric, malic, propionic and tartaric acids. Malonic, fumaric, citric, and trans‐aconitic acids were found at the lowest concentrations. Very good resolution, short analysis time, and low detection limits make the GC method very suitable for routine determinations of low molecular organic acids in rhizosphere soil.

Interaction of the bHLH-zip domain of c-Myc with H1-type peptides. Characterization of helicity in the H1 peptides by NMR.

The carboxyl-terminal 92 residues of c-Myc-92 display site-specific DNA binding specificity for the consensus sequence 5'-CACCACGTGGTG-3' (Blackwell, T. K., Kretzner, L., Blackwood, E. M., Eisenman, R. N., and Weintraub, H. (1990) Science 250, 1149-1151). Size exclusion high pressure liquid chromatography analysis of the purified fusion protein, glutathione S-transferase-c-Myc-92, indicates that c-Myc-92 is tetrameric with a dissociation constant of < 60 nM. Helix-1 (H1) and leucine zipper peptides from the basic-helix-loop-helix-zipper domain of c-Myc and Max were assayed as potential inhibitors of c-Myc-92 DNA binding. H1 peptides with substitutions that confer greater helicity are found to inhibit c-Myc-92 DNA binding. The mechanism of inhibition involves the cooperative binding of H1 peptides with tetrameric c-Myc-92 as determined by a spectrophotometric assay employing 2,4-dinitrophenyl-H1-F8A. NMR structural characterization reveals a correlation between helicity and inhibition. In a partially hydrophobic environment, H1-Mx (from Max) is a random coil, while H1-WT, H1-F8A, and H1-F8A,S6A (from c-Myc) display differing degrees of helicity. Structure determination on the basis of nuclear Overhauser effect data indicates that the H1-F8A helix is significantly more ordered than H1-WT. Analysis on the basis of the Max x-ray structure (Ferré-D'Amaré, R., Prendergast, G. C., Ziff, E. B., and Burley, S. K. (1993) Nature 363, 38-45) suggests that H1 peptide binding to c-Myc-92 may occur through an alteration in the packing of helix-1 in c-Myc-92 or through an interaction with an exposed hydrophobic cluster of residues at each H1-H2 interface. This binding site for H1 peptides may be of significance in the interaction of c-Myc with proteins involved in transcriptional regulation.