C18 Reversed-phase HPLC Research Articles

Lumbricin I, a novel proline-rich antimicrobial peptide from the earthworm: purification, cDNA cloning and molecular characterization

A novel antimicrobial peptide was isolated and characterized from the earthworm, Lumbricus rubellus. The antimicrobial peptide was purified to homogeneity by a heparin-affinity column and C18 reverse-phase HPLC, and named lumbricin I. Lumbricin I was a proline-rich antimicrobial peptide of 62 amino acids (15% proline in molar ratio; molecular mass, 7231 Da), whose complete sequence was determined by a combination of peptide sequence and cDNA analysis. The peptide and cDNA sequence analysis revealed that lumbricin I was produced as a precursor form consisting of 76 amino acids, with 14 residues in a presegment and 62 residues in mature lumbricin I. Lumbricin I showed antimicrobial activity in vitro against a broad spectrum of microorganisms without hemolytic activity. In addition, a 29-amino acid peptide, named lumbricin I(6–34), which was derived from residues 6–34 of lumbricin I, showed marginally stronger antimicrobial activity than lumbricin I. Northern blot analysis on total RNA revealed that expression of lumbricin I gene was not induced by bacterial infection, but was constitutively expressed. Furthermore, the expression of lumbricin I gene was specific in adult L. rubellus: Lumbricin I mRNA was detected only in adult L. rubellus, but not in eggs and young L. rubellus.

Characterization of Aplysia Attractin, the First Water-borne Peptide Pheromone in Invertebrates

Although animals in the genus Aplysia are solitary during most of the year, they form breeding aggregations during the reproductive season. The aggregations contain both mating and egg-laying animals and are associated with masses of egg cordons. The egg cordons are a source of pheromones that establish and maintain the aggregation, but none of the pheromonal factors have been chemically characterized. In these studies, specimens of Aplysia were induced to lay eggs, the egg cordons collected and eluted, and the eluates fractionated by C18 reversed-phase HPLC. Four peak fractions were bioassayed in a T-maze. All four increased the number of animals attracted to a nonlaying conspecific and were thus subjected to compositional and microsequence analysis. Each contained the same NH2-terminal peptide sequence. The full-length peptide ("attractin") was isolated from the albumen gland, a large exocrine organ that packages the eggs into a cordon. The complete 58-residue sequence was obtained, and it matched that predicted by an albumen gland cDNA. Mass spectrometry showed that attractin is 21 wt.% carbohydrate as the result of N-linked glycosylation. T-maze bioassays confirmed that the full-length peptide is attractive. Attractin is the first water-borne peptide pheromone characterized in molluscs, and the first in invertebrates.

Investigation of far red dyes for use in peroxyoxalate chemiluminescence detection and analysis of the cy5 derivative of amantadine hydrochloride in human plasma.

Peroxyloxalate chemiluminescence is well established as a tool for improvement of selectivity and sensitivity for chemiluminophores and their derivations in HPLC eluates. Chemiluminescence in the far-red spectral region was investigated in this work to further enhance the sensitivity of chemiluminescence through more efficient singlet excitation energy transfer and to enhance the selectivity of the approach through a reduction in matrix and scatter interference. A number of fluorescent compounds that can be excited in the UV, visible and far red spectral regions were investigated for chemiluminescence yield using the bis(2,4,6-trichlorphenyl) oxylate reaction. It was found that a trend of increasing chemiluminescence with increasing excitation wavelength could be observed with cy5 providing the most efficient chemiluminescence. The succinate ester of cy5 was used to derivatize amantadine hydrochloride, an antiparkinsons drug, to form the derivative. The derivative was separated from reaction by products by C18 reversed phase HPLC and detected using a Soma S-3400 chemiluminescence detector. The detection limit for the diluted derivative was 200 femtomoles on column and sufficient for plasma analysis. Selectivity in plasma was demonstrated through derivatization of extracts of plasma that had been spiked with amantadine hydrochloride.

Automated analytical/preparative high-performance liquid chromatography–mass spectrometry system for the rapid characterization and purification of compound libraries

A fully automated analytical and preparative HPLC–MS system has been developed and applied to the characterization and purification of compound libraries derived by parallel synthesis. Our automated LC–MS system incorporates fast, reversed-phase C18 HPLC (5–10 min analyses) and electrospray ionization mass spectrometry (ESI–MS). Post-data acquisition purity assessment of compound libraries is performed automatically using applescripts. Compounds falling below a threshold level of purity (≪90%) are subjected to automated on-line preparative HPLC–MS (i.e., PrepLCMS) purification. PrepLCMS is a method that we developed and is the first mass spectrometry-based system that permits automated and rapid purification of multimilligram quantities of compound libraries using a mass spectrometer to `signal' fraction collection. The method utilizes real-time mass spectrometric ion signals to trigger fraction collection and only the mass(es) of the compound(s) identified by the user in the data acquisition method are collected. This one-sample–one-fraction format means that `batches' of compounds can be purified without the need for excessively large fraction collector beds. Further, this eliminates the need for post-purification analysis or pooling of fractions collected, typically associated with other preparative purification techniques. This technique was designed for use by the chemist in an `open access'-like environment, and has been successfully implemented for automated and unattended batch purifications of large numbers of compound libraries derived by parallel synthetic strategies. The results for several compound libraries, synthesized and purified at the multimilligram level by automated PrepLCMS are presented.

Purification and Characterization of a Recombinant Human Cripto-1 Protein

Cripto-1 (CR-1) is a novel protein that contains a modified EGF-like motif and that does not directly bind to any of the known erb B type-1 receptor tyrosine kinase receptors. To more clearly define the biological effects of CR-1 and to more adequately compare the structure-function relationships of CR-1 with other members of the EGF family of growth factors, we have expressed a modified, full-length recombinant human CR-1 protein (rhCR-1) in E. coli and have devised a procedure for the solubilization, refolding and purification of a biologically active form of this protein. We have generated the mature form of hCR-1 from computer assisted predictions of potential signal peptide cleavage sites. Expression of the modified rhCR-1 protein in E. coli was limited to the inclusion bodies. The rhCR-1 protein was found to be expressed at high levels in bacterial cells when fused to a histidine-tag sequence. Refolding of rhCR-1 was found to be difficult because of the large number of cysteine residues in the protein which results in protein aggregation. By chemically modifying the cysteine residues in the rhCR-1 protein with 3-trimethyl-ammoniopropyl methanethiosulfonate, additional positive charges have been introduced into the protein by this disulfiding reagent. This modification facilitates solubilization of the protein when rhCR-1 is denatured. The solubilized, denatured protein was then purified by CM cation exchange and C4 reverse phase HPLC chromatography and refolded in a redox buffer. The refolded, modified rhCR-1 protein was found to be biologically active by its ability to inhibit β-casein expression, to stimulate the tyrosine phosphorylation of She and the activation of MAPK and by its capacity to facilitate branching growth of mouse mammary epithelial cells in type I collagen gels.

Purification of Endogenous Digitalis-Like Factors from Normal Human Urine

We detected two candidates for endogenous digitalis-like factors in human urine based on the inhibition of 3H-ouabain binding to human erythrocytes. Two ouabain-displacing compound(ODC)s were consistently eluted off the C18reverse phase HPLC column with 18% and 31% acetonitrile. The more-polar ODC-1 was ubiquitously found in mammals, markedly increased after acute and chronic salt loading in humans, and was thought to be a natriuretic factor with vasoactive property. ODC-1 mostly resembled ouabain in biological, physicochemical, and chromatographic properties and may correspond to ouabainlike compound purified by other investigators. The less-polar ODC-2 was indistinguishable from digoxin in proton nuclear magnetic resonance(NMR) and fast atom bombardment(FAB) mass spectrum

Affinity cleavage at the metal-binding site of phosphoenolpyruvate carboxykinase.

Chicken liver phosphoenolpyruvate carboxykinase (PEPCK) was rapidly inactivated by micromolar concentrations of ferrous sulfate in the presence of ascorbate at pH 7.4. Omitting ascorbate or replacing the Fe2+ with Mn2+ or Mg2+ gives no inactivation. Mn2+, Mg2+, or Co2+ at 100-fold molar excess over Fe2+ offered complete protection from Fe2+/ascorbate-induced inactivation. The substrates PEP and GTP, but not OAA, GDP, or CO2, offered full protection from inactivation. The addition of 5 mM EDTA stopped further inactivation of the enzyme. Thermodynamic studies indicate that the inactive enzyme no longer binds Mn2+ but still had high affinity for GTP indicating that the inactivation process was specific for the metal site. A decrease in cysteine content was observed over time following PEPCK treatment with Fe2+ and ascorbate. The apparent first-order rate constant for free sulfhydryl loss (0.085 +/- 0.005 min-1) is similar to the apparent first-order rate constant for inactivation (0.067 +/- 0.005 min-1). Amino acid composition analysis revealed that cysteic acid was generated upon Fe2+/ascorbate addition to PEPCK. Native chicken liver PEPCK has an Mr of 67 kDa. SDS-PAGE of the inactivated enzyme showed the presence of two new bands at 31.7 and 35.3 kDa indicating that PEPCK was specifically cleaved at a single site. The rate of cleavage was slower than the rate of inactivation and fully inactivated enzyme was only 50% cleaved. The Fe2+/ascorbate-catalyzed inactivation was not solely due to protein cleavage. The protein fragments generated by cleavage were separated by C4 reverse phase HPLC. The cleavage exposed a new N-terminus which was identified to be the 35.3 kDa C-terminal half of PEPCK. Sequencing of the fragments indicated that the site of cleavage was between Asp296 and Ile297. These results indicate that Asp296 is involved in metal chelation. This agrees with previous studies [Hlavaty, J. J., & Nowak, T. (1997) Biochemistry 36, 3389-3403] that suggested that Asp295 and Asp296 are involved in metal binding.

Human kallikrein hK2 has low kininogenase activity while prostate-specific antigen (hK3) has none

In the present paper, we determined the kinin-releasing activity of human prostatic kallikrein hK2 and compared it to one of the kallikreins hK1 and prostate specific antigen (hK3). Kinin-like substances active on the rabbit jugular vein were progressively produced when nanomolar concentrations of hK2 were incubated with heated plasma. However in these experiments, hK1 appeared much more potent than hK2 while hK3 was totally inactive. When hK2 was incubated with purified high molecular weight kininogen, several peptides were generated as shown by the analysis on C18 reverse-phase HPLC. Kinin activity was localized exclusively in a small peak having an elution time identical to that of bradykinin while the only important peak obtained with hK1 corresponded to Lys-bradykinin. Finally, the rate of kinin production of hK2 was found to be more than a thousandfold lower than that of hK1. These experiments show that kallikreins hK2 has only a low kininogenase activity. However, it is not excluded that some of the peptides produced by hK2 action could have other types of biological activity.

Purification, Amino Acid Sequence, Synthesis, and Receptor Selectivity of Alligator Gastrin

Gastrin-like immunoreactive peptides were extracted from the gastric antrum of the American alligator (Alligator mississippiensis) and purified by fractionation using C18Sep-Paks, Sephadex G-50, pH stable C8reversed-phase HPLC, and C18reversed-phase HPLC. Three major immunoreactive peaks were purified and found to correspond to 49, 45, and 34 residue peptides by microsequence analysis. The amino acid sequence of the largest peptide was DWLASLSQDQ KHLISKFLPH IYGELAN QEN YWQEDDALHD HDYPGWMDF-amide. The two smaller peptides corresponded to carboxyl-terminal 45 and 34 residue fragments of the 49 residue peptide. The putative proteolysis of the 49 residue peptide to the two shorter peptides occurs at cleavage sites that are unusual in biosynthetic processing. Mass spectral analysis confirmed the molecular weights that were predicted from the amino acid sequences, thus revealing the absence of any post-translational modifications, such as sulfation. Although the three alligator gastrins resemble mammalian cholecystokinin in having a tyrosine residue in the seventh position from the carboxyl terminus, this tyrosine is apparently nonsulfated as in turtle gastrin. When tested by radioreceptor assay, a synthetic replicate of alligator gastrin-49 exhibited a gastrin-like pattern of biological activity on mammalian CCK-A and CCK-B receptors. Comparison of the amino acid sequences of known peptides revealed that alligator gastrin is most similar to turtle gastrin (76% identical), followed by frog gastrin (51% identical), chicken gastrin (49% identical), and human gastrin (12% identical). These similarities closely reflect vertebrate phylogeny and support the hypothesis that functionally distinct gastrins evolved from CCK in early tetrapods. However, gastrin evolved via different mechanisms in the mammalian lineage (mechanism unknown) versus the amphibian and reptilian/avian lineages, in which two different single nucleotide base changes can account for the separate evolution of amphibian gastrin and reptilian/avian gastrin.

Abstract

We detected two candidates for endogenous digitalis-like factors in human urine based on the inhibition of 3H-ouabain binding to human erythrocytes. Two ouabain-displacing compound(ODC)s were consistently eluted off the C18reverse phase HPLC column with 18% and 31% acetonitrile. The more-polar ODC-1 was ubiquitously found in mammals, markedly increased after acute and chronic salt loading in humans, and was thought to be a natriuretic factor with vasoactive property. ODC-1 mostly resembled ouabain in biological, physicochemical, and chromatographic properties and may correspond to ouabainlike compound purified by other investigators. The less-polar ODC-2 was indistinguishable from digoxin in proton nuclear magnetic resonance(NMR) and fast atom bombardment(FAB) mass spectrum

Structural‐based differences in ecotoxicity of benzoquinoline isomers to the zebra mussel (Dreissena polymorpha)

AbstractEffects of four benzoquinoline isomers on the filtration rate of the zebra mussel ( Dreissena polymorpha) were analyzed, to study the effect of minor differences in chemical structure on adverse biological effects. Filtration rates were measured after 48 h of exposure to different concentrations of acridine, phenanthridine, benzo[f]quinoline, and benzo[h]quinoline in the water. The 50% effective concentration (EC50) values for filtration rate of the four isomers differed significantly. Effects increased in the order benzo[f], ‐[h], ‐[b], and ‐[c]quinoline, and the difference between the most toxic isomer and the least toxic isomer amounted to a factor of 30. Attempts were made to relate these differences in toxicity to the structure of the isomers. Size‐ or topology‐related molecular descriptors provided insufficient resolution to distinguish between the benzoquinoline isomers, and none of the electronic descriptors separately provided a significant correlation with the observed effects. In an alternative approach, molecular shape, accessibility, and minimum agent–macromolecule distance were used to represent repulsive and attractive forces between the benzoquinoline isomers and biological membranes. This approach could tentatively explain the observed effects and is supported by a high correlation between the EC50 data and the reversed‐phase C18‐HPLC behavior of the benzoquinolines (k0), which is likely to be governed by similar processes.

3H](azidophenyl)ureido taxoid photolabels peptide amino acids 281-304 of alpha-tubulin.

The taxoid binding site on porcine brain tubulin was covalently labeled, in the presence or absence of Taxotere, with the photoaffinity reagent [3H]-p-(azidophenyl)ureido taxoid derivative [3H]TaxAPU [Combeau, C., Commercon, A., Mioskowski, C., Rousseau, B., Aubert, F., & Goeldner, M. (1994) Biochemistry 33, 6676-6683]. After disulfide reduction and carboxymethylation, the alkylated tubulin samples were treated with trypsin and the mixtures of peptides were first fractionated by gel filtration over Sephadex G50. Anion exchange chromatography of the radioactive areas showed, for one area, three major radioactive signals which were further analyzed by reversed phase C18 HPLC, leading to well-resolved radioactive peaks. Microsequencing of these different peaks gave a complete sequence of a tryptic fragment on alpha-tubulin (alpha-281-304) and two partial peptide sequences of a tryptic fragment on beta-tubulin (beta-217-229) in addition to sequences of mixture of peptides. The radioactive signals were lost while concentrating the samples for microsequencing, preventing the identification of the modified amino acids. These results identify the first peptide on alpha-tubulin which binds to the taxoids and confirm the involvement of both alpha- and beta-tubulin in the taxoid binding site.

QUANTITATIVE CONCORDANCE BETWEEN GLOBIN mRNAs AND SYNTHESIS OF FETAL HEMOGLOBIN (HbF) AND ADULT HEMOGLOBIN (HbA) DURING HYPOXEMIC STRESS ERYTHROPOIESIS. † 1293

Increased production of HbF can be considered a marker for hypoxemia, since increased levels of HbF synthesis have been found in newborn infants following maternal hypoxemia, placental insufficiency, and diabetes, as well as in infants with broncho-pulmonary dysplasia and children with congenital cyanotic heart disease. To examine if a quantitative relationship exists between the mRNAs encoding globins and their translation into proteins during hypoxemic erythropoietic stress, the relative amounts of the mRNAs and the relative amounts of the α,β,γ-globin synthesis in samples obtained from cord blood from infants of insulin-dependent diabetic mothers, intrauterine growth-retarded infants of preeclamptic mothers and older infants with cyanotic heart disease were measured and compared. The synthesis of globins in reticulocytes was measured by the incorporation of [3H]-leucine followed by the separation and quantitation of the polypeptides on a C4-reverse phase HPLC. The relative proportions of the mRNAs of globins were determined by RNase protection assay. Total RNA isolated from blood (50μl) was incubated with [32P]-labelled cRNA probes ofα,β,γ-globins, followed by RNase A/T1 digestion, separation of the protected fragments and densitometry of the radioactive bands by phosphorimaging. The study population showed an increase in HbF synthesis, compared to controls. There was a strong correlation (r2=0.993; n=16) between the ratio of mRNAs encoding HbF and HbA and the ratio of de novo synthesis of HbF and HbA. Therefore, under conditions of stress erythropoiesis there is a close correlation between quantitation of globin mRNAs and HbF& HbA synthesis. As well, globin mRNA quantitation could replace the usual method of HbA & HbF synthesis determination.

Studies on the Substrate Binding Segments and Catalytic Action of Lanosterol Synthase. Affinity Labeling with Carbocations Derived from Mechanism-Based Analogs of 2,3-Oxidosqualene and Site-Directed Mutagenesis Probes

Four 2,3-oxidosqualene analogs, 3, 4, 5, and 6, which are irreversible, time-dependent inhibitors of the enzyme lanosterol synthase, were found to attach covalently within the 231−236 (yeast numbering) segment (Figure 3). The attachment was determined by tryptic digestion of the inactivated enzyme, separation of the tryptic cleavage products by C18 reverse phase HPLC, and fragment identification by mass spectroscopy or Edman degradation. W232 and H234 are the targets of the chemical inactivation by cations derived from analogs 3−6. 2,3-Oxidosqualene analogs 7, 8, and 9 inactivated the enzyme with covalent attachment to the 486−512 segment (Figure 3), which is in a domain that is predicted to be an amphipathic α-helix. Site-directed mutagenesis of various amino acid residues (76 total) in lanosterol synthase which are conserved in five different species has revealed that residues D456, H146, and H234 are essential for catalytic activity. These and other data permit the formulation of a hypothetical working model of some aspects of the activation and binding of 2,3-oxidosqualene by lanosterol synthase. The model is depicted in Figure 4. In that model D456 and protonated H146 initiate cyclization, and the domains containing 231−236 and 486−512 make contact with the reacting substrate.

Mass spectroscopic identification of in vitro glycated sites of MIP

PURPOSE. Earlier reconstitution studies showed that glycation of the major intrinsic peptide (MIP) of the lens affected the permeability of liposomes. This study is aimed to identify in vitro glycated sites. METHODS. Urea- and alkali-washed calf lens membranes were incubated with 0 and 1 M glucose for 5 days. Following the incubation, MIP was purified by size exclusion HPLC. The C-terminus peptide of MIP was then cleaved by cyanogen bromide (CNBr) and purified by C4 reversed-phase HPLC. The CNBr peptide was analyzed, either directly or after digestion with trypsin, by electrospray ionization mass spectrometry (ESIMS) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). RESULTS. The ESIMS and MALDI-MS analyses of the intact C-terminus peptide from 1 M glucose incubated samples showed a mass shift equivalent to one, two and three glucose adducts. The MALDI-MS of the tryptic digest of the same sample showed peptides with mass shift equivalent to one or more glucose adducts. Sequence assignment confirmed that these glycated peptides contained lys238 and lys259. Although the intact C-terminus peptide showed up to three glucose adducts, we could not assign any tryptic peptide that contained glycated lys228. Samples incubated with 0 M glucose did not show any protein modifications. CONCLUSIONS. The data suggest that in vitro glycation sites of calf lens MIP are lys238 and lys259, and possibly lys228.

Regulation of ovarian ecdysteroid production in the housefly,Musca domestica

Highly purified Musca ecdysteroidogenin (ESG) was obtained by extracting housefly heads with an acid-ethanol-urea procedure followed by desalting on a Sephadex G25F column and fractionation on a Sephadex G50F column. Further fractionation was done on a semi-preparative C18 reverse-phase HPLC column with an acetonitrile gradient in 0.1% trifluoroacetic acid in water. Ecdysteroidogenic activity eluted from the column with 35% acetonitrile. I-125 size exclusion HPLC revealed activity in a fraction with a molecular mass of 8.1 kDa. ESG from the C18 column at a concentration of 1.1 μg/fly stimulated ovarian development to late vitellogenesis when injected into flies without the corpus allatum-cardiacum complex and stimulated maximal ovarian ecdysteroid production in vitro at a concentration of 40 μg/μl. Concentrations of ESG outside these ranges were less effective. Ovaries produced about 500 pg each of ecdysone, 20-hydroxyecdysone, and makisterone A when exposed to ESG in vitro. However, control ovaries produced about a tenth the amount of ecdysone and 20-hydroxyecdysone and half the amount of makisterone A. An exposure of 4 h or more to ESG was required by ovaries before they would make ecdysteroid, with the greatest rate of ecdysteroid production occurring in the 12–24 h interval of exposure. Ecdysteroid production ceased from 36–50 h after ESG was removed from the medium. A juvenile hormone analog (JHA) did not stimulate ovarian ecdysteroid production, but ovaries from flies without the corpus allatum-cardiacum complex required JHA to produce high levels of ecdysteroid during incubation with ESG. ESG activity was recovered from both male and female heads. Arch. Insect Biochem. Physiol. 35:135–148, 1997. © 1997 Wiley-Liss, Inc.1

Regulation of ovarian ecdysteroid production in the housefly, Musca domestica

Highly purified Musca ecdysteroidogenin (ESG) was obtained by extracting housefly heads with an acid-ethanol-urea procedure followed by desalting on a Sephadex G25F column and fractionation on a Sephadex G50F column. Further fractionation was done on a semi-preparative C18 reverse-phase HPLC column with an acetonitrile gradient in 0.1% trifluoroacetic acid in water. Ecdysteroidogenic activity eluted from the column with 35% acetonitrile. I-125 size exclusion HPLC revealed activity in a fraction with a molecular mass of 8.1 kDa. ESG from the C18 column at a concentration of 1.1 μg/fly stimulated ovarian development to late vitellogenesis when injected into flies without the corpus allatum-cardiacum complex and stimulated maximal ovarian ecdysteroid production in vitro at a concentration of 40 μg/μl. Concentrations of ESG outside these ranges were less effective. Ovaries produced about 500 pg each of ecdysone, 20-hydroxyecdysone, and makisterone A when exposed to ESG in vitro. However, control ovaries produced about a tenth the amount of ecdysone and 20-hydroxyecdysone and half the amount of makisterone A. An exposure of 4 h or more to ESG was required by ovaries before they would make ecdysteroid, with the greatest rate of ecdysteroid production occurring in the 12–24 h interval of exposure. Ecdysteroid production ceased from 36–50 h after ESG was removed from the medium. A juvenile hormone analog (JHA) did not stimulate ovarian ecdysteroid production, but ovaries from flies without the corpus allatum-cardiacum complex required JHA to produce high levels of ecdysteroid during incubation with ESG. ESG activity was recovered from both male and female heads. Arch. Insect Biochem. Physiol. 35:135–148, 1997. © 1997 Wiley-Liss, Inc. 1 This article is a US Government work and, as such, is in the public domain in the United States of America.

A Combined High-Performance Liquid Chromatographic–Microbiological Assay for Serum Folic Acid

An assay of folic acid in human serum is described involving HPLC fractionation of deproteinized serum prior toLactobacillus caseiassay. High specific activity [3H]folic acid is added as internal standard to 1 ml serum prior to deproteinization with perchlorate and an aliquot is applied to a C18reverse-phase HPLC column. The folic acid fraction was assayed byL. caseimicrotiter plate assay. Intra- and interassay variations (CV) were 5.5 and 7.5% respectively for sera (n= 10) spiked with folic acid. The percentage recovery for folic acid additions of 10 and 20 ng/liter were 105.2 and 103.7%, respectively. The lower limit of detection was 1 ng folic acid/ml serum. Folates other than folic acid and 5-methyltetrahydrofolate were not detected. The assay permitted serial measurements to be made of folates in serum following an oral dose.

Isolation of the murine S100 protein MRP14 (14 kDa migration-inhibitory-factor-related protein) from activated spleen cells: characterization of post-translational modifications and zinc binding

MRP14 (macrophage migration-inhibitory factor-related protein of molecular mass 14 kDa) is an S100 calcium binding protein constitutively expressed in human neutrophils which may be associated with cellular activation/inflammation. Murine MRP14 expression was up-regulated following concanavalin A activation of spleen cells, and the protein was isolated from conditioned medium in high yield (approx. 500 ng/ml). MRP14 had a mass of 12972 +/- 2 Da by electrospray ionization MS, whereas the theoretical mass derived from the cDNA sequence, after removal of the initiator Met, was 12918 Da, suggesting that the protein was post-translationally modified. We identified four post-translational modifications of MRP14: removal of the N-terminal Met, N-terminal acetylation, disulphide bond formation between Cys79 and Cys90, and 1-methylation of His106; the calculated mass was then 12971.8 Da. Methylation of His106 was further characterized after incubation of spleen cells with L-[methyl-3H]Met during concanavalin A stimulation. Sequential analysis of a peptide (obtained by digestion with Lys C) containing methylated His indicated that > 80% of the label in the cycle corresponded to His106, suggesting that the methyl residue was transferred from S-adenosyl-L-methionine. Comparison of the C18 reverse-phase HPLC retention times of phenylthiocarbamoyl derivatives of a hydrolysed digest peptide of MRP14 with those of standards confirmed methyl substitution on the 1-position of the imidazole ring. MRP14 bound more 85Zn2+ than the same amounts of the 10 kDa chemotactic protein (CP10) or S100 beta. Ca2+ decreased Zn2+ binding in S100 beta but it did not influence binding to MRP14, suggesting that the Zn2+ binding site was distinct from and independent of the two Ca2+ binding domains.

QUANTITATIVE CONCORDANCE BETWEEN GLOBIN mRNA AND SYNTHESIS OF FETAL AND ADULT HEMOGLOBIN DURING HEMOGLOBIN SWITCHOVER IN PERINATAL PERIOD. • 1725

During the perinatal period the switchover from fetal (HbF) to adult hemoglobin (HbA) synthesis in humans, γ-globin levels decline as the proportion of β-globin increases under the influence of a developmental clock. In order to examine if a quantitative relationship existed between the mRNAs encoding globins and their translation into proteins during the period of switchover, the relative amounts of the mRNAs and the relative amounts ofα, β, γ-globin synthesis in cord blood samples were measured and compared. The synthesis of globins in reticulocytes was measured by the incorporation of [3H]-leucine followed by the separation and quantitation of the polypeptides on a C4-reverse phase HPLC equipped with an integrator. The relative proportions of the mRNAs of globins were determined by RNase protection assay. Total RNA isolated from cord blood was incubated with [32P]-labelled cRNA probes (of equivalent specific activity) of α, β, γ-globins overnight at 50°C, followed by RNAse A (10 μg/ml) /T1 (200 U/ml) digestion at 25°C, separation of the protected fragments on 6% urea-polyacrylamide gels and densitometry of the radioactive bands by phosphorimaging. A comparison cord blood samples from 18 newborn infants of different gestational ages (27-40 weeks; birth weight: 850-4035 g) with no congenital anomalies, revealed a strong and significant correlation (r2= 0.934) between the ratio of mRNAs encoding HbF and HbA and the ratio of de novo synthesis of HbF and HbA. There was a linear relationship between the proportions of mRNAs encoding HbF and HbA with the proportional synthesis of HbF and HbA throughout the gestational age studied. This information can be useful for studies furthering the understanding of the mechanisms regulating globin gene expression.