Kevin L. Duffin

Generation of hemoglobin peptides in the acidic digestive vacuole of Plasmodium falciparum implicates peptide transport in amino acid production

Intraerythrocytic malaria parasites avidly consume hemoglobin as a source of amino acids for incorporation into parasite proteins. An acidic organelle, the digestive vacuole, is the site of hemoglobin proteolysis. Early events in hemoglobin catabolism have been well studied. Two aspartic proteases, plasmepsins I and II, and a cysteine protease, falcipain, cleave hemoglobin into peptides. While it has been presumed that hemoglobin peptide fragments are degraded to individual amino acids by exopeptidase activity in the digestive vacuole, this hypothesis lacks experimental support. Incubation of human hemoglobin with P. falciparum digestive vacuole lysate generated a series of discrete peptide fragments with cleavage sites an average of 8.4 amino acids apart. No free amino acids could be detected and there was no evidence of peptide heterogeneity due to exopeptidase trimming. These sites correspond to points of cleavage previously established for plasmepsin I, plasmepsin II, and falcipain as well as some novel sites that suggest the existence of an additional endoproteinase. By colorimetric assay, P. falciparum has abundant aminopeptidase activity but this activity is not found in the digestive vacuoles and the parasite lacks detectable carboxypeptidase activity altogether. These data support a model for hemoglobin catabolism wherein small peptides are formed from cleavage of hemoglobin by the enzymes of the digestive vacuole and then are transported through the membrane of the digestive vacuole to the cytoplasm. There, exopeptidase activity converts the peptides to individual amino acids for parasite growth and maturation.

Altered Arachidonic Acid Metabolism in Urate Crystal Induced Inflammation

Gout is an acute rheumatic disorder that occurs in connection with the deposition of monosodium urate (MSU) crystals in the joints. This disease is characterized by intermittent episodes of severe pain and inflammatory joint swelling which are seemingly driven by prostaglandins. In this study we investigated the effect of MSU crystals on arachidonic acid (AA) metabolism in the mouse. We have demonstrated that prostaglandins and other AA metabolites were transiently formed after MSU crystal injection with peak levels occurring after 10 min. In contrast, free AA levels remained high for 2–4 hours after MSU crystal injection. By contrast, when exogenous AA was administered instead of MSU crystals, both the eicosanoids and AA diminished at the same high rates. The metabolism of exogenously administered AA to eicosanoids was inhibited by pretreatment with MSU crystals. No inhibition of AA metabolism was observed when mice were pretreated with AA itself, Ca2+ ionophore (A23187), or zymosan. We conclude that the MSU crystal treatment of mice results in a transient eicosanoid production which is followed by attenuated AA metabolism. It could be that MSU crystals similarly inhibit AA metabolism in gout and thereby limit the duration of gout attacks.

Identification of components in waste streams by electrospray and tandem mass spectrometry

Highly polar, non-gas-chromatographable compounds have few unambiguous analysis protocols for environmental applications. A recent environmental investigation, concerning the identification of a non-gas-chromatographable yellow component in chemical waste water and in effluents from a biological wastewater treatment plant required the use of a number of analytical approaches. Electrospray mass spectrometry, tandem mass spectrometry, high-performance liquid chromatography, nuclear magnetic resonance, and molecular spectroscopy of commercial and synthesized chlorodinitrophenol isomers were required in order to identify the specific isomer causing the color. The present report summarizes the electrospray ionization and tandem mass spectrometric studies that were used. The mass spectrometric study shows that two different isomers of chlorodinitrophenol exhibit very different collision-induced dissociation (CID) spectra. Differences in the tandem mass spectra can be attributed to the different structures of the anions formed from these two different isomers. Instrumentation that uses electrospray ionization and produces CID mass spectra and optical absorption spectra in a single analysis may be required in order to produce highly specific information on non-gas-chromatographable compounds found in the environment.

Suicide Inactivation of Leukotriene A4 Hydrolase/Aminopeptidasea

Leukotrienes (LT) are lipid mediators of inflammation.’” LTJ3, stimulates adhesion of circulating neutrophils to vascular endothelium, directs their migration to sites of inflammation: catalyzes the release of their granule constituents, and produces hyperalge~ia.~ The sulfidopeptide LTs (LTC,, LTD4, and LTE,) cause vascular permeability and constrict bronchial smooth muscle.’” Leukotriene biosynthesis originates from sequential enzymatic reactions involving: (i) phospholipases, which liberate arachidonic acid from membrane phospholipids; (ii) 5-lipoxygenase (5-L0), which produces S(S)-hydroperoxyeicosatetraenoic acid (5-JXPETE) and converts it into LTA.,, the pivotal intermediate in this biosynthetic pathway: (iii) LTC, synthase, which conjugates reduced glutathione and LTA,, yielding LTC,; and (iv) LTA, hydrolase, which hydrates LT& stereospecifically, producing LTB4. Nonenzymatic hydration yields biologically inactive 5,12or 5,6-dihydroxyeicosatetraenoic acids (diHETE). Pathological actions of leukotrienes are best understood in terms of their roles in immediate hypersensitivity and asthma.b9

Carboxy-terminal Protein Sequence Analysis using Carboxypeptidase P and Electrospray Mass Spectrometry

Publisher Summary This chapter discusses carboxy-terminal protein sequence analysis using carboxypeptidase P (CPP) and electrospray mass spectrometry (ESMS). To test the applicability of the ESMS analysis method for the CPP digestion of proteins, recombinant E. coli derived human IL-3 was chosen as a model. IL-3 has a C-terminal sequence of …T-T-L-S-L-A-I-F-OH. The digestion conditions were used to digest 2.7 nmoles of IL-3. Aliquots of 500 pmoles were removed at several time points—10 min, 20 min, and 50 min—and analyzed by ESMS. At the 10 min time point, the appearance of multiply-charged ions representing consecutive losses of five C-terminal amino acids was observed in the mass spectrum. The average molecular weight of each truncated species can be determined through deconvolution of the mass spectrum. The chapter shows a closeup view of the (M+9H)+9 charge state of CPP-digested IL-3. The mass differences of these ions reflect the sequential loss of amino acids from full-length IL-3.

Isolation of Escherichia coli synthesized recombinant proteins that contain ε-N-acetyllysine

Publisher Summary This chapter describes the presence of significant amounts of ɛ-N-acetyllysine in rpST and rbST, eukaryotic proteins expressed in a prokaryotic system. ESMS, amino acid sequencing and amino acid analyses demonstrate the presence of ɛ-N-acetyllysine in two recombinant proteins; bovine placental lactogen and human tissue factor pathway inhibitor. These data establish that this modified amino acid is present in several distinct recombinant eukaryotic proteins expressed in E.coli . Formation of ɛ-N-acetyllysine in eukaryotic systems involves a post-translational mechanism in which the acetyltransferase uses acetyl-CoA as the source of the acetyl group. It may be possible that acetylation of lysines occurs by a chemical mechanism with acetyl-CoA or some other metabolic intermediate providing the source of the acetyl group. The chapter explains that acetylation of lysines is an important modification, which can occur during the expression of recombinant proteins expressed in E.coli. However, investigations into the effects of fermentation conditions on the level of ɛ-N-acetyllysine formation may lead to a better understanding of this event.

Determination of the disulfide bond pairings in human tissue factor pathway inhibitor purified fromEscherichia coli

The disulfide bond assignments of human alanyl tissue factor pathway inhibitor purified fromEscherichia coli have been determined. This inhibitor of the extrinsic blood coagulation pathway possesses three Kunitz-type inhibitor domains, each containing three disulfide bonds. The disulfide bond pairings in domains 1 and 3 were determined by amino acid sequencing and mass spectrometry of peptides derived from a thermolysin digest. However, thermolysin digestion did not cleave any peptide bonds within domain 2. The disulfide bond pairings in domain 2 were determined by isolating it from the thermolysin treatment and subsequently cleaving it with pepsin and trypsin into peptides which yielded the three disulfide bond pairings in this domain. These results demonstrate that the disulfide pairings in each of the three domains of human tissue factor pathway inhibitor purified fromEscherichia coli are homologous to each other and also to those in bovine pancreatic trypsin inhibitor.