Approximately 70% of the initial ferriprotoporphyrin IX polymerizing activity in cell-free preparations of erythrocytes infected with Plasmodium berghei was recovered in a chloroform extract. No polymerizing activity remained in the residue. In studies to identify substances that promote FP polymerization, arachidonic, linoleic, oleic, and palmitoleic acids, 1-mono- and di-oleoylglycerol, and the detergents, SDS, Tween 80, and n-octyl-glucopyranoside, were active. Tri-oleoylglycerol, cholesterol, di-oleoylphosphatidylethanolamine, and stearic and palmitic acids were inactive. The model lipid, mono-oleoylglycerol (250 nmol), co-precipitated with FP from a 0.09 M acetate medium at pH 5 and promoted the polymerization of 215 nmol (61%) of the ferriprotoporphyrin IX in the precipitate during a 24-h incubation at 37 degrees C. Polymerization was maximal at pH 5, it was approximately linear for 2 h, and it continued at a decreasing rate for 24 h. The polymer contained exclusively ferriprotoporphyrin IX (97+/-1.3%, mean+/-S.E., n=4) and exhibited the solubility and the electronic absorption and infrared spectral characteristics of the sequestered ferriprotoporphyrin IX of hemozoin. Detergents presumably promote polymerization in an acid medium by helping to dissolve monomeric FP. We suggest that unsaturated lipids co-precipitate with FP in the parasite's acidic food vacuole and also dissolve sufficient monomeric FP to allow polymerization.

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Human pulmonary surfactant protein D (SP-D) is a collagenous C-type lectin with high binding specificity to α-d-glucosyl residues. It is composed of four regions: a short NH2-terminal noncollagen sequence, a collagenous domain, a short linking domain (“neck” region), and a COOH-terminal carbohydrate recognition domain (CRD). Previous studies demonstrated that SP-D is chemotactic for inflammatory cells. To test which domain of SP-D might play a role in this function, a mutant that contains only neck and CRD regions was expressed in Escherichia coli and purified by affinity chromatography on maltosyl-agarose. A 17-kDa recombinant SP-D CRD was identified by two antibodies (antisynthetic SP-D COOH-terminal and neck region peptides) but not by synthetic SP-D NH2-terminal peptide antibody. The recombinant SP-D CRD was confirmed by amino acid sequencing. Gel-filtration analysis found that 84% of CRD was trimeric and the rest was monomeric. Analysis of the chemotactic properties of the trimeric CRD demonstrated that the CRD was chemotactic for neutrophils (polymorphonuclear leukocytes), with peak activity at 10−10 M equal to the positive control [formyl-Met-Leu-Phe (fMLP) at 10−8 M]. The chemotactic activity was abolished by 20 mM maltose, which did not suppress the chemotactic response to fMLP. The peak chemotactic activity of the CRD is comparable to the activity of native SP-D, although a higher concentration is required for peak activity (10−10 vs. 10−11 M). The chemotactic response to CRD was largely prevented by preincubation of polymorphonuclear leukocytes with SP-D, and the response to SP-D was prevented by preincubation with CRD. These preincubations did not affect chemotaxis to fMLP. These results suggest that trimeric CRD accounts for the chemotactic activity of SP-D.

Unesterified fatty acids were measured in mouse erythrocytes infected either with chloroquine-susceptible (CS) or with chloroquine-resistant (CR) lines of Plasmodium berghei. This work was undertaken to identify candidates for the lipid involved in ferriprotoporphyrin IX (FP) polymerization. Linoleic, oleic, palmitic, and stearic acids were quantified by gas chromatography/mass spectrometry. In total, they increased 4-fold with CS infections and 6-fold with CR infections. Treating infected mice with chloroquine did not affect the amounts of unesterified fatty acids in erythrocytes. Of the four fatty acids, only linoleic acid increased disproportionately to the total. It increased 16-fold for the CS line and 35-fold for the CR line. The method could detect monoglycerides but they were below the limit of detection. It could not detect diglycerides, triglycerides or phospholipids. Triglycerides and phospholipids have been tested previously, however, and found to be ineffective at promoting FP polymerization. Therefore, other than linoleic acid, the lipids most likely to be involved in FP polymerization are diglycerides. We tested dilinoleolyglycerol in the present work and found it to be an effective promoter of FP polymerization. These results suggest that linoleic acid or a diglyceride containing it has the critical role of promoting FP polymerization in malaria parasites.

Mice infected with the NYU-2 strain of Plasmodium berghei were used to study the effect of chloroquine on masking of a lipid that promotes ferriprotoporphyrin IX dimerization. More than 40% of this lipid was masked and unable to promote dimerization in membrane ghosts from erythrocytes of untreated, infected mice. Thus, preparations of membrane ghosts dimerized 57 ؎ 6 nmol of ferriprotoporphyrin IX during a 2-h incubation, whereas the lipids extracted from these preparations dimerized 101 ؎ 11 nmol of ferriprotoporphyrin IX (means ؎ S.D. for four experiments). Exposure of membrane ghosts to sonication or cold significantly increased the extent of masking. In addition, chloroquine treatment of infected mice increased the extent of masking to ϳ90%. The lipid could be unmasked by extracting it into acetone or by aging erythrocyte membrane ghosts from untreated or chloroquine-treated, infected mice for 24 h at pH 7.4 and 25°C. These findings indicate that masking and unmasking of a lipid is central to the regulation of ferriprotoporphyrin IX dimerization in malaria parasites. They also indicate that chloroquine impairs the function of this regulatory process.When mice infected with Plasmodium berghei are treated with chloroquine, the parasites lose 80% or more of their ability to produce ␤-hematin (1), a dimer of ferriprotoporphyrin IX (FP) 1 (2). Consequently, undimerized, toxic FP accumulates (3) and, presumably, kills the parasites (4, 5). The biochemical basis for the effect of chloroquine on the production of betahematin in vivo presently is unknown. It is true that chloroquine and other quinoline derivatives, including quinine and mefloquine, bind to FP and inhibit dimerization in vitro (3, 6), but something else occurs in vivo. In fact, inhibition of FP dimerization by chloroquine is antagonized by quinine and mefloquine in vivo (3). These and other observations indicate that chloroquine acts by affecting a regulatory process rather than by directly inhibiting FP dimerization (7). To understand the biochemical basis for the antimalarial action of chloroquine, therefore, it is desirable to know more about the process that regulates FP dimerization.Current knowledge of FP dimerization in malaria parasites can be briefly summarized as follows. The process is promoted only by the lipid fraction of parasitized erythrocytes (8). Apparently, the lipid serves to concentrate monomeric FP and keep it in a state favorable for dimerization (8). Once formed, FP dimers spontaneously aggregate in an acidic milieu (2) such as occurs in the digestive vacuoles of malaria parasites. These aggregates are insoluble at physiologic pH or lower (5), and, in contrast to undimerized FP, they appear to be nontoxic for malaria parasites.Since malaria parasites are enriched with linoleic acid, it is likely that linoleate or a linoleate derivative is the lipid that promotes FP dimerization (9). The linoleate probably is released from the inner membranes of hemoglobin-laden endocytic vesicles as they are processed into digestive vacuoles (...

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