Activation of phospholipase A2 by temporin B: Formation of antimicrobial peptide-enzyme amyloid-type cofibrils

Phospholipases A2 have been shown to be activated in a concentration dependent manner by a number of antimicrobial peptides, including melittin, magainin 2, indolicidin, and temporins B and L. Here we used fluorescently labelled bee venom PLA2 (PLA2D) and the saturated phospholipid substrate 1,2-dipalmitoyl-glycero-sn-3-phosphocholine (L-DPPC), exhibiting a lag-burst behaviour upon the initiation of the hydrolytic reaction by PLA2. Increasing concentrations of Cys-temporin B and its fluorescent Texas red derivative (TRC-temB) caused progressive shortening of the lag period. TRC-temB/PLA2D interaction was observed by Förster resonance energy transfer (FRET), with maximum efficiency coinciding with the burst in hydrolysis. Subsequently, supramolecular structures became visible by microscopy, revealing amyloid-like fibrils composed of both the activating peptide and PLA2. Reaction products, palmitic acid and 1-palmitoyl-2-lyso-glycero-sn-3-phosphocholine (lysoPC, both at > 8 mol%) were required for FRET when using the non-hydrolysable substrate enantiomer 2,3-dipalmitoyl-glycero-sn-1-phosphocholine (D-DPPC). A novel mechanism of PLA2 activation by co-fibril formation and associated conformational changes is suggested.     

Orientation and dynamics of transmembrane peptides: the power of simple models

In this review we discuss recent insights obtained from well-characterized model systems into the factors that determine the orientation and tilt angles of transmembrane peptides in lipid bilayers. We will compare tilt angles of synthetic peptides with those of natural peptides and proteins, and we will discuss how tilt can be modulated by hydrophobic mismatch between the thickness of the bilayer and the length of the membrane spanning part of the peptide or protein. In particular, we will focus on results obtained on tryptophan-flanked model peptides (WALP peptides) as a case study to illustrate possible consequences of hydrophobic mismatch in molecular detail and to highlight the importance of peptide dynamics for the experimental determination of tilt angles. We will conclude with discussing some future prospects and challenges concerning the use of simple peptide/lipid model systems as a tool to understand membrane structure and function.     

Effect of phospholipids and a transmembrane peptide on the stability of the cubic phase of monoolein: implication for protein crystallization from a cubic phase

The cubic phase of monoolein has successfully been used for crystallization of a number of membrane proteins. However, the mechanism of protein crystallization in the cubic phase is still unknown. It was hypothesized, that crystallization occurs at locally formed patches of bilayers. To get insight into the stability of the cubic phase, we investigated the effect of different phospholipids and a model transmembrane peptide on the lipid organization in mixed monoolein systems. Deuterium-labeled 1-oleoyl-rac-[(2)H(5)]-glycerol was used as a selective probe for (2)H NMR. The phase behavior of the phospholipids was followed by (31)P NMR. Upon incorporation of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, or phosphatidic acid, the cubic phase of monoolein transformed into the L(alpha) or H(II) phase depending on the phase preference of the phospholipid and its concentration. The ability of phospholipids to destabilize the cubic phase was found to be dependent on the phospholipid packing properties. Electrostatic repulsion facilitated the cubic-to-L(alpha) transition. Incorporation of the transmembrane peptide KALP31 induced formation of the L(alpha) phase with tightly packed lipid molecules. In all cases when phase separation occurs, monoolein and phospholipid participate in both phases. The implications of these findings for protein crystallization are discussed.     

Membrane interaction of the glycosyltransferase MurG: a special role for cardiolipin

MurG is a peripheral membrane protein that is one of the key enzymes in peptidoglycan biosynthesis. The crystal structure of Escherichia coli MurG (S. Ha, D. Walker, Y. Shi, and S. Walker, Protein Sci. 9:1045-1052, 2000) contains a hydrophobic patch surrounded by basic residues that may represent a membrane association site. To allow investigation of the membrane interaction of MurG on a molecular level, we expressed and purified MurG from E. coli in the absence of detergent. Surprisingly, we found that lipid vesicles copurify with MurG. Freeze fracture electron microscopy of whole cells and lysates suggested that these vesicles are derived from vesicular intracellular membranes that are formed during overexpression. This is the first study which shows that overexpression of a peripheral membrane protein results in formation of additional membranes within the cell. The cardiolipin content of cells overexpressing MurG was increased from 1 +/- 1 to 7 +/- 1 mol% compared to nonoverexpressing cells. The lipids that copurify with MurG were even further enriched in cardiolipin (13 +/- 4 mol%). MurG activity measurements of lipid I, its natural substrate, incorporated in pure lipid vesicles showed that the MurG activity is higher for vesicles containing cardiolipin than for vesicles with phosphatidylglycerol. These findings support the suggestion that MurG interacts with phospholipids of the bacterial membrane. In addition, the results show a special role for cardiolipin in the MurG-membrane interaction.     

The Possible Role of Glutamate Uptake in Metaphit-Induced Seizures

In a study of the possible mechanism of action of metaphit and phencyclidine in the brain, the uptake of glutamate at the luminal side of the blood-brain barrier (BBB) was studied by means of an in situ brain perfusion technique in normal guinea pigs and in those pretreated with metaphit. Metaphit, an isothiocyanate analog of phencyclidine (PCP), induces time-dependent epileptogenic changes in the electroencephalogram in guinea pig, reaching a maximum 18–24 h after metaphit administration (50 mg/kg IP). In metaphit-pretreated animals a significant reduction of glutamate BBB uptake was found, in comparison with that of controls. Reduction of glutamate transport from blood to brain ranged from 77% to 79% in all brain structures studied. This inhibition was probably due to changes in the properties of saturable components responsible for transport of glutamate across the BBB. Kinetic measurements revealed a saturable amino acid influx into the parietal cortex, caudate nucleus, and hippocampus, with a Km between 3.1 and 5.1 M, and the V_max ranging from 14.3 to 27.8 pmol^–1 g^–1. The nonsaturable component, K_id, was statistically different from zero, ranging from 1.47 to 2.00 M min^–1 g^–1. Influx of glutamate into the brain was not altered in the presence of 1 mM D-aspartate, but it was significantly inhibited in the presence of 1 mM L-aspartate. We conclude that the cerebrovascular permeability of circulating glutamate is due to the presence of a higher-capacity saturable receptor and/or a carrier-mediated transport system (75%) and also a low-capacity diffusion transport system (25%) for the glutamate located at the luminal side of the BBB. The glutamate transport system is probably fully saturated at physiological plasma glutamate concentrations.     

5alpha-reductase isoenzymes 1 and 2 in the rat testis during postnatal development

The pubertal initiation of spermatogenesis is reliant on androgens, and during this time, 5alpha-reduced androgens such as dihydrotestosterone (DHT) are the predominant androgens in the testis. Two 5alpha-reductase (5alphaR) isoenzymes (5alphaR1 and 5alphaR2) have been identified, which catalyze the conversion of testosterone to the more potent androgen DHT. The present study aimed to investigate the developmental pattern of 5alphaR isoenzymes and their relationship to the production of 5alpha-reduced androgens in the postnatal rat testis. Both 5alphaR1 and 5alphaR2 isoenzyme mRNAs were measured by real-time polymerase chain reaction, isoenzyme activity levels by specific assays, and testicular androgens by radioimmunoassay after high-performance liquid chromatographic separation. Both 5alphaR1 and 5alphaR2 mRNAs and activity levels were low in the 10-day-old (prepubertal) testis, peaked between Days 20 and 40 during puberty, and then declined to low levels at 60-160 days of age. The developmental pattern of both 5alphaR isoenzyme activity levels was mirrored by the testicular production of 5alpha-reduced metabolites. Although 5alphaR1 was greater than 5alphaR2 at all ages, it is likely, given the substrate preferences of the two, that both isoenzymes contribute to the pubertal peak of 5alpha-reduced androgen biosynthesis. The peak in 5alphaR isoenzymes and 5alpha-reduced metabolite production coincided with the first wave of spermatogenesis in the rat, suggesting a role for 5alpha-reduced metabolites in the initiation of spermatogenesis. This was explored by acute administration of a 5alphaR inhibitor (L685,273) to immature rats. The L685,273 markedly suppressed testicular 5alphaR activity during puberty by 75%-86%. However, a marked increase was observed in testicular testosterone levels (in the absence of changes in LH), and no decrease was observed in the absolute levels of 5alpha-reduced metabolites. Therefore, whether the formation of DHT in the presence of low testosterone levels in the pubertal testis is required for the initiation of spermatogenesis cannot be tested using 5alphaR inhibitors. We conclude that both 5alphaR1 and 5alphaR2 isoenzymes are involved in the peak of 5alpha-reduced androgen biosynthesis in the testis during the pubertal initiation of spermatogenesis.     

Lipid dependence of membrane anchoring properties and snorkeling behavior of aromatic and charged residues in transmembrane peptides

31P NMR spectroscopy was used to investigate the effects of transmembrane alpha-helical peptides with different flanking residues on the phase behavior of phosphatidylethanolamine and phosphatidylethanolamine/phosphatidylglycerol (molar ratio 7:3) model membranes. It was found that tryptophan-flanked (WALP) peptides and lysine-flanked (KALP) peptides both promote formation of nonlamellar phases in these lipid systems in a mismatch-dependent manner. Based on this mismatch dependence, it was concluded that the effective hydrophobic length of KALP peptides is considerably shorter than that of the corresponding WALP peptides. Peptides with other positively charged residues showed very similar effects as KALP. The results suggest that the peptides have a well-defined effective hydrophobic length, which is different for charged and aromatic flanking residues, but which is independent of the precise chemical nature of the side chain. Strikingly, the effective length of KALP peptides in the lipid systems investigated here is much smaller than that previously found for the same peptides in phosphatidylcholine. This suggests that snorkeling of lysine side chains, as proposed to occur in phosphatidylcholine, does not occur in lipid systems that are prone to form nonlamellar phases by themselves. This suggestion was supported by using peptides with shortened lysine side chains and by investigating the effects of mixtures of WALP and KALP peptides. The lipid dependency of the snorkeling behavior is explained by considering the free energy cost of snorkeling in relation to the free energy cost of the formation of nonlamellar phases.     

Unit gravity sedimentation separation of cells comprising the caput epididymidis of the rat

Summary Cells from the rat caput epididymidis were separated by unit gravity sedimentation. Purest 12-ml fractions contained 88–95% basal cells or 64–76% principal cells. Ultrastructure of separated cells was similar to that of cells in intact tissue. Viability of separated cells was excellent as determined by dye exclusion tests and cellular ATP content. By combining fractions pools containing 4.0±0.9 × 10⁶ cells (86±8% basal cells) and 1.4±0.4 × 10⁶ cells (56±7% principal cells) were obtained. Thus, studies on the function of basal and principal cells from the rat caput epididymidis should be possible.This research was supported by NIH Grant HD-07244 and was authorized for publication as Paper No. 5231 in the Journal Series of the Pennsylvania Agricultural Experiment Station. We thank Mrs. J. Eastman for expert technical assistance     

Species-specific expression of microsomal prostaglandin E synthase-1 and cyclooxygenases in male monkey reproductive organs

We investigated the tissue distribution and cellular localization of microsomal PGE synthase-1 (mPGES-1) and cyclooxygenase (COX)-1 and -2 in male monkey reproductive organs. Western blotting revealed that monkey mPGES-1 was expressed most intensely in the seminal vesicles, moderately in the testis, and weakly in the epididymis and vas deferens. The tissue distribution profile was quite different from those profiles for rats, rabbits, and pigs, e.g., rat mPGES-1 was the most abundant in the vas deferens, and the rabbit and pig enzymes, in the testis. Immunohistochemical staining with mouse monoclonal anti-human mPGES-1 antibody revealed that monkey mPGES-1 was localized in spermatogonia, Sertoli cells, and primary spermatocytes of testis and in epithelial cells of the epididymis, vas deferens, and seminal vesicles. In monkeys, COX-1 was localized in epithelial cells of the epididymis and vas deferens, whereas COX-2 was dominantly found in epithelial cells of the seminal vesicles.