Latexin, a carboxypeptidase A inhibitor, is expressed in rat peritoneal mast cells and is associated with granular structures distinct from secretory granules and lysosomes.

Latexin, a protein possessing inhibitory activity against rat carboxypeptidase A1 (CPA1) and CPA2, is expressed in a neuronal subset in the cerebral cortex and cells in other neural and non-neural tissues of rat. Although latexin also inhibits mast-cell CPA (MCCPA), the expression of latexin in rat mast cells has not previously been confirmed. In the present study we examined the expression and subcellular localization of latexin in rat peritoneal mast cells. Western blot and reverse-transcriptase-mediated PCR analyses showed that latexin was contained and expressed in the rat peritoneal mast cells. Immunocytochemically, latexin immunofluorescence was localized on granular structures distinct from MCCPA-, histamine- or cathepsin D-immunopositive granules. Immunoelectron microscopy revealed that latexin was associated with a minority population of granules. The latexin-associated granules were separated from MCCPA- or histamine-containing granules on a self-generating density gradient of polyvinylpyrrolidone-coated silica-gel particles (Percoll). Treatments with high ionic strength and heparinase released latexin from the granules, suggesting that latexin is non-covalently associated with a heparin-like component of the granules. MCCPA and histamine were released from the mast cells after non-immunological and immunological stimulation with compound 48/80, A23187 and anti-IgE antibody, whereas latexin was not released. These results show that latexin is synthesized in rat peritoneal mast cells and suggest that it is associated with a unique type of intracellular granules distinct from MCCPA- and histamine-containing secretory granules and lysosomes.

Phospholipids stabilize the secondary structure of the sodium-coupled branched-chain amino acid carrier of Pseudomonas aeruginosa.

For functional reconstitution of bacterial cotransporters (carriers or permeases) including the sodium-coupled branched-chain amino acid carrier (LIV-II carrier) of Pseudomonas aeruginosa, the presence of phospholipid is required through the process of solubilization and purification of the transporters from the bacterial membranes, suggesting the possibility that phospholipid may stabilize the structure of the cotransporter proteins to be in a functional form. In this study, this possibility was examined by studying the effect of denaturant on the secondary structure of the LIV-II carrier purified in the absence and presence of phospholipid using circular dichroism (CD) spectroscopy. CD spectra of the purified LIV-II carrier solubilized in n-octyl-beta-D-glucopyranoside (OG), OG/dioleoylphosphatidylethanolamine (DOPE)/dioleoylphosphatidylglycerol (DOPG) mixture, and dispersed into DOPE/DOPG small unilamellar vesicles were measured in the absence of denaturant. The three spectra were very similar and had a trough at 222 nm with mean residue molar ellipticity of -23000 deg.cm(2)/dmol and a shoulder at 208 nm. CD spectral analyses with three different methods (S.W. Provencher, J. Glöckner, Estimation of globular protein secondary structure from circular dichroism, Biochemistry 20 (1981) 33-37; J.Y. Yang, C.-S.C. Wu, H.Z. Martinez, Calculation of protein conformation from circular dichroism, Methods Enzymol. 130 (1986) 208-269; N. Sreerama, R.W. Woody, A self-consistent method for the analysis of protein secondary structure from circular dichroism, Anal. Biochem. 209 (1993) 32-44) revealed that the LIV-II carrier solubilized in OG/DOPE/DOPG mixture contained 69-75% alpha-helix and 0-9% beta-sheet. Addition of 6 M guanidine hydrochloride decreased 48% of the amplitude at 222 nm of the CD spectrum of the carrier solubilized in OG alone and 9-14% of the CD amplitude of the carrier solubilized in OG/DOPE/DOPG or OG/dioleoylphosphatidylcholine mixture and dispersed in liposomes composed of DOPE/DOPG. These results show that the ordered secondary structure of the LIV-II carrier is partially unfolded in OG without phospholipid by denaturant but is greatly stabilized with phospholipids with oleoyl chains independently of their polar head group composition and suggest that the alpha-helical structure of the carrier is mainly embedded in the lipid environment.

Cerebral cortical specification by early potential restriction of progenitor cells and later phenotype control of postmitotic neurons.

Neurons expressing latexin, a carboxypeptidase A inhibitor, are restricted to lateral areas in the cerebral cortex of adult and early postnatal rats. To address the precise timing of cortical regional specification at the cellular level, we monitored latexin expression in developing cortical cells under specific conditions in vitro. Individual cortical cells were labeled with 5-bromo-2'-deoxyuridine in vivo, dissociated and exposed to a defined new environment in a monolayer or a reaggregated-cell culture system. While a substantial fraction of early progenitor cells derived from the lateral cerebral wall became latexin-expressing neurons in both systems, far fewer progenitors from dorsal cortex did so under the same environmental conditions, indicating early establishment of cortical regional specification at the progenitor cell level. Furthermore, it was shown that the probability for postmitotic cells within lateral cortex to become latexin-expressing neurons was influenced by temporally regulated regional environmental signals. These findings suggest that developing cortical cells are progressively specified for a regional molecular phenotype during both their proliferative and postmitotic periods.

Latexin expression in smaller diameter primary sensory neurons in the rat.

Most of the smaller diameter neurons of dorsal root and trigeminal ganglia in adult rats expressed latexin, which has the inhibitor activity of carboxypeptidase A. Most of the dorsal root ganglion (DRG) neurons containing either calcitonin gene-related peptide (CGRP), substance P (SP) or somatostatin (SST) coexpressed latexin. Latexin was widely distributed in the cytoplasm of the cell body and in axonal fibers of cultured DRG neurons which were sensitive to capsaicin. In addition, latexin-immunoreactivity was observed throughout lamina II of the spinal cord in normal animals, but was lost following sciatic nerve-axotomy, suggesting the presence of latexin-immunoreactive axonal fibers and/or terminals from DRG neurons. Immunoelectron microscopy indeed revealed latexin-immunoreactive axonal terminals and thinly myelinated and unmyelinated axonal fibers within the dorsal horn. These observations suggest that latexin may be involved in nociceptive information transmission or its modulation.

Intracortical regionality represented by specific transcription for a novel protein, latexin.

The monoclonal antibody (mAb) PC3.1 recognizes a subset of neurons distributed in the infragranular layers of the lateral neocortex of the rat. Immunoaffinity chromatography with mAb PC3.1 showed that this antibody specifically binds a peptide epitope on a 29 kDa protein named latexin. To study the molecular details of the protein, we isolated four independent cDNA clones for latexin from cDNA libraries of the rat cerebral cortex and whole brain using the amino acid sequences of latexin fragments. Analysis of these cDNA clones showed that the predicted primary structure of latexin consists of 223 amino acids, and has no strict homology to any sequences so far known. Western and Northern blots demonstrated that the latexin and its mRNA were expressed predominantly in neural tissues with some expression in non-neural tissues. The gene that encodes latexin in the rat appeared to have homologues in other mammalian species and in the chick. In situ hybridization showed that latexin mRNA is synthesized in a subset of neurons in the lateral but not the dorsal neocortex, and that the distribution profile of these neurons is quite similar to that of neurons expressing latexin. These results indicate that latexin is a novel class of neuronal protein which represents intracortical regionality, and suggest that the regional specification of the neocortex involves selective parcellation of neurons which express a particular gene.

Early regional specification for a molecular neuronal phenotype in the rat neocortex.

The timing of neocortical regional specification was examined using a monoclonal antibody, designated PC3.1, that binds a 29-kDa polypeptide and recognizes a neuronal subpopulation located in the lateral but not dorsomedial neocortex in the rat. When lateral cortical tissue fragments at embryonic days 12 and 16 were maintained in an organotypic culture system, a substantial number of neurons became PC3.1-immunopositive. In marked contrast, considerably fewer, if any, PC3.1-positive neurons were observed in cultures of dorsal cortical tissue. The selective appearance of PC3.1-immunopositive neurons was also observed in dissociated cultures derived from the lateral, but not dorsal, cortical primordium at embryonic day 13 and later. In light of previous reports showing that the interactions between developing neocortical neurons and cortical afferents begin at embryonic day 14 or later, our findings imply that some regional specification occurs well before these interactions and suggest the importance of elements intrinsic to the neocortex in establishing neocortical regional specificity. Furthermore, [3H]thymidine birth-dating experiments revealed that the majority of presumptive PC3.1-immunopositive neurons underwent their final mitosis around embryonic day 15, suggesting that the regional specification events for these neurons occur before their neurogenesis.

Immunoaffinity purification and reconstitution of sodium-coupled branched-chain amino acid carrier of Pseudomonas aeruginosa.

The gene product of braB encoding the Na+(Li+)-coupled carrier protein for L-leucine, L-isoleucine, and L-valine (LIV-II carrier) of Pseudomonas aeruginosa PML strain was identified and overexpressed using a T7 RNA polymerase/promoter plasmid system. The gene product was pulse-labeled with [35S]methionine as a protein of an apparent Mr of 34,000 on a sodium dodecyl sulfate-polyacrylamide gel. Cell membranes overproducing the LIV-II carrier were solubilized with n-dodecyl beta-D-maltopyranoside. The carrier protein was purified from the detergent extract by two purification steps: (i) immunoaffinity column chromatography using purified polyclonal antibody directed against synthetic 13-mer peptide corresponding to the carboxyl terminus region of the carrier and (ii) subsequent DEAE-cellulose column chromatography. The detergent was replaced by n-octyl beta-D-glucopyranoside prior to the first elution and phospholipid was present during purification. Proteoliposomes reconstituted with the purified LIV-II carrier exhibited Na+ or Li+ concentration gradient-driven transport of leucine, isoleucine, and valine. These results show that the LIV-II carrier was purified to be in a functional form.

Isolation of the braZ gene encoding the carrier for a novel branched-chain amino acid transport system in Pseudomonas aeruginosa PAO.

The braZ gene for a novel branched-chain amino acid transport system in Pseudomonas aeruginosa PAO was isolated and characterized. Determination of the nucleotide sequence showed that the braZ gene comprises 1,311 nucleotides specifying a protein of 437 amino acids. Hydropathy analysis suggested that the product is an integral membrane protein with 12 membrane-spanning segments. The amino acid sequence showed extensive homology to those of the braB and brnQ gene products, branched-chain amino acid carriers of P. aeruginosa and Salmonella typhimurium, respectively. By using the T7 RNA polymerase-promoter system, the braZ gene product was identified as a protein of an apparent Mr of 34,000 on a sodium dodecyl sulfate-polyacrylamide gel. Properties of the transport system encoded by braZ were studied by using P. aeruginosa PAO3537, defective in both the high- and low-affinity branched-chain amino acid transport systems (LIV-I and LIV-II, respectively). The transport system encoded by braZ was found to be another effective branched-chain amino acid transport system in P. aeruginosa PAO and was thus designated as LIV-III. This system is specific for isoleucine and valine, giving the same Km value of 12 microM for these amino acids. The system was found, however, to have a very low affinity for leucine, with a Km value of 150 microM, which contrasts with the substrate specificities of LIV-I and LIV-II.

Cloning and nucleotide sequence of the gene braB coding for the sodium-coupled branched-chain amino acid carrier in Pseudomonas aeruginosa PAO.

The gene braB, encoding the Na(+)-coupled carrier for branched-chain amino acids in Pseudomonas aeruginosa PAO, was cloned on cosmid pMMB34. The cosmid clones carrying the braB gene were identified as those that restored growth at low leucine concentration and Na(+)-dependent leucine transport activity to P. aeruginosa PAO3536 defective in the transport of branched-chain amino acids. Determination of the nucleotide sequence of the DNA fragment shows that the braB gene comprises 1311 bp and encodes a hydrophobic protein of 437 amino acids with a calculated Mr of 45,279. The hydropathy profile suggests that there exist in the carrier protein 12 hydrophobic segments long enough to traverse the membrane. The amino acid sequence shows a high degree of homology with the brnQ product, a branched-chain amino acid carrier of Salmonella typhimurium, while no homology in the nucleotide sequences is found in the braB and brnQ genes.

Difference in sodium requirement of branched chain amino acid carrier between Pseudomonas aeruginosa PAO and PML strains is due to substitution of an amino acid at position 292.

Sodium dependence of leucine transport, a measure of the Na+-coupled leucine-isoleucine-valine II (LIV-II) transport system in Pseudomonas aeruginosa, was compared between two wild-type strains, PAO and PML. The leucine transport activity was saturated at 0.1 mM NaCl for PAO and at 5.0 mM for PML. From kinetics experiments, the apparent Km value for Na+ with respect to leucine transport was estimated to be 3 microM for PAO and 95 microM for PML. The Km value for leucine was 6 microM for PAO and 13 microM for PML. The LIV-II carrier gene (braB) of PML was isolated for comparison of its amino acid sequence with that of the PAO carrier cloned previously. The Km values for Na+ and leucine of the cloned LIV-II carriers of PAO and PML expressed in LIV-II defective mutants were similar to those in wild-type strains. Determination of the nucleotide and deduced amino acid sequences of the LIV-II carrier gene of PML showed an amino acid difference at position 292 between the PAO and PML carriers. The amino acid was threonine for PAO and alanine for PML. These results indicate that the substitution of the amino acid at position 292 of the LIV-II carrier causes a difference in the sodium requirement of the carriers of the PAO and PML strains.

Na+(Li+)/branched-chain amino acid cotransport in Pseudomonas aeruginosa.

A transport system for branched-chain amino acids (designated as LIV-II system) in Pseudomonas aeruginosa requires Na+ for its operation. Coupling cation for this system was identified by measuring cation movement during substrate entry using cation-selective electrodes. Uptakes of Na+ and Li+ were induced by the imposition of an inwardly-directed concentration gradient of leucine, isoleucine, or valine. No uptake of H+ was found, however, under the same conditions. In addition, effects of Na+ and Li+ on the kinetic property of the system were examined. At chloride salt concentration of 2.5 mM, values of apparent Km and Vmax for leucine uptake were larger in the presence of Na+ than Li+. These results indicate that the LIV-II transport system is a Na+(Li+)/substrate cotransport system, although effects of Na+ and Li+ on kinetics of the system are different.

Conformation of antithrombin III with defective biological functions derived from a thrombophilic patient.

Antithrombin III (AT III) with defective biological functions and altered antigenicity has been found in a thrombophilic patient. Circular dichroism (CD) spectra of antithrombin III purified from the patient's plasma were measured in near and far-ultraviolet (UV) wavelength regions. The far-UV CD spectrum of the patient's AT III was similar at room temperature to that of normal AT III derived from healthy adult males. Mean residue ellipticity at 221 nm of the patient's AT III, however, decreased its magnitude by 6% gradually as temperature increased up to 74 degrees C, whereas that of the normal AT III reduced its magnitude sharply at around 63-64 degrees C and by 18%. The near-UV CD spectrum of patient's AT III was different from that of normal AT III. These results indicate that the secondary structure of patient's AT III is similar to that of normal AT III at room temperature, while local conformation of aromatic amino acid residues in the abnormal protein is different from that in the normal one and that the secondary structure of the patient's AT III is heat-stable, while that of the normal one is heat-labile.

Effect of lipid acyl chain length on activity of sodium-dependent leucine transport system in Pseudomonas aeruginosa.

The sodium-dependent leucine transport system of Pseudomonas aeruginosa was reconstituted into liposomes of binary lipid mixtures of dilauroylphosphatidylethanolamine (di(12:0)PE)/phosphatidylcholine (PC) with cis-monounsaturated fatty acid chains (di(n:1)PC) (n = 14-22) or dioleoylphosphatidylethanolamine (di(18:1)PE)/di(n:1)PC (n = 14-22). Leucine carrier proteins can be activated with phosphatidylethanolamine, whereas activation does not occur in PC-reconstituted vesicles (Uratani, Y., and Aiyama, A. (1986) J. Biol. Chem. 261, 5450-5454). Na+-dependent counterflow was measured at 30 degrees C as reconstituted transport activity. Proteoliposomes containing di(12:0)PE exhibited high counterflow activity at the PC acyl carbon number (n) of 18 and 20 but no or low activity at n = 14, 16, and 22. On the other hand, proteoliposomes containing di(18:1)PE exhibited higher transport activity than those vesicles with di(12:0)PE and corresponding di(n:1)PC. A lipid mixture of di(18:1)PE and di(16:1)PC supported maximal activity. These results show that the leucine transport system of P. aeruginosa is dependent on the lipid acyl chain length and suggest that there exists optimal bilayer thickness for maximal carrier activity.

Effect of phospholipid composition on activity of sodium-dependent leucine transport system in Pseudomonas aeruginosa.

The sodium-dependent leucine transport system of Pseudomonas aeruginosa was reconstituted into liposomes of specific polar head group composition. Na+-dependent counterflow and Na+ gradient-driven transport were measured as reconstituted transport activities. Proteoliposomes containing phosphatidylethanolamine exhibited increased transport activities. Phosphatidylglycerol, second to phosphatidylethanolamine, also enhanced the reconstituted transport activities. Proteoliposomes composed of phosphatidylcholine did not accumulate leucine. The enhanced transport activity by phosphatidylethanolamine was significantly influenced by its fatty acid composition. Dioleoylphosphatidylethanolamine was more effective in stimulating counterflow activity than dilauroylphosphatidylethanolamine. These results show that the leucine transport system of P. aeruginosa is sensitive to not only the polar head group composition but also the acyl group composition of phospholipids.

Solubilization and reconstitution of sodium-dependent transport system for branched-chain amino acids from Pseudomonas aeruginosa.

The sodium-dependent transport system for branched-chain amino acids of Pseudomonas aeruginosa was solubilized with n-octyl-beta-D-glucopyranoside and reconstituted into liposomes by a detergent-Sephadex G-50 gel filtration procedure. The reconstituted proteoliposomes exhibited Na+-dependent counterflow and Na+-gradient-driven transport of L-leucine, L-isoleucine, and L-valine. The leucine counterflow was specifically inhibited by only branched-chain amino acids and the uphill transport of two species of amino acids among the three was induced by counterflow of the other substrate. These results show that the transport system for branched-chain amino acids was reconstituted into liposomes from P. aeruginosa cells and strongly suggest that three branched-chain amino acids are transported by a common carrier system.

Dependence of the conformation of a colicin E1 channel-forming peptide on acidic pH and solvent polarity.

The secondary structure content of the COOH-terminal tryptic peptide of colicin E1 has been measured by analysis of UV circular dichroism spectra as a function of pH in aqueous medium and in the presence of the nonionic detergents octyl glucoside and Triton X-100. The alpha-helical content of the peptide increased by approximately 10%, from 45-47% to 56-57%, in the presence of the nonionic detergents, but not in aqueous medium, as the pH was decreased from 4.5 to 3.5. This pH dependence of conformation is similar to that reported elsewhere for the in vitro activity and binding of this peptide. A smaller increase in helical content was observed for the peptide in aqueous medium or in Triton X-100 as the pH was decreased from 6.5 to 4.5. The letter change in helical content was not seen in octyl glucoside which was present at a detergent:peptide stoichiometry 100 times that of Triton. The mean residue ellipticity measured at 222 nm for peptide added to asolectin vesicles by a freeze-thaw treatment was slightly larger at pH 3.5, and substantially larger at pH 4.5, than found at these pH values in the detergent solutions. Changes in helical content at the former, but not the latter pH, could be attributed to peptide insertion. It appears that protonation of one or more acidic amino acid residues in the COOH-terminal region of the molecule causes a conformational change that can be attributed to an extra helical domain that is stabilized in a nonpolar environment. From the similar pH dependence of the conformational change and in vitro binding and activity, it is inferred that interaction of this domain with the membrane is essential for binding and insertion.

Pyocin R1 inhibits active transport in Pseudomonas aeruginosa and depolarizes membrane potential.

Pyocin R1, a bacteriocin of Pseudomonas aeruginosa, inhibited active transport of proline in the presence of high concentrations of malate and magnesium salt. Pyocin R1 did not affect the respiration of sensitive cells nor induce cell lysis, but it caused a decrease in the intracellular ATP level. In addition, a passive inflow of [14C]thiocyanate anion, a probe of membrane potential, was induced by pyocin R1, showing a depolarization of the cytoplasmic membrane. It is considered that membrane depolarization is a primary action of pyocin R1.