Modifications of cysteine residues in the solution and membrane-associated conformations of phosphatidylinositol transfer protein have differential effects on lipid transfer activity.

The alpha isoforms of mammalian phosphatidylinositol transfer protein (PITP) contain four conserved Cys residues. In this investigation, a series of thiol-modifying reagents, both alkylating and mixed disulfide-forming, was employed to define the accessibility of these residues and to evaluate their role in protein-mediated intermembrane phospholipid transport. Isolation and analysis of chemically modified peptides and site-directed mutagenesis of each Cys residue to Ala were also performed. Soluble, membrane-associated, and denatured preparations of wild-type and mutant rat PITPs were studied. Under denaturing conditions, all four Cys residues could be detected spectrophotometrically by chemical reaction with 4,4'-dipyridyl disulfide or 5,5'-dithiobis(2-nitrobenzoate). In the native protein, two of the four Cys residues were sensitive to some but not all thiol-modifying reagents, with discrimination based on the charge and hydrophobicity of the reagent and the conformation of the protein. With the soluble conformation of PITP, achieved in the absence of phospholipid vesicles, the surface-exposed Cys(188) was chemically modified without consequence to lipid transfer activity. Cys(188) exhibited an apparent pK(a) of 7.6. The buried Cys(95), which constitutes part of the phospholipid substrate binding site, was covalently modified upon transient association of PITP with a membrane surface. The Cys-to-Ala mutations showed that neither Cys(95) nor Cys(188) was essential for lipid transfer activity. However, chemical modification of Cys(95) resulted in the loss of lipid transfer activity. These results demonstrate that the Cys residues of PITP can be assigned to several different classes of chemical reactivity. Of particular interest is Cys(95), whose sulfhydryl group becomes exposed to modification in the membrane-associated conformation of PITP. Furthermore, the inhibition of PITP activity by thiol-modifying reagents is a result of steric hindrance of phospholipid substrate binding.

Structure of a multifunctional protein. Mammalian phosphatidylinositol transfer protein complexed with phosphatidylcholine.

Eukaryotic phosphatidylinositol transfer protein is a ubiquitous multifunctional protein that transports phospholipids between membrane surfaces and participates in cellular phospholipid metabolism during signal transduction and vesicular trafficking. The three-dimensional structure of the alpha-isoform of rat phosphatidylinositol transfer protein complexed with one molecule of phosphatidylcholine, one of its physiological ligands, has been determined to 2.2 A resolution by x-ray diffraction techniques. A single beta-sheet and several long alpha-helices define an enclosed internal cavity in which a single molecule of the phospholipid is accommodated with its polar head group in the center of the protein and fatty acyl chains projected toward the surface. Other structural features suggest mechanisms by which cytosolic phosphatidylinositol transfer protein interacts with membranes for lipid exchange and associates with a variety of lipid and protein kinases.

Macular translocation: histopathologic findings in swine eyes.

PURPOSE: Macular translocation has been proposed as an alternative technique in the treatment of some cases of choroidal neovascularization. The purpose of the paper is to report the histopathologic findings in the retina of swine eyes undergone macular translocation. METHODS: Ten eyes of ten Yucatan pigs underwent posterior pars plana vitrectomy and scleral imbrication to achieve macular translocation. Mattress sutures were preplaced at the equator of the eyes. After a pars plana vitrectomy, balanced saline solution was injected under the temporal retina to produce a retinal detachment. Scleral imbrication was achieved by tightening the mattress sutures. An air-fluid exchange was performed and the eye was filled with sulfur hexafluoride 18%. The eyes were enucleated 2, 4, 8 and 12 weeks after surgery and analyzed under light and electron microscopy. RESULTS: Macular translocation was achieved in all cases. The major findings consist of a minimal decrease in the number of photoreceptors outer segments; also a change in the morphology was noted. This included some degree of loss of vertical alignment and an increase in the interphotoreceptor space. There was a recovery in the morphology of the photoreceptors over time. CONCLUSIONS: Minimal changes in the photoreceptors and retinal pigment epithelium are observed when macular translocation is performed with recovery of these changes over time. Scleral imbrication is an effective technique to achieve translocation of the fovea.

X-ray analysis of crystals of rat phosphatidylinositol-transfer protein with bound phosphatidylcholine.

Phosphatidylinositol-transfer protein (PITP) is a soluble, ubiquitously expressed, highly conserved protein encoded by two genes in humans, rodents and other mammals. A cDNA encoding the alpha isoform of the rat gene was expressed to high levels in Escherichia coli, the protein purified and the homogeneous protein used for crystallization studies. Crystals of rat PITP-alpha were obtained by vapor-diffusion techniques using the sitting-drop method. Crystals grow within two weeks by vapor-diffusion techniques in the presence of polyethylene glycol 4000. Both crystal forms pack in the monoclinic space group P21. Crystal form I has unit-cell parameters a = 44.75, b = 74.25, c = 48.32 A and beta = 114.14 degrees. Unit-cell parameters for crystal form II are a = 47.86, b = 73.59, c = 80.49 A and beta = 98.54 degrees. Crystal form I has a Vm of 2.295 A3 Da-1 and an estimated solvent content of 46.4% with one molecule per asymmetric unit, while crystal form II has a Vm of 2.196 A3 Da-1 and an estimated solvent content of 44.0%, assuming two molecules per asymmetric unit.

The C-terminus of phosphatidylinositol transfer protein modulates membrane interactions and transfer activity but not phospholipid binding.

Rat phosphatidylinositol transfer protein (PITP) is a 32 kDa protein containing 271 amino acids. It is involved in a number of cell functions including secretion and cell signaling. To further characterize structure/activity relationships of PITP, two C-terminal truncated derivatives, PITP(1-259) and PITP(1-253), were produced in Escherichia coli and purified to homogeneity. PITP(1-259) had transfer activity equal to 30-40% to that of native PITP in transfer of either phosphatidylcholine (PC) or phosphatidylinositol (PI) when transfer was measured using 95/5 mol% PC/PI donor and acceptor vesicles; PITP(1-253) had only slight transfer activity, even under the most favorable assay conditions. Thus, amino acids 254-258 are critical for transfer activity. The transfer activity of PITP(1-259) was strongly dependent on the composition of the donor and acceptor vesicles. With 100 mol% PC donor and acceptor vesicles, PITP(1-259) transfer activity ranged from 70 to 100% to that of PITP. The presence of 2 mol% phosphatidic acid (PA) in either donor or acceptor vesicles reduced transfer activity to between 10 and 20% that of full-length PITP under the same conditions. If both donor and acceptor contained 2% PA, PITP(1-259) was essentially inactive, though the activity of PITP was not affected significantly under these conditions. PITP(1-253) and PITP(1-259) bind much more avidly to vesicles than does PITP, and this enhanced binding reflects increased electrostatic interactions. Thus, the C-terminal residues modulate the affinity of PITP for vesicles and the efficiency of phospholipid transfer.

Importance of phospholipid in the folding and conformation of phosphatidylinositol transfer protein: comparison of apo and holo species.

The significance of noncovalently bound phospholipid as a structural component of phosphatidylinositol transfer protein (PITP) and its role in acquisition and maintenance of the native conformation of the protein have been addressed by studying the refolding of PITP after exposure to 6 M guanidinium chloride (GdnCl). Protein conformations were characterized by (1) the intrinsic tryptophan fluorescence, circular dichroism, and absorbance spectroscopy, (2) the degree of binding of the fluorescent probe 1,8-ANS, and (3) limited proteolytic digestion. When the GdnCl concentration was reduced 100-fold by rapid dilution at 25 degrees C, practically all of the native transfer activity was regained within 20 min. Endogenous phospholipid demonstrated a strong interaction with the native PITP. Separation of the phospholipid from the protein by chromatography on a lipophilic matrix was achieved only under denaturing conditions and resulted in spontaneous oxidation of the apo-protein, accompanied by almost complete loss of recoverable transfer activity. Under reducing conditions, however, apo-PITP recovered more than 80% of the native transfer activity and was similar to holo-PITP in the kinetics of phospholipid transfer. Renatured apo-PITP demonstrated a significant relaxation of the tertiary structure, compared to native and renatured holo-PITP. Incubation of apo-PITP with phospholipid vesicles resulted in a more compact protein conformation. We conclude that the polypeptide can spontaneously fold to a native-like conformation, sufficient for interaction with a lipid membrane and acquisition of a phospholipid ligand. Binding of a phospholipid ligand brings about the final adjustments of protein conformation to the more compact native structure.

Competing strategically through market orientation.

As organizations seek to better understand their customers, competitors, and environments, the marketing function effectively serves as the support mechanism for these activities in many industries. Accordingly, in many organizations the marketing concept has been elevated to the stature of a strategic weapon, manifested in the form of market orientation. Market-oriented firms emphasize the collection, organization, and dissemination of information regarding both customers and competitors. This strategy is especially well-suited for the health care industry where customer knowledge is of paramount importance, customer interaction is instantaneous, and customer satisfaction is essential. Results of a national study of general service hospitals suggest that firms which embrace a market orientation benefit through enhanced customer satisfaction and quality.

Truncations of the C-terminus have different effects on the conformation and activity of phosphatidylinositol transfer protein.

Contributions of the C-terminus toward the conformation and activity of phosphatidylinositol transfer protein (PITP) were studied by comparing properties of the 271 amino acid, full-length protein, PITP(1-271), and two truncated species, PITP(1-259) and PITP(1-253). Using recombinant proteins and an in vitro phospholipid transfer assay with phosphatidylcholine vesicles, the activities of PITP(1-271) and PITP(1-259) were identical, while the activity of PITP(1-253) was almost totally abolished. By most physical and chemical criteria, however, PITP(1-259) and PITP(1-253) were virtually indistinguishable and differed significantly from the full-length protein. Results of second derivative analysis of absorbance spectra were consistent with an additional two Tyr residues being exposed to the solvent in PITP(1-259) and PITP(1-253) in comparison to PITP(1-271). Only one out of four Cys residues in PITP(1-271) reacted with dithiobisnitrobenzoic acid, while two Cys residues were accessible in both truncated species. Quenching of intrinsic Trp fluorescence by acrylamide demonstrated an increase in exposure of Trp residues in both PITP(1-259) and PITP(1-253); binding of the fluorescence probe 1,8-ANS to these proteins was also significantly higher compared to PITP(1-271). These results describe a more relaxed overall tertiary structure brought about by the C-terminal truncations. This altered structure did not affect the stability of the truncated proteins, as indicated by equilibrium unfolding in guanidinium chloride. Refolding of the denatured PITP(1-259), however, was considerably slower than that of full-length PITP. Our study suggests a critical role of the C-terminal residues 254-259 in transfer activity of PITP. Residues 260-271, on the other hand, appear to be more important for the rapid folding and maintenance of a compact native conformation of the protein.

Limited proteolysis of rat phosphatidylinositol transfer protein by trypsin cleaves the C terminus, enhances binding to lipid vesicles, and reduces phospholipid transfer activity.

Rat phosphatidylinositol transfer protein (PITP) is a 32-kDa protein of 271 amino acids that transfers phosphatidylinositol and phosphatidylcholine between membranes. The alpha isoform of rat PITP was expressed in Escherichia coli and purified in high yields. The purified protein contained 1 mol of phosphatidylglycerol and had a transfer activity for phosphatidylinositol and phosphatidylcholine equal to or greater than that of PITP purified from mammalian brain. Limited protease digestion was used to further define structure, activity, and function relationships in PITP. PITP alone is relatively resistant to digestion by chymotrypsin, trypsin, and Staphylococcus V8 protease but is readily cleaved by subtilisin. Phospholipid vesicles containing phosphatidic acid enhance susceptibility to digestion by all four proteases. In the presence of vesicles, PITP, which migrates as a 36-kDa protein in SDS-polyacrylamide gel electrophoresis, is cleaved rapidly by trypsin to a form that appears to be 2-3 kDa smaller than the native form. The tryptic fragment retains partial phospholipid transfer activity and shows an enhanced affinity for phospholipid vesicles containing phosphatidic acid. Analysis of the tryptic digestion products by immunoblotting, N-terminal sequencing, and electrospray mass spectrometry showed that trypsin cleaves the C terminus of PITP at Arg253 and Arg259. Thus, removal of the C terminus enhances the affinity of PITP for vesicles and results in a dimunition of transfer activity. Overall, the data show that PITP undergoes conformation changes and that the C terminus becomes more accessible to trypsin when bound to vesicles. Hence, the C terminus is not an essential component of the membrane binding site and may be located distal to it.

Mechanics of motility: distinct dynein binding domains on alpha- and beta-tubulin.

Microtubules (MTs) interact with force-generating proteins to generate a variety of intracellular movements, including intracellular particle transport, ciliary-flagellar beating, and chromosome-spindle movements during mitosis-meiosis. Relatively little is known about the mechanics of these motor-MT interactions, in part because the motor binding domains of the MT and the corresponding MT binding domains of the motor have not been well characterized. Using a flagellar motility assay, we report that the MT subunits, alpha- and beta-tubulin, each contain a dynein binding domain located near the C-termini of their respective tubulin subunits. Blocking either alpha- or beta-tubulin binding domains of dynein attenuates motility in demembranated sea urchin sperm up to 50%. Interestingly, blocking both alpha- and beta-tubulin binding domains on dynein produces much greater decreases in motility. These data suggest that flagellar dynein binds to both subunits of the MT polymer, alpha- and beta-tublin. In addition, the two subunits appear to contribute equivalent, but functionally separate, roles to flagellar motility.

Refolding and release of tubulins by a functional immobilized groEL column.

Denatured tubulins form stable complexes with groEL upon dilution into refolding buffer. These complexes are retained on an immunoaffinity column which contains chemically immobilized antibodies to groEL. Tubulin remains bound to the immobilized groEL column after extensive washing and is released upon incubation with groES and ATP. Similar results were obtained with glutamine synthetase. These data suggest that groEL can function while it is attached to a solid support system.

Sequence of a human cDNA encoding phosphatidylinositol transfer protein and occurrence of a related sequence in widely divergent eukaryotes.

Phosphatidylinositol (PtdIns) transfer protein (PtdInsTP) is a phospholipid transfer protein that has been detected in all mammalian tissues examined. It catalyzes the transfer in vitro of PtdIns and phosphatidylcholine between membranes in a number of natural and artificial membrane systems and may be involved in secretion in vivo. In previous studies, we isolated and sequenced a cDNA encoding a rat PtdInsTP. A rat cDNA probe was used to isolate clones from a lambda gt11 human testis cDNA library which encoded full-length human PtdInsTP. The cDNA sequence defines a 270-amino-acid, 31.8-kDa protein whose sequence shares 98.9% identity to that of rat, making it one of the most conserved proteins known between the two species. DNA blot hybridization studies suggest that there may be more than one gene encoding this protein in humans. A comparison of rat and human PtdInsTP cDNAs revealed strong sequence similarity (88 and 84%) in portions of the corresponding 5'- and 3'-untranslated regions (UTR) of the rat and human mRNAs.

Localization of the gene encoding human phosphatidylinositol transfer protein (PITPN) to 17p13.3: a gene showing homology to the Drosophila retinal degeneration B gene (rdgB).

The human gene for phosphatidylinositol transfer protein (PITPN) has previously been shown to share sequence and functional homology to part of the Drosophila retinal degeneration B gene (rdgB). In view of the possible involvement of the PITPN locus in the etiology of retinal disease, the gene has been mapped to human chromosome 17p13.3 and mouse Chromosome 11.

Expression of chick and yeast beta-tubulin-encoding genes in insect cells.

A chick cDNA encoding the beta 2 isotype of tubulin (beta 2Tub) was cloned into a baculovirus expression vector designed to produce unfused proteins, and several recombinant viruses (re-viruses) were isolated. Immunoblotting studies of homogenates of insect cells infected with re-virus showed a 50-kDa protein that reacted with antibodies specific for beta Tub. Cells infected with the re-virus appeared to contain much higher levels of beta Tub than uninfected control cells, perhaps as much as five- to tenfold higher. Isotype-specific antibody for beta 2Tub showed little reaction in uninfected cells or cells infected with wild-type virus; strong reaction was found with cells infected with re-virus. Analysis by gel filtration of extracts of cells infected with re-virus showed that almost all beta Tub eluted in the column void volume, suggesting that it was aggregated or associated with other cell proteins. Recombinant baculoviruses producing Saccharomyces cerevisiae beta Tub were also isolated. Immunoblotting studies using antibodies specific for yeast beta Tub showed a 50-kDa protein which was absent in uninfected cells or cells infected with wt virus. Immunofluorescence studies suggest that yeast beta Tub is incorporated poorly, if at all, into the insect cell cytoskeleton.

Straight ileoanal anastomosis after longitudinal strip myectomy in the swine model.

In an attempt to improve the function of the straight ileoanal anastomosis, an experimental study was performed using the swine model. The terminal ileum was altered by completely removing two longitudinal strips of muscle prior to performing a straight ileoanal anastomosis. The intent of the study was to determine whether muscle stripping was technically possible and whether bowel thus treated would remain viable to passively form a pelvic reservoir. The length of time required for formation of the reservoir was noted. All animals survived the procedure to allow evaluation. The muscle stripping was not difficult to perform. Viability was not a problem since the myectomy animals thrived well and demonstrated continence, weight gain, and reservoir formation. The results are encouraging. It appears that strips of muscularis propria can be removed from the terminal ileum without jeopardizing its viability. This seems to disrupt sufficiently the tonus of the bowel to allow better function of the straight ileoanal anastomosis through formation of a passive pelvic reservoir within a month's time in the swine model, and it may have application in the human.

Conserved beta-tubulin binding domain for the microtubule-associated motors underlying sperm motility and fast axonal transport.

An antiserum against tubulin, NS20, has been previously shown to inhibit anterograde and retrograde axonal transport by 50% in vivo and in vitro. We report here that Protein A purified NS20 antibodies also attenuate sperm motility by 50% in demembranated sea urchin sperm. This inhibition is absorbed out by preincubating the NS20 antibodies with a biochemically purified porcine microtubule preparation, with recombinant Trypanosoma beta- (but not alpha-) tubulin and most specifically, with a 37 amino acid (a.a.) synthetic peptide corresponding to a domain near (but not including) the porcine beta-tubulin C terminus. Furthermore, addition of this beta-tubulin peptide alone is sufficient to attenuate motility by 50% in demembranated sperm, indicating that this critical 37a.a. NS20 antigen is a motor binding domain. Together, the results suggest that at least two phenotypically distinct forms of microtubule-based motility, axonal transport and flagellar beating, are homologous at the fundamental level of the microtubule domains (the beta-tubulin peptide and we suggest a distinct but similarly located alpha-tubulin domain) mediating the attachment of tubulin-associated motors.

Effects of age on myosin and creatine kinase isoforms in left ventricles of Fischer 344 rats.

Left ventricles of hearts from male Fischer 344 rats of 2, 8 and 23 months of age were analyzed to determine if aging results in significant alterations in the isoform distribution of myosin and creatine kinase protein and mRNAs. Left ventricles of maturing (2-month) rats contained almost exclusively alpha-myosin heavy chain (MHC) mRNA and protein. In adults (8 months) there was a 5-fold increase in beta-MHC (fetal isoform) and an approximate 10% decrease in alpha-MHC mRNA levels, relative to 2 months. By 23 months (senescence), beta-MHC mRNA levels had increased by 11-fold and alpha-MHC mRNA levels had decreased by about 30%. These changes corresponded to an increase in the relative proportion of beta-MHC protein, from undetectable levels at 2 months, to about 40% by 8 months and to about 60% by 23 months. Increased levels of beta-MHC and its mRNA in older rats correlated with decreased serum thyroid hormone levels. The specific activity of creatine kinase in crude homogenates decreased with age, as has been reported previously. Relative to 2-month controls, the specific activity of creatine kinase had decreased by 21% at 8 months and by 37% at 23 months. Analysis of creatine kinase activity showed no large increase in levels of the fetal (B) isoform with age, as was found for myosin. Levels of mRNAs encoding the B and M isoforms of creatine kinase were significantly reduced in senescent rats. Thus, the decreased levels of creatine kinase in aging rats is correlated with decreased levels of mRNA encoding the BCK and MCK isoforms but not an isoform shift.