Protein Of Apparent Molecular Weight Research Articles

The SP1 antigen in subplate neurons of the developing cat cortex is an immunoglobulin-like molecule.

Monoclonal antibody subplate-1 (mAb SP1) specifically stains somata, dendrites and axons of spiny inverted pyramidal neurons in the subplate zone in the early postnatal kitten neocortex. The SP1 antigen has been previously identified as a cytosolic protein of apparent molecular weight 56 kDa. We have now employed immune-affinity chromatography to further characterize this antigen. An antigen with SP1-like immunoreactivity (ir) is present in various organs, and is particularly enriched in blood plasma. Exsanguination of the organs prior to protein extraction reduces the SP1-ir band dramatically, indicative of a blood-borne molecule. The 56 kDa SP1-ir antigen was purified from plasma by affinity chromatography and subjected to Edman degradation. The first 20 N-terminal amino acids show 80% homology to the N-terminus of immunoglobulin heavy chain of man, the mouse and the dog. If the 56 kDa SP1-ir antigen in plasma is an immunoglobulin, and if an immunoglobulin-like molecule is present in the subplate, then antisera against cat immunoglobulins should stain subplate neurons. A polyclonal antiserum against cat IgG intensely stains the somata and dendrites of subplate neurons. On protein blots, this antiserum recognizes the 56 kDa band, and an additional band of approximately 27 kDa, corresponding in size to immunoglobulin light chains. Preabsorbing mAb SP1 with cat immunoglobulin G abolishes the immunoreactivity in sections of kitten cortex. Further, it dramatically reduces the reactivity on protein blots. The results suggest that the 56 kDa SP1-ir antigen in cortical subplate neurons belongs to the immunoglobulin superfamily.

CDNA Cloning Identified a Calmodulin-Binding Protein in Bovine Seminal Plasma as Bovine C-Type Natriuretic Peptide

A basic protein of apparent molecular weight 15 kD, designated bSVSP15, was purified from bovine seminal vesicle secretion to homogeneity, employing affinity absorption to calmodulin-Sepharose and reverse-phase HPLC. Immunoblotting identified bSVSP15 in bovine seminal plasma and seminal vesicle secretion, but it was not present either in extracts of bovine ampulla, epididymis, and testis or in serum or follicular fluid. When added to cAMP phosphodiesterase, bSVSP15 inhibited the activation of enzymatic activity by calmodulin in a reversible manner. Immunoscreening of a lambda gt11 expression cDNA library from bovine seminal vesicle tissue yielded two positive clones, pSVS4 and pSVS5, which were characterized by sequencing. Both sequences are identical, except for the 3' region. Because the derived amino acid sequence comprises, with an identity of 81%, the amino-terminal 21 residues of bSVSP15, cDNA clones pSVS4 and pSVS5 represent bSVSP15-specific mRNAs. The mature protein bSVSP15 contains 101 residues and is preceded by 25 residues of a signal sequence, characteristic for secretory proteins. Northern analysis identified two bSVSP15-specific mRNAs of 900 bp and 1200 bp, respectively. Sequence comparison yielded high homologies to human C-type natriuretic peptide. We conclude from this result that bSVSP15 is identical with the hitherto unknown bovine C-type natriuretic peptide.

Purification and Biochemical Characterization of the Protein Kinase Encoded by the US3 Gene of Herpes Simplex Virus Type 2

Post-ribosomal cytoplasmic fractions from Vero cells mock-infected or infected with wild-type herpes simplex virus type 2 (HSV-2) or a US3 gene-disrupted mutant of HSV-2 were fractionated with DEAE-cellulose chromatography, and the peak fraction of the protein kinase which was detectable only in the extract of wild-type virus-infected cells was subjected to succesive chromatography. The enzyme was purified more than 1000-fold from the post-ribosomal supernatant, and the final preparation contained one major protein of apparent molecular weight 66 kilodalton (K), which was phosphorylated in the autophosphorylation reaction. Western blotting analysis showed that antibodies to an synthetic peptide corresponding to the 15 amino acids of the predicted HSV-2 US3 protein sequence strongly reacted with a 66 K protein in the enzyme fractions. On Superose 12 HR chromatography, the protein kinase activity was eluted as a single major peak at a position corresponding to an apparent molecular mass of approximately 60 K. These results suggest that the 66 K protein is the protein kinase encoded by the US3 gene of HSV-2 and that it acts as a monomer. The HSV-2 protein kinase was relatively resistant to high concentrations of salt, but KCl above 400 mM exerted a significant inhibitory effect. When the substrate specificity was investigated using synthetic oligopeptides, the peptides containing arginyl residues on the amino-terminal side of the target seryl residue were found to be the best substrates for the protein kinase. However, the replacement of the seryl residue to threonine markedly reduced the rate of phosphorylation by this enzyme, suggesting that threonine is a poor phosphate acceptor of the protein kinase. The enzyme was resistant to heparin, a potent inhibitor of casein kinase II, but was moderately sensitive to H-9 (N-(2-aminoethyl)-5-isoquinolinesulfonamide dihydrochloride), a potent inhibitor of cyclic nucleotide-dependent protein kinases and protein kinase C. Quercetin, a bioflavonoid, also inhibited the protein kinase and the inhibitory effect was competitive towards ATP (Ki = 10 μM). The results indicate that the biochemical properties of the HSV-2 US3 protein kinase are very similar to those of the HSV-1 counterpart and pseudorabies virus-encoded 38-kDa protein kinase, but are different from those in several respects.

FL-160 proteins of Trypanosoma cruzi are expressed from a multigene family and contain two distinct epitopes that mimic nervous tissues.

The partial sequence of a gene encoding the COOH terminus of a protein of apparent molecular weight of 160 kD associated with the flagellum of trypomastigotes of Trypanosoma cruzi (FL-160 now renamed to FL-160-1) has been previously reported. The COOH terminus of FL-160-1 has an epitope, defined by 12 amino acids, which molecularly miMics a nervous tissue antigen of 48 kD found in myenteric plexus, sciatic nerve, and a subset of cells in the central nervous system. We now report that FL-160 is a family of highly related genes. The sequence has been determined for the entire open reading frame (ORF) of one of the members of the FL-160 gene family (FL-160-2) and three other partial ORFs. Sequence analysis reveals the various members of the FL-160 gene family to be approximately 80% homologous in the predicted amino acid sequence, but all retain the 12-amino acid molecular mimicry epitope on the COOH terminus. Comparison of the sequence of FL-160-2 to other sequences demonstrates amino acid homology to bacterial sialidase (27%), members of the SA85 gene family (25-30%) and the shed acute-phase antigen/neuraminidase/trans-sialidase gene family (25-30%). Quantitative hybridization at high stringency suggests 750 copies of FL-160 are present in the DNA of each parasite. Reverse transcription and sequence analysis demonstrates that at least five of the members of the FL-160 gene family are transcribed. The NH2 terminus of one of the FL-160 gene products was expressed and antibodies prepared. Antibodies directed to either the COOH or the NH2 terminus of FL-160 bind a 160-kD T. cruzi protein. Both antibodies bind the surface membrane in the flagellar pocket of the trypomastigote. Antibodies to the NH2 terminus bind epineurium and scattered linear densities in sciatic nerve in a pattern distinct from the pattern with antibodies to the COOH terminus. Thus, there are at least two distinct molecular mimicry epitopes on the FL-160 molecule and both mimic epitopes found in nervous tissues. FL-160 may be involved in the generation of autoimmunity to nervous tissues by molecular mimicry, observed in chronic Chagas' disease.

Cloning and Expression of Rabbit Pancreatic Phospholipase A 2

Pancreatic phospholipase A 2 (PLA 2; E.C. 3.1.1.4) has been cloned from a gt11 library made from poly A + RNA of adult rabbit pancreas. PLA 2 catalyzes the hydrolysis of the 2-acyl ester bond of 3-sn-phophoglycerides. As the rabbits are classically used for the study of diet induced changes in lipid metabolism, as a prelude to studying the diet and age dependent changes in this enzyme, we have undertaken to clone it from a rabbit pancreatic library. Three full length clones were obtained from the rabbit pancreatic library when probed with a synthetic oligonucleotide derived from the conserved portion of the molecule. One of these clones is completely sequenced and analyzed. The sequence consists of 606 nucleotides with an open reading frame of 441 nucleotides, coding for 147 amino acids which include a 15 amino acid leader peptide and a seven amino acid propeptide. Northern blot analysis revealed a major mRNA band at 600bp. When compared to phospholipases A 2 of other species, rabbit PLA 2 exhibited considerable conservation both at nucleotide and the protein level. In vitro translation of synthetic mRNA obtained from T 7 polymerase transcription of the cDNA yielded a protein of apparent molecular weight of 15kd similar to that predicted from its primary structure.

Biochemical characterization and solubilization of human NK2 receptor expressed in Chinese hamster ovary cells.

The human ileum neurokinin NK2 receptor has been stably expressed in Chinese hamster ovary (CHO) cells using the dihydrofolate reductase (DHFR) expression system. Amplified cell populations expressing approximately 7 x 10(5) NK2 receptors/cell were selected in the presence of the DHFR inhibitor methotrexate. Cross-linking of [125I]NKA to NK2 receptor transfected cells revealed a specifically labeled protein of apparent molecular weight 64 kDa by SDS-polyacrylamide gel electrophoresis. This protein was deglycosylated by the enzymes N-glycosidase F and endoglycosydase F to a protein of apparent molecular weight of 39 kDa. The NK2 receptor was solubilized in an active form from CHO cell membranes using the zwitterionic detergent CHAPS. This method represents a valuable approach for the production of significant amounts of NK2 receptor protein from mammalian cells.

Purification and characterization of fructan: fructan fructosyltransferase from Jerusalem artichoke (Helianthus tuberosus L.)

SUMMARYFructan: fructan fructosyltransferase activity (FFT, EC 2.4.1.100) from Helianthus tuberosus L. was purified 221‐fold by a basic procedure involving ammonium sulphate precipitation, lectin chromatography and ion‐exchange chromatography. The resulting FFT preparation was separated into three protein bands, of apparent molecular weight 72800, 60 500 and 56 200, by denaturing polyacrylamide gel electrophoresis. These proteins showed affinity to sucrose‐Eupergit. FFT proteins with a molecular weight of 72800 were isolated by preparative native gel electrophoresis, and yielded six distinguishable forms on separation by analytical isoelectric focusing. Proteins from the basic purification were separated by preparative isoelectric focusing into several forms with isoelectric points between pH 4.3 and 4.5. Samples from the gel with FFT activity were analyzed by denaturing polyacrylamide gel electrophoresis. Three samples contained only protein with the molecular weight 72800, and one sample contained only protein of apparent molecular weight 60500. The remaining samples contained a mixture of proteins with molecular weights of 72800, 60500 and 56 200.FFT was detected by 1‐kestose‐dependent nystose production. The enzyme was most active at pH 65, and up to 80 % of the activity was retained on pre‐incubation (1 h) at temperatures of up to 40 °C. FFT transferred fructosyl groups from oligofructans [degree of polymerization (DP) 3–8] of the inulin series. No glycosyl transfer occurred with 6‐kestose, neokestose, maltose, raffinose and maltotriose as the sole substrate. Sucrose efficiently accepted fructosyl units from oligofructans with a Km of approximately 0.2 mM. The rate of fructosyl transfer increased with degree of polymerization (from DP 4). 1‐kestose was shown to be an efficient donor of fructosyl units to sucrose.

A Sensitive Peroxidase Staining Immunoblotting Method for Measuring Total Protein S in Human Plasma

SummaryIn plasma, protein S is found in its free form and as a complex with C4b-binding protein. After 125I-protein S was added tonormal human plasma and applied to SDS-8% polyacrylamide gel electrophoresis, the autoradiogram of the gel showed only one single band at free protein S position. Applying this evidence, we have developed a peroxidase staining Western Blotting method to quantitate total protein S in human plasma which consists of sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by blotting to nitrocellulose membrane and a sensitive avidin-biotinylated peroxidase staining method (ABC technique). The measurement of protein S by the immunoblotting was reproducible and the coefficient of variation was 7%. As little as 1 ng of protein S could be detected. C4b-binding protein did not affect the measurement of protein S. Compared to other immunoassays, this peroxidase staining immunoblotting method has the advantage of directly estimating the apparent molecular weight of protein of interest, eliminating nonspecific stain and having high sensitivity without using radioisotope.

Salicylate-inducible antibiotic resistance in Pseudomonas cepacia associated with absence of a pore-forming outer membrane protein.

The most common mechanism of antibiotic resistance in multiply resistant Pseudomonas cepacia is decreased porin-mediated outer membrane permeability. In some gram-negative organisms this form of antibiotic resistance can be induced by growth in the presence of weak acids, such as salicylates, which suppress porin synthesis. To determine the effects of salicylates on outer membrane permeability of P. cepacia, a susceptible laboratory strain, 249-2, was grown in 10 mM sodium salicylate. Antibiotic susceptibility and uptake, as well as outer membrane protein patterns, were compared between strain 249-2 grown with and without salicylates. The MICs of chloramphenicol, trimethoprim, ciprofloxacin, and ceftazidime were compared between organisms grown in standard and salicylate-containing medium and are as follows: chloramphenicol, 12.5 versus 100 micrograms/ml; trimethoprim, 0.78 versus 3.125 micrograms/ml; ciprofloxacin, 0.4 versus 1.56 micrograms/ml; ceftazidime, 3.125 versus 3.125 micrograms/ml. The permeability of beta-lactam antibiotics was calculated from the rate of hydrolysis of the chromogenic cephalosporin, PADAC. There was no significant difference between strains grown in the presence and absence of salicylate. By using high-pressure liquid chromatography quantitation of loss from culture medium, the effect of 10 mM salicylate on the cellular permeability of chloramphenicol was measured in strain 249-2 by introduction of a plasmid which encodes production of chloramphenicol acetyltransferase. After 1 h of incubation, 18.5% +/- 1.54% versus 70.1% +/- 3.52%, and after 2 h, 4.20% +/- 1.65% versus 41.90% +/- 2.16% remained in supernatants from organisms grown in the absence and presence of 10 mM salicylate, respectively. Outer membrane protein pattern analysis demonstrated the absence of a protein of apparent molecular weight of 40,000 when strain 249-2 was grown in the presence of 10 mM salicylate. To determine whether this protein functioned as a porin, reconstituted membrane vesicles were constructed to assess antibiotic permeability. Vesicles constructed with this salicylate-suppressible outer membrane protein (OpcS) were permeable to chloramphenicol but not to penicillin G. These findings suggest that OpcS is a selective, antibiotic-permeable porin which can be suppressed by growth in the presence of salicylate. Further investigation will be required to determine the biochemical effects of salicylate on porin synthesis.

Bacteriophage T4 gene 21 encodes two proteins essential for phage maturation

The T4 prohead protease (T4 Pase) is the key enzyme in the morphopoietic pathway of the T4 phage head. It is responsible for the proteolytic processing of all head proteins allowing protein rearrangement and head expansion. To study its biochemistry and gene regulation, T4 gene 21 was cloned into an expression vector under the control of the inducible tac promoter. Two proteins of apparent molecular weights of 21.5 and 27.5 kDa were detected after induction. These proteins are synthesized using two different start codons in the same reading frame. Destruction of either start codon resulted in the loss of the respective protein. Complementation experiments with bacteriophage T4 21 −-infected cells showed that both proteins are functional in vivo and essential for T4 phage assembly.

Characterization and toxicity to mosquito larvae of four Bacillus sphaericus strains isolated from Brazilian soils

Four Bacillus sphaericus strains, S1, S2, S5, and L2, isolated from Brazilian soils, were found to be toxic to larvae of the mosquitoes Culex pipiens and Anopheles stephensi at a level similar to that of strain 2362 which is now used operationally. Like strain 2362, the four strains belonged to the serotype H5 and produced major proteins of apparent molecular weights of 125, 110, 56, and 43 kDa. These latter two proteins were immunologically related to toxins of the same molecular weight as B. sphaericus 2362. Although the four Brazilian strains were very similar to strain 2362, gas chromatography analysis of the fatty acids revealed that these strains were different from strain 2362 and from each other, except for a possible similarity between strains S1 and S5.

Expression of “Dehydrin-Like” Proteins in Embryos and Seedlings of Zizania palustris and Oryza sativa during Dehydration

Proteins inducible by dehydration and abscisic acid (ABA), termed dehydrins or RAB (Responsive to ABA) proteins, have been identified in a number of species and have been suggested to play a role in desiccation tolerance, particularly during seed development. Seeds (caryopses) of North American wild rice (Zizania palustris var interior [Fassett] Dore) are tolerant of dehydration to <10% moisture content (fresh weight basis) only under restricted dehydration and rehydration conditions. In comparison, seeds of paddy rice (Oryza sativa L.) readily tolerate desiccation to <5% water content. Expression of "dehydrin-like" proteins in Zizania and Oryza seedlings and embryos was examined to investigate the relationship between the presence of such proteins and desiccation tolerance. [(35)S]Methionine labeling of newly synthesized proteins showed that seedlings (first leaf stage) of both Zizania and Oryza synthesized a novel "heat-stable" protein of apparent molecular weight = 20,000 when dehydrated to <75% of their initial fresh weight. ABA (100 micromolar) induced synthesis of a protein with similar electrophoretic mobility in both species. Western blots using antiserum raised against maize (Zea mays L.) dehydrin detected a protein band from dehydrated Zizania shoots and mature embryonic axes that comigrated with the labeled 20-kilodalton polypeptide. Northern blots using a cDNA for an ABA-responsive protein from Oryza (rab 16a) showed that both seedlings and excised embryonic axes of Zizania accumulated RNA similar in sequence to rab 16a in response to water loss. Zizania seedlings and embryonic axes were also capable of ABA accumulation during dehydration. The intolerance of Zizania seeds to dehydration at low temperature is apparently not due to an absence of dehydrin-like proteins or an inability to accumulate ABA.

Isolation and characterization of polyferredoxin from Methanobacterium thermoautotrophicum The mvhb gene product of the methylviologen-reducing hydrogenase operon

The methylviologen-reducing hydrogenase operon of Methanobacterium thermoautotrophicum contains an open reading frame. mvhB, the product or which was predicted to have a molecular weight of 44 kDa and to contain as many as 48 iron atoms in 12 [4Fe-4S] clusters, and was therefore suggested to be a polyrerredoxin. We have now, for the first time, isolated this polyfarradoxin. Its identity with the mvhB gene product was evidenced by a comparison of the N-terminal amino acid sequence. The dark-brown protein of apparent molecular weight 44 kDa was found to contain 53 mol Fe and 43 mol acid-labile sulfur per mol. The UV/visible spectrum showed two maxima at 280 nm and 390 nm, and a shoulder at 308 nm. The A 390/ A 280 ratio was 0.73. The molar extinction coefficient at 390 nm was 170,000 M −1 cm −1. In the dithionite reduced State the protein displayed an EPR spectrum like that of [4Fe-4S] clusters. The results indicate that the mvhB gene product is indeed a polyferredoxin.

Characterization of prolactin and growth hormone immuno- and bioactivities in the pituitary gland and serum of the squirrel monkey (Saimiri boliviensis boliviensis).

The immunological and biological activities of growth hormone (GH) and prolactin (PRL) in the pituitary gland and serum of the squirrel monkey (Saimiri boliviensis boliviensis) have been studied. Proteins in pituitary homogenates were solubilized in 1% SDS, electrophoresed on 12% polyacrylamide gels, and transferred to nitrocellulose. Squirrel monkey GH and PRL were identified by immunoblotting with anti-human GH antibodies and a monoclonal antibody to ovine PRL, 6F11, respectively. Squirrel monkey GH appeared predominantly as two proteins of apparent molecular weight 22 and 20 kD, corresponding to native and variant forms of human GH. Squirrel monkey PRL appeared as two proteins of apparent molecular weight 24 and 26.5 kD, which co-migrated with native and glycosylated forms of ovine PRL. The cross-reactivity of neutralizing antibodies to human GH and PRL with squirrel monkey GH and PRL were examined using the Nb2 lymphoma bioassay. One of three monoclonal antibodies to human GH (2A1) neutralized squirrel monkey GH with an apparent affinity for squirrel monkey GH (IC50 = 70 ng IgG/ml) which was fourfold lower than for human GH (IC50 = 15 ng IgG/ml). Both polyclonal [AR38-5(1)] and monoclonal (9C3) antibodies to human PRL inhibited the activity of squirrel monkey PRL, athough their affinities for squirrel monkey PRL were four- and twentyfold lower than for human PRL. The activities of antibodies 2A1 and 9C3 on GH and PRL in squirrel monkey serum were also examined by the Nb2 bioassay. The anti-glucocorticoid RU486 was used in all incubations with squirrel monkey serum to eliminate the effect of high glucocorticoid levels on Nb2 cell growth. The mitogenic activity of squirrel monkey serum in the Nb2 assay was completely eliminated in the presence of 2A1 and 9C3. This study represents the first description of the biochemistry of GH and PRL in the squirrel monkey.

GTP-related effects and binding by differentiating fetal rabbit type II alveolar cells in vitro

Type II alveolar cells were isolated from fetal rabbit lungs and used to determine the effect of GTP-binding protein activation on release of surfactant-related material. Cells were prelabelled with [ 3H]choline for 24 hours. NaF, a G-protein activator and GTPγS, a nonhydrolysable analogue of GTP, both loaded by hypoosmotic shock treatment, stimulated release of radioactive disaturated phosphatidylcholine. Localization of the cellular binding of [ α- 32P]GTP in fetal type II cells which were induced to differentiate by exposure to fetal lung fibroblast conditioned medium showed that two proteins of apparent molecular weights of 39.6kd and 17.9kd bound [ α- 32P]GTP. These proteins were detected only in the cells exposed to the conditioned medium. These results suggest GTP-binding proteins are involved in DSPC secretion and differentiation of fetal type II cells is accompanied by changes in GTP binding characteristics.

Culture of pea embryo axes for studies on the reactivation of the cell cycle at germination

A method for culturing excised pea embryo axes, which allows growth of large batches of embryo axes in a reduced volume of medium and is suitable for performing biochemical analyses and early treatment or labeling of embryos with exogenous substances, has been devised. Pea embryo axes were excised from the seeds after 3 h of imbibition and cultured in a mineral medium containing sucrose, vitamins, and casamino acids. Under these conditions, the reactivation of the cell cycle occurs much earlier than in intact seeds: with flow cytometry and bromodeoxyuridine labeling, cells in the S phase were found in the root meristems after only 12 h from the beginning of imbibition. The transition of cells from a quiescent to a proliferative state is accompanied by changes in the electrophoretic pattern of nuclear proteins, particularly with regard to two proteins of apparent molecular weight of 90 and 46 kDa, as shown previously in normally germinating seeds.

Identification of a 220 kDa membrane-associated plant cell protein immunologically related to human β-spectrin

Electrophoretic analysis of low ionic strength extracts of tomato plant leaves revealed the presence of two proteins of apparent molecular weights of 240 kDa and 220 kDa which co-migrated with purified human erythrocyte α- and β-spectrin subunits. Immunochemical analyses employing an affinity-purified polyclonal antibody to human erythrocyte β-spectrin reacted specifically with the 220 kDa plant cell protein. Immunofluorescence microscopy indicated that the β-spectrin antibody recognized an antigen which was primarily restricted to the peripheral areas of the cells. Collectively, these results suggest that the cells of higher plants contain polypeptides related to the spectrin family of proteins. It is proposed that the plant cell possesses a membrane skeleton which is structurally and perhaps functionally analogous to that of the animal cell.

An ontogenically regulated 48-kDa protein is a component of the Na(+)-bile acid cotransporter of rat liver

Recent evidence suggests that the Na(+)-coupled carrier mechanism for bile acids on the hepatocyte basolateral plasma membrane is a polypeptide in the molecular weight range of 48,000-50,000. In this study we used a strategy for the identification and isolation of this transport protein based on the observation that Na(+)-dependent transport activity is abruptly expressed in fetal rat liver just before birth [Suchy et al. Am. J. Physiol. 251 (Gastrointest. Liver Physiol. 14): G665-G673, 1986]. Analysis of basolateral plasma membranes by SDS-PAGE revealed that a protein of apparent molecular weight 48,000 was absent from fetal rat liver on day 19 of gestation, barely detectable on day 20, and thereafter increased progressively with postnatal development. Monospecific, polyclonal antibodies raised against the 48-kDa protein but not preimmune antibodies significantly inhibited the initial rate of Na(+)-dependent taurocholate uptake by isolated rat hepatocytes. In contrast, Na(+)-independent taurocholate transport and uptake of another anion, 35SO4(2-), were not affected by antibody treatment. When an extract containing the total complement of basolateral proteins was incorporated into asolectin liposomes, Na+ gradient-dependent uptake of taurocholate was observed, including a 2- to 2.5-fold accumulation of substrate above its equilibrium concentration (overshoot). However, if the membrane extract was first selectively depleted of the 48-kDa protein by immunoprecipitation with the anti-48-kDa antibody before reconstitution, Na(+)-dependent stimulation of taurocholate transport was completely abolished. These studies indicate that an ontogenically regulated 48-kDa protein is a component of the basolateral Na(+)-dependent transport system for bile acids.

An Inherited Defect of Neutrophil Motility and Microfilamentous Cytoskeleton Associated With Abnormalities in 47-Kd and 89-Kd Proteins

AbstractA 2-month-old male Tongan infant presented with fever, severe skin and mucosal infections, hepatosplenomegaly, thrombocytopenia, and normal neutrophil counts. While polymorphonuclear neutrophil (PMN) morphology was normal, several neutrophil motile functions were found to be altered in the patient. Furthermore, two siblings had died in infancy with a similar clinical picture, raising the possibility of an inherited neutrophil defect. Random migration and chemotaxis, assessed by the under agarose method, were profoundly impaired. Actin polymerization, as measured by flow cytometry of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallaci- din (NBD-phallacidin)-stained PMNs, showed lower basal F-actin and a 1.75-fold increase in response to 10'7 mol/L formyl-methionyl-leucyl-phenylalanine (FMLP) compared with a 4.51-fold increase in control. Microscopic examination of NBD-phallacidin-stained PMN spread on glass showed decreased area of spreading and F-actin-rich filamentous projections distinct from control. The early phase of FMLP- induced right angle light scattering was absent, similar to the effect caused by cytochalasin-B (CB), an inhibitor of actin polymerization. Accordingly, FMLP induced secretion of elastase without the addition of CB. Staphylococcus aureus killing was 50% of control whereas superoxide production in response to FMLP and surface expression of CD11b were greater than twice normal. Partial defects in actin polymerization and scatter were seen in the parents and release of elastase, in the absence of CB, was also increased in both parents. Sodium dodecyl sulfate-polyacrylamide electrophoresis of whole cell proteins from the patient showed a marked decrease in an 89-Kd protein (8% of control) and a marked increase in a 47-Kd protein (4.2-fold). Both mother and father had decreased 89-Kd (77% and 42% of control) and increased 47-Kd proteins (2- and 3.4-fold), although neither had recurrent infections or chemotactic defects. These studies describe a new inherited actin dysfunction syndrome associated with severe propensity to fungal infection and draw attention to the proteins of apparent molecular weights of 89 Kd and 47 Kd, which may be of great importance in the regulation of actin polymerization in human PMNs.

An inherited defect of neutrophil motility and microfilamentous cytoskeleton associated with abnormalities in 47-Kd and 89-Kd proteins

A 2-month-old male Tongan infant presented with fever, severe skin and mucosal infections, hepatosplenomegaly, thrombocytopenia, and normal neutrophil counts. While polymorphonuclear neutrophil (PMN) morphology was normal, several neutrophil motile functions were found to be altered in the patient. Furthermore, two siblings had died in infancy with a similar clinical picture, raising the possibility of an inherited neutrophil defect. Random migration and chemotaxis, assessed by the under agarose method, were profoundly impaired. Actin polymerization, as measured by flow cytometry of N-(7-nitrobenz-2-oxa- 1,3-diazol-4-yl)phallacidin (NBD-phallacidin)-stained PMNs, showed lower basal F-actin and a 1.75-fold increase in response to 10(-7) mol/L formyl-methionyl-leucyl-phenylalanine (FMLP) compared with a 4.51- fold increase in control. Microscopic examination of NBD-phallacidin- stained PMN spread on glass showed decreased area of spreading and F- actin-rich filamentous projections distinct from control. The early phase of FMLP-induced right angle light scattering was absent, similar to the effect caused by cytochalasin-B (CB), an inhibitor of actin polymerization. Accordingly, FMLP induced secretion of elastase without the addition of CB. Staphylococcus aureus killing was 50% of control whereas superoxide production response to FMLP and surface expression of CD11b were greater than twice normal. Partial defects in actin polymerization and scatter were seen in the parents and release of elastase, in the absence of CB, was also increased in both parents. Sodium dodecyl sulfate-polyacrylamide electrophoresis of whole cell proteins from the patient showed a marked decrease in an 89-Kd protein (8% of control) and a marked increase in a 47-Kd protein (4.2-fold). Both mother and father had decreased 89-Kd (77% and 42% of control) and increased 47-Kd proteins (2- and 3.4-fold), although neither had recurrent infections or chemotactic defects. These studies describe a new inherited actin dysfunction syndrome associated with severe propensity to fungal infection and draw attention to the proteins of apparent molecular weights of 89 Kd and 47 Kd, which may be of great importance in the regulation of actin polymerization in human PMNs.