Immunodetection of biotinylated lymphocyte-surface proteins by enhanced chemiluminescence: a nonradioactive method for cell-surface protein analysis.

Biotinylation and radioiodination have been compared for labeling lymphocyte-surface proteins and the labeled proteins symmetrically immunoprecipitated with antibodies recognizing major lymphocyte markers such as the murine Thy-1, CD25 (the alpha subunit of the interleukin-2 receptor), CD45, and human CD2 glycoproteins. The detection of biotinylated proteins by enhanced chemiluminescence after transfer to nitrocellulose was found to be fast and as efficient as the detection of iodinated proteins by autoradiography. The vectoriality of cell-surface biotinylation was ascertained by two-dimensional electrophoresis of the cellular extract in which the major cytoplasmic proteins were not found biotinylated. This nonradioactive labeling procedure offers a convenient and efficient alternative to radiolabeling of cell surfaces for the biochemical analysis of extracellular domains of membrane proteins.     

Flow-injection determination of proteins using enhanced peroxyoxalate chemiluminescence applied to the determination of immunoglobin G and albumin in serum.

The intensity of the chemiluminescence (CL) signal from an aqueous peroxyoxalate CL reaction can be significantly enhanced in the presence of various proteins with hydrophobic sites. A flow-injection measurement for various hydrophobic proteins based on this CL enhancement was developed. The enhancement is due to the inclusion of the CL species in the favorable environment provided by the protein's hydrophobicity, which results in efficient light production. Various protein structures were evaluated; the degree of enhancement depends on the protein structure and CL reaction conditions. The CL enhancement measurement in the flow-injection system is made after the introduction of the protein solution to the main phosphate buffer stream followed by the addition of the CL reagent streams: (1) hydrogen peroxide in water and (2) 8-anilino-1-naphthalene sulfonic acid and 4,4'-oxalylbis-(trifluoromethylsulfonylimino)ethylene bis(4-methyl morpholinium trifluoromethane sulfonate) in acetonitrile. Although prior separation of proteins is required before the measurement, the advantage of this approach is increased sensitivity without derivatization of the protein. The enhancement was demonstrated for several proteins, including antibodies, which suggests that this approach may be generally applicable to a variety of measurements, including immunoassay determinations. This CL enhancement was used to develop a simple and accurate flow-injection measurement for the determination of albumin and IgG in human serum.     

Large scale characterization of plant plasma membrane proteins.

After a brief review of the strategies used to date to identify systematically plasma membrane (PM) proteins, emphasis was given to the proteomic approach of PM proteins from the model plant Arabidopsis thaliana. Comparative analysis of two-dimensional gels from PM and cytosolic fractions was used to assess the cellular origin of proteins found in PM fraction. The classification obtained was confirmed by protein sequencing that showed, in addition, that most analyzed proteins were peripheral proteins. A large proportion of these appeared to correspond to PM-constitutive proteins that were present in the PM from different plant organs, but were not uniquely located at the PM depending on the organ. In addition, the presence of organ-specific sets of PM-specific proteins was also demonstrated. Additional procedures were developed to identify integral PM proteins. The combined use of PM washes with alkaline carbonate buffer or Triton X-100/KBr, and of a new detergent to solubilize protein, resulted in improved recovery of hydrophobic proteins on gels. Results are discussed in terms of construction of comprehensive proteomes for PM and other membranes and organelles.     

A nonradioactive screening method for cloning genes encoding sequence-specific DNA binding proteins.

We have developed a novel nonradioactive screening method for cloning genes encoding sequence-specific DNA binding proteins. This method is derived from previously described protocols developed for the same purpose by using radioactively labeled DNA probes containing protein recognition sequences. This nonradioactive strategy relies upon the use of a small hapten, digoxigenin. Fusion proteins expressed from the recombinant bacteriophage lambda gt11/lambda ZAP are immobilized on nitrocellulose filters and probed with digoxigenin-labeled double-stranded DNA as a ligand. The specifically bound DNA probes can be detected through sequential incubations with antibody-enzyme conjugate and enzyme substrates. This technique has been successfully utilized to isolate several cDNA clones encoding DNA binding proteins.     

A nonradioactive method for isolating complementary DNAs endocing calmodulin-binding proteins

Calmodulin labeled with¹²⁵I or³⁴S has been used to screen expression libraries to isolate cDNAs encoding calmodulin-binding proteins (CBPs) from several eukaryotic systems. The use of radiolabeled calmodulin has, however, several disadvantages. We have developed a nonradiactive method to isolate cDNAs for CBPs using biotinylated calmodulin. Screening of a cDNA library in an expression vector with biotinylated calmodulin resulted in the isolation of cDNAs encoding CBPs. Avidin and biotin blocking steps, prior to incubation of the filters with biotinylated calmodulin, are found to be essential to eliminate the cDNAs that code for biotin-containing polypeptides. The cDNA clones isolated using this nonradioactive method bound calmodulin in a calcium-dependent manner. The binding of biotinylated calmodulin to these clones was completely abolished by ethylene glycolbis(\-aminoethylether)-N,N′-tetraacetic acid (EGTA), a calcium chelator. Furthermore, the isolated cDNAs were confirmed by probing the clones with³⁵S-labeled calmodulin. All the isolated clones bound to radiolabeled calmodulin in the presence of calcium but not in the presence of EGTA. The method described here is simple, fast, and does not involve preparation and handing of radiolabeled calmodulin. All the materials used in this method are commercially available; hence, this procedure should be widely applicable to isolate cDNAs encoding CBPs from any eukaryotic organism.     

Isolation and characterization of major intrinsic microsomal membrane proteins.

Treatment of the membrane matrix derived from hepatic microsomes with buffered 1 M urea resulted in the selective extraction of a group of proteins together with a portion of the membrane lipid. Thorough chemical characterization of this fraction has been performed, and the proteins have been fractionated by two different procedures. The first of these, preparative polyacrylamide gel electrophoresis, has produced five highly homogeneous membrane proteins which have been characterized with regard to molecular weight, electrophoretic behavior in five different polyacrylamide systems, NH2 terminus, relative carbohydrate content, isoelectric point, and amino acid composition. The five proteins of this group fell in the molecular weight range of 54,000 to 96,000 and had isoelectric points ranging from pH 4.9 to pH 6.7. Further fractionation of the urea-soluble proteins by gel filtration in a sodium dodecyl sulfate-containing medium resulted in the isolation of four homogeneous molecular weight classes of proteins which have been characterized with respect to various physicochemical parameters. The major membrane glycoprotein (apparent molecular weight, 171,000) was isolated by this procedure and found to contain approximately equal amounts of NH2-terminal glycine and serine. suggesting the presence of at least two polypeptide chains in this molecular weight region. From the urea-insoluble fraction of the membrane comprising approximately 80% of the total protein, five intrinsic polypeptides designated S-5 through S-9 were isolated. S-5 (54,000) and S-6 (49,000) represent the most prominent components in the microsomal membrane, accounting for close to 30% of the total protein. Also isolated and characterized is the smallest membrane protein (S-9), a hydrophobic polypeptide of molecular weight 16,000. All of the urea-insoluble proteins are glycoproteins, and S-7 (35,000) gives the second most intense stain for carbohydrate of all proteins in the microsomal membrane.     

Immunodot detection of nisin Z in milk and whey using enhanced chemiluminescence

A highly specific antisera was produced in New Zealand white rabbits against nisin Z, a 3400 Da bacteriocin produced by Lactococcus lactis ssp. lactis biovar. diacetylactis UL 719. A dot immunoblot assay was then developed to detect nisin Z in milk and whey. As few as 1·5 10⁻¹ international units per ml (IU ml⁻¹), corresponding to 0·003 μg ml⁻¹ of pure nisin Z, were detected in carbonate-bicarbonate buffer within 6 h using chemiluminescence. When milk and whey samples were tested, approximately 0·155 μg ml⁻¹ (7·9 IU ml⁻¹) of nisin Z was detected. The detection limit obtained was lower than that of traditional methods including microtitration and agar diffusion.     

Cloning, expression, and biochemical characterization of hexahistidine-tagged terminase proteins.

The terminase enzyme from bacteriophage lambda is composed of two viral proteins (gpA, 73.2 kDa; gpNu1, 20.4 kDa) and is responsible for packaging viral DNA into the confines of an empty procapsid. We are interested in the genetic, biochemical, and biophysical properties of DNA packaging in phage lambda and, in particular, the nucleoprotein complexes involved in these processes. These studies require the routine purification of large quantities of wild-type and mutant proteins in order to probe the molecular mechanism of DNA packaging. Toward this end, we have constructed a hexahistidine (hexa-His)-tagged terminase holoenzyme as well as hexa-His-tagged gpNu1 and gpA subunits. We present a simple, one-step purification scheme for the purification of large quantities of the holoenzyme and the individual subunits directly from the crude cell lysate. Importantly, we have developed a method to purify the highly insoluble gpNu1 subunit from inclusion bodies in a single step. Hexa-His terminase holoenzyme is functional in vivo and possesses steady-state and single-turnover ATPase activity that is indistinguishable from wild-type enzyme. The nuclease activity of the modified holoenzyme is near wild type, but the reaction exhibits a greater dependence on Escherichia coli integration host factor, a result that is mirrored in vivo. These results suggest that the hexa-His-tagged holoenzyme possesses a mild DNA-binding defect that is masked, at least in part, by integration host factor. The mild defect in hexa-His terminase holoenzyme is more significant in the isolated gpA-hexa-His subunit that does not appear to bind DNA. Moreover, whereas the hexa-His-tagged gpNu1 subunit may be reconstituted into a holoenzyme complex with wild-type catalytic activities, gpA-hexa-His is impaired in its interactions with the gpNu1 subunit of the enzyme. The results reported here underscore that a complete biochemical characterization of the effects of purification tags on enzyme function must be performed prior to their use in mechanistic studies.     

Proteomics characterization of abundant Golgi membrane proteins

A mass spectrometric analysis of proteins partitioning into Triton X-114 from purified hepatic Golgi apparatus (84% purity by morphometry, 122-fold enrichment over the homogenate for the Golgi marker galactosyl transferase) led to the unambiguous identification of 81 proteins including a novel Golgi-associated protein of 34 kDa (GPP34). The membrane protein complement was resolved by SDS-polyacrylamide gel electrophoresis and subjected to a hierarchical approach using delayed extraction matrix-assisted laser desorption ionization mass spectrometry characterization by peptide mass fingerprinting, tandem mass spectrometry to generate sequence tags, and Edman sequencing of proteins. Major membrane proteins corresponded to known Golgi residents, a Golgi lectin, anterograde cargo, and an abundance of trafficking proteins including KDEL receptors, p24 family members, SNAREs, Rabs, a single ARF-guanine nucleotide exchange factor, and two SCAMPs. Analytical fractionation and gold immunolabeling of proteins in the purified Golgi fraction were used to assess the intra-Golgi and total cellular distribution of GPP34, two SNAREs, SCAMPs, and the trafficking proteins GBF1, BAP31, and alpha(2)P24 identified by the proteomics approach as well as the endoplasmic reticulum contaminant calnexin. Although GPP34 has never previously been identified as a protein, the localization of GPP34 to the Golgi complex, the conservation of GPP34 from yeast to humans, and the cytosolically exposed location of GPP34 predict a role for a novel coat protein in Golgi trafficking.     

Selective solubilization and biochemical analysis of R. prowazekii outer membrane proteins]

Solubilization of proteins from total membranes (a mixture of cytoplasmic and outer membranes) of Rickettsia prowazekii, a typical gram-negative bacterium, was studied using three different detergents. It was shown that isolated outer membranes and sarkosyl-insoluble material contain major polypeptides of 134, 31, 29.5 and 25 kDa as well as minor polypeptides of 78, 60, 42, and 17 kDa, while the total membranes--the same plus a great number of additional minor proteins. The material solubilized by octyl glucoside in the presence of MgCl2 contains exclusively major proteins (134, 31, 29.5, and 25 kDa). No differential solubilization takes place upon membrane treatment with octyl glucoside in the absence of Mg2+ or with Triton X-100. Rickettsial proteins are insensitive to trypsin in both whole cells and total membranes, unless the latter are presolubilized with octyl glucoside. Proteinase K degrades all of the total membrane proteins but only the 134 kDa polypeptide of whole cells. Upon immunoblotting predominantly the major outer membrane proteins (134, 31, and 20.5 kDa) and, to a lesser extent, the minor proteins (60, 42, and 17 kDa) interact with human convalescent serum.     

Regular structures in unit membranes. II. Morphological and biochemical characterization of two water-soluble membrane proteins isolated from the suckling rat ileum

Specialized plasma membranes from the endocytic complex of ileal epithelial cells of suckling rats were isolated by differential flotation. Thin-section and negative-stain electron microscopy showed the luminal surfaces of these membranes to be covered by an ordered array of particles 14.5-nm separations in long rows. This particulate coating was released from the membrane surfaces by 10 mM CaCl2 and recovered free of membranes after dialysis against 0.5 mM EGTA and high- speed centrifugation. Two proteins were resolved by gel filtration to be in supernate: n-acetyl-beta-glucosaminidase and a filamentous protein which attaches n-acetylglucosaminidase to the membrane surface thereby providing bidirectionality to the array of enzyme. We believe that the filamentous protein has not been previously described. Therefore we have called it ligatin from the latin ligare, which translates "to bind together". Furthermore, we suggest that the membranes of the endocytic complex contain sites for the extracellular digestion of carbohydrate moieties in the maternal milk.     

Identification and preliminary characterization of Vibrio cholerae outer membrane proteins.

Outer membrane proteins of Vibrio cholerae were purified by sucrose density centrifugation and Triton X-100 extraction at 10 mM Mg2+. The proteins were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. V. cholerae outer membrane proteins presented a unique pattern when compared with the patterns of other gram-negative rods. There were 8 to 10 major bands (Mr 94,000 to 27,000), with most of the protein located in band 5 (Mr approximately 45,000), which thus appears to be the major structural protein of the outer membrane. Lipid and carbohydrate were associated with band 6.     

Detection of Legionella pneumophila serogroup 1 urinary antigen using an enhanced chemiluminescence ELISA

An enhanced chemiluminescence enzyme-linked immunosorbent assay has been developed for the detection of soluble antigen in the urine of patients with Legionnaires' disease (LD). In the assay antigen(s) in the urine samples are captured by a rabbit anti-L. pneumophila antibody coated onto microtitre strips. A fluorescein-isothiocyanate (FITC) conjugate of the same antibody is then added which binds to the captured antigen. Any immobilized FITC-labelled antibody is then detected with a horseradish peroxidase (HRP) conjugate of a monoclonal anti-FITC antibody. HRP activity is monitored after oxidation of luminol in the presence of H₂O₂ and iodophenol. The resulting luminescence is recorded using a camera luminometer. Urine specimens were available for testing from 31 patients with evidence of ongoing L. pneumophila serogroup 1 infection. A positive result was obtained in the cases of 12/12 specimens from culture-proven LD patients, and 16/19 specimens from patients with serological evidence of LD. Thus the sensitivity is estimated to be 28/31 (90%) The specificity was estimated using urine specimens from eight patients with non-L. pneumophila pneumonias of known aetiology. All eight specimens gave a negative result.     

Arabidopsis 22-kilodalton peroxisomal membrane protein. Nucleotide sequence analysis and biochemical characterization.

We sequenced and characterized PMP22 (22-kD peroxisomal membrane protein) from Arabidopsis, which shares 28% to 30% amino acid identity and 55% to 57% similarity to two related mammalian peroxisomal membrane proteins, PMP22 and Mpv17. Subcellular fractionation studies confirmed that the Arabidopsis PMP22 is a genuine peroxisomal membrane protein. Biochemical analyses established that the Arabidopsis PMP22 is an integral membrane protein that is completely embedded in the lipid bilayer. In vitro import assays demonstrated that the protein is inserted into the membrane posttranslationally in the absence of ATP, but that ATP stimulates the assembly into the native state. Arabidopsis PMP22 is expressed in all organs of the mature plant and in tissue-cultured cells. Expression of PMP22 is not associated with a specific peroxisome type, as it is detected in seeds and throughout postgerminative growth as cotyledon peroxisomes undergo conversion from glyoxysomes to leaf-type peroxisomes. Although PMP22 shows increased accumulation during the growth of young seedlings, its expression is not stimulated by light.     

Immunochemical characterization of brain synaptic membrane glutamate-binding proteins

Two glutamate-binding proteins (71 and 63 kDa) were previously purified from synaptic plasma membranes (Chen, J.-W., Cunningham, M.D., Galton, V., and Michaelis, E. K. (1988) J. Biol. Chem. 263, 417-426). These proteins may play a role in glutamate neurotransmission in brain. Polyclonal antibodies were raised against the denatured glutamate-binding proteins in rabbits, including sets of antibodies against each of the binding proteins. The antibodies reacted specifically against both 71- and 63-kDa proteins. The antibodies recognized the denatured form of the proteins in Western blots and the native state of the proteins in enzyme-linked immunosorbent assays and in immunoaffinity chromatography and extraction procedures. All antibodies labeled most strongly the 71-kDa protein in Western blots, but extracted both proteins from solubilized synaptic membrane preparations. These findings indicate that the two proteins are closely related immunologically but the reactivity on Western blots differs between these two proteins. Immunoextraction of the 71- and 63-kDa proteins led to a approximately 60% decrease in L-[3H]glutamate-binding activity associated with synaptic membrane proteins. Of the brain subcellular fractions examined, the isolated synaptic plasma membranes had the strongest reaction in enzyme-linked immunosorbent assays toward the antiglutamate-binding protein antisera. Electron microscopy combined with gold particle immunohistochemistry revealed the sites labeled by the antibodies as entities present either on the surface or within the postsynaptic membranes and the associated densities of brain nerve ending particles (synaptosomes). Immunohistochemical procedures of gold labeling with silver enhancement of labeled sites revealed selective neuronal labeling in brain regions enriched in glutamate neurotransmitter pathways such as the hippocampus. Labeling was along dendrites and around cell bodies of pyramidal neurons. Based on the pattern of histochemical labeling, the distribution of immune reactivity in synaptic membranes, and the extractions of a major component of membrane glutamate-recognizing proteins by the antibodies, the glutamate-binding proteins must play a role in glutamate neurotransmission.     

Systematic characterization of sperm-specific membrane proteins in swine

To establish a systematic strategy for characterizing fertilization proteins of sperm cells, we prepared alloantisera by immunizing gilts with salt-washed membranes from boar spermatozoa. The antisera recognized a unique subset of sperm membrane proteins that migrated with M(r) 7500-66,000 in SDS-PAGE under nonreducing conditions. The antisera did not recognize proteins of erythrocyte membranes, and tissue absorption experiments further confirmed that the alloantigens were sperm-specific proteins. Each of these sperm-specific membrane proteins (SSMPs) possessed one or more disulfide bonds that were essential for its interaction with alloantibody. Enzymatic deglycosylation revealed that most of the SSMPs were glycoproteins, and their alloantigenicity was not dependent on the presence of N-linked oligosaccharides. The presence of disulfide bonds and glycosylation indicated that the SSMPs identified each comprise at least one extracellular domain. Two-dimensional electrophoresis resolved at least 14 distinct SSMPs, 13 of which possessed acidic pIs (range 4.2-4.8). By indirect immunofluorescence, the SSMPs localized to the cell surface overlying all major regions of the sperm cell. We conclude that the repertoire of immunodominant SSMPs in the pig is relatively small, which makes feasible the systematic elucidation of their functions in fertilization.