Identification of calcium-binding proteins associated with the lymphocyte plasma membrane

The Nonidet P40 insoluble fraction of lymphocyte plasma membrane contains three polypeptides of about 68,000-, 33,000- and 28,000-Mr which are solubilised by Ca2+-chelators. As judged by various criteria the 33,000-Mr polypeptide is homologous to the 36,000-Mr pp60src kinase substrate of chicken fibroblasts and the 68,000-Mr polypeptide is related to the 67,000-Mr "calelectrin" of bovine liver. The 28,000-Mr polypeptide may also be related to calelectrin.     

Antigenic relatedness and N-terminal sequence homology define two classes of periplasmic flagellar proteins of Treponema pallidum subsp. pallidum and Treponema phagedenis

The periplasmic flagella of many spirochetes contain multiple proteins. In this study, two-dimensional electrophoresis, Western blotting (immunoblotting), immunoperoxidase staining, and N-terminal amino acid sequence analysis were used to characterize the individual periplasmic flagellar proteins of Treponema pallidum subsp. pallidum (Nichols strain) and T. phagedenis Kazan 5. Purified T. pallidum periplasmic flagella contained six proteins (Mrs = 37,000, 34,500, 33,000, 30,000, 29,000, and 27,000), whereas T. phagedenis periplasmic flagella contained a major 39,000-Mr protein and a group of two major and two minor 33,000- to 34,000-Mr polypeptide species; 37,000- and 30,000-Mr proteins were also present in some T. phagedenis preparations. Immunoblotting with monospecific antisera and monoclonal antibodies and N-terminal sequence analysis indicated that the major periplasmic flagellar proteins were divided into two distinct classes, designated class A and class B. Class A proteins consisted of the 37-kilodalton (kDa) protein of T. pallidum and the 39-kDa polypeptide of T. phagedenis; class B included the T. pallidum 34.5-, 33-, and 30-kDa proteins and the four 33- and 34-kDa polypeptide species of T. phagedenis. The proteins within each class were immunologically cross-reactive and possessed similar N-terminal sequences (67 to 95% homology); no cross-reactivity or sequence homology was evident between the two classes. Anti-class A or anti-class B antibodies did not react with the 29- or 27-kDa polypeptides of T. pallidum or the 37- and 30-kDa T. phagedenis proteins, indicating that these proteins are antigenically unrelated to the class A and class B proteins. The lack of complete N-terminal sequence homology among the major periplasmic flagellar proteins of each organism indicates that they are most likely encoded by separate structural genes. Furthermore, the N-terminal sequences of T. phagedenis and T. pallidum periplasmic flagellar proteins are highly conserved, despite the genetic dissimilarity of these two species.     

A novel type of putrescine (diamine)-acetylating enzyme from the nematode Ascaris suum.

Component polypeptides of both the bovine lens and pituitary multicatalytic proteinase complexes demonstrate different immunoreactivities with a polyclonal antiserum raised against the purified pituitary enzyme. Four (Mr 24000, 26000, 34000 and 38000) of eight bands that have been resolved by SDS/polyacrylamide-gel electrophoresis are stained in immunoblot experiments. Monospecific antibodies obtained from this antiserum by affinity purification from the 38000- and 34000-Mr bands of the lens enzyme bound equally well to either band, but showed little or no binding to the 26000- and 24000-Mr bands upon immunoblotting. Antibody affinity-purified from the 24000-Mr band showed comparable binding to the 24000-, 34000- or 38000-Mr band. One explanation of these results is that the 24000-Mr polypeptide is derived from the higher-Mr polypeptide(s) and has lost some of the common immunodeterminants.     

Structure of the Mouse Mammary Tumor Virus: Polypeptides and Glycoproteins

The polypeptide and glycoprotein compositions of the mouse mammary tumor virus virion from primary monolayer cultures of BALB/cfC3H mouse mammary tumor cells were studied by polyacrylamide gel electrophoresis by using internal and external labeling and Coomassie blue and periodic acid Schiff (PAS) staining. Twelve polypeptides were reproducibly resolved by the combined methods. Five major polypeptides were demonstrable with estimated molecular weights of 52,000, 36,000, 28,000, 14,000, and 10,000. Seven minor polypeptides were also consistently detected and had estimated molecular weights of 70,000, 60,000, 46,000, 38,000, 30,000, 22,000, and 17,000. Carbohydrate was associated with five of these polypeptides as measured by PAS stain or [(3)H] glucosamine labeling, or both. These glycoproteins had estimated molecular weights of 70,000, 60,000, 52,000, 36,000 and 10,000. The majority of the PAS stain and glucosamine was found in the 52,000 and 36,000 dalton peaks.     

Structural homology among calf thymus alpha-polymerase polypeptides.

A sample of highly purified calf thymus alpha-polymerase contained an abundant 118,000 Mr polypeptide as well as five lower molecular weight polypeptides in the range of 54,000- to 64,000-Mr. This 118,000-Mr polypeptide was capable of DNA polymerase activity, as revealed by in situ assay after SDS-polyacrylamide gel electrophoresis. Tryptic peptide mapping indicated that the 118,000-Mr polypeptide shared extensive primary structure homology with 57,000-, 58,000- and 64,000-Mr polypeptides and some limited homology with 54,000- and 56,000-Mr polypeptides. This is the first evidence that lower and higher Mr polypeptides of purified calf thymus alpha-polymerase share sequence homology; these results are interpreted in the context of a model that predicts the existence of a common precursor with molecular weight greater than 140,000.     

The 95000-Mr gelatin-binding protein in human serum and plasma.

A gelatin-binding 95000-Mr protein was detected in human serum and plasma by immunoblotting using antibodies against the 95000-Mr gelatin-binding protein, a major secretory component of cultured adherent human monocyte/macrophages. Serum and plasma were prepared by incubating blood at 4, 22 or 37 degrees C for different periods of time, and gelatin-binding proteins were isolated from 200 microliter portions by gelatin-Sepharose affinity chromatography. The bound material was analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. In protein-stained gels, fibronectin and some minor polypeptides were seen, but not the 95000-Mr protein. In immunoblotting of identical serum samples the antibodies detected apparently two closely spaced polypeptide bands at Mr95000, and in plasma samples a single band at the position of the faster-migrating one of the two above-mentioned bands. The immunoperoxidase reaction was stronger when serum and plasma were prepared by incubating for longer periods of time (up to 8 h) or at higher temperatures (up to 37 degrees C). In samples made from plasma, the immunoperoxidase reactions were weaker than in those from serum, indicating a lower quantity of the protein. The results suggest that the 95000-Mr protein is released from monocytes and granulocytes during the incubation of blood and, more likely, when they possibly interact with the blood clot and may become adherent.     

Storage protein precursor polypeptides in cotyledons of Pisum sativum L. Identification of, and isolation of a cDNA clone for, an 80000-Mr legumin-related polypeptide

An 80 000-Mr polypeptide, which bound to anti-legumin IgG, was detected among labelled polypeptides from cotyledons at late stages of development. When poly(A)-containing RNA from similar cotyledons was translated in a cell-free protein-synthesizing system, an 80 000-Mr polypeptide was also detected. Immunoprecipitation of translation products with anti-legumin IgG showed that, in addition to the major legumin precursor polypeptides of Mr approximately 60 000, the 80 000-Mr polypeptide was specifically immunoprecipitated. A cDNA clone, pCD32, was found to select an RNA coding for an 80 000-Mr polypeptide in hybrid-selection experiments. Additional minor polypeptides of Mr 63 000 and 65 000 were present in translation products of RNA selected by pCD32; all three polypeptides were immunoprecipitated by anti-legumin IgG. Thermal elution of RNAs bound to pCD32 showed that the affinity of pCD32 to the RNA coding for the 80 000-Mr polypeptide was greater than to the RNAs coding for the 63 000-Mr and 65 000-Mr polypeptides. In similar hybrid-selection experiments, another cDNA clone, pCD40, selected RNAs coding predominantly for polypeptides of Mr 63 000 and 65 000. A minor polypeptide of Mr 80 000 was also detected among these products; again all three polypeptides were immunoprecipitated by anti-legumin IgG. Peptide mapping revealed close similarities between the 80000-Mr polypeptide and the 63 000-Mr/65 000-Mr polypeptides obtained by translation of RNAs selected by pCD32. There were similarities also between maps obtained from translation products of RNA selected by pCD32 and those obtained from anti-legumin IgG immunoprecipitates of total translation products of poly(A)-containing RNA.     

Expression of the divergent tricarboxylate transport operon (tctI) of Salmonella typhimurium.

Membrane-associated gene products of shock-sensitive bacterial transport operons are often difficult to detect. A 4.5-kilobase DNA fragment, known to completely encode the Salmonella typhimurium tctI operon, was cloned in both orientations behind the T7 phage promoter phi 10 and expressed by using the T7 polymerase-promoter system of Tabor and Richardson (S. Tabor and C. C. Richardson, Proc. Natl. Acad. Sci. USA 82:1074-1078, 1985). Under these conditions, five proteins were clearly demonstrated. One DNA strand was shown to encode the periplasmic (29,000-Mr) C protein (as a 31,000-Mr precursor), a 19,000-Mr protein, and a 40,000- to 45,000-Mr protein which ran as a diffuse band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The opposite strand carried the information for two additional proteins of 29,000 and 14,000 Mr. By Tn5 mutagenesis, subcloning of Tn5 insertions, and subcloning of various deletion mutants it was shown that the tctI system is divergently transcribed. The periplasmic binding protein (C protein) is the first product of one operon, followed by the 19,000-Mr and 45,000-Mr integral inner membrane proteins. On the opposite strand only the 29,000-Mr protein was essential for tctI function, and it was found to be weakly attached to the inner membrane. Thus tctI encodes four proteins, one periplasmic, two integral, and one peripheral to the cytoplasmic membrane, with the genes arranged as tctA tctB tctC tctD.     

Comparison of indirect immunofluorescence (IF) test with enzyme-linked immunosorbent assay (ELISA) in screening of hybridomas to very virulent Marek's disease virus.

For identifying virus-specific antigens of Marek's disease virus (MDV), monoclonal antibodies (MAbs) against strain Md5 of serotype 1, which is known to be a very virulent MDV (vvMDV), were isolated. Fifty-eight hybridoma clones that secreted MAbs against vvMDV were obtained. Of these MAbs, 36 gave positive reactions in an immunofluorescence (IF) test, and 22 gave positive reactions on enzyme-linked immunosorbent assay (ELISA). None of these MAbs gave positive reactions in both the IF test and ELISA. Of the MAbs that gave positive reactions in the IF test, 33 clones reacted with MDV1-specific epitopes, the other three reacting with MDV1-HVT intertypic epitopes. None of the clones reacted with MDV1-MDV2 intertypic epitopes. Three virus-specific polypeptides were identified by radioimmunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or immunoblotting. These polypeptides were recognized by 12 MAbs giving positive reactions by IF, but by none of those giving positive reactions by ELISA. In addition, size heterogeneity of the MDV1-specific phosphorylated polypeptides in the MDV1 strains was shown using the MAbs against Md5.     

The purification and polypeptide composition of avian infectious bronchitis virus.

Purification of egg-grown infectious bronchitis virus (IBV) by sucrose density gradient centrifugation alone, or sucrose density gradient centrifugation plus pH 8.0 treatment, concanavalin A precipitation or metrizamide density gradient centrifugation, failed to produce any differences in the virus polypeptide pattern following polyacrylamide gel electrophoresis in the presence of SDS(SDS-PAGE). SDS-PAGE of purified IBV on 7.5% acrylamide gels separated 16 polypeptides which were detectable by staining with Coomassie blue or measurement of radioactivity following electrophoresis of (3H)-leucine labelled IBV. The molecular weights of the polypeptides were within the range 15,000-135,000. The polypeptides of egg and chick kidney (CK) cell-grown IBV were identical in both size and number but quantitative differences were detected. In particular the relative proportion of the major 52,000 molecular weight polypeptide was greatly reduced in IBV grown in CK cells. SDS-PAGE of purified IBV and staining with Schiff's reagent to detect carbohydrate revealed four.bands with molecular weights of 128,000, 86,000, 67,500 and 37,000. The 128,000 band did not correspond to any of the detected polypeptides. Use of 5% acrylamide gels for SDS-PAGE of IBV failed to resolve all the minor polypeptides and only seven bands were detected.     

Nuclear matrix of HeLa S3 cells. Polypeptide composition during adenovirus infection and in phases of the cell cycle

A subnuclear fraction has been isolated from HeLa S3 nuclei after treatment with high salt buffer, deoxyribonuclease, and dithiothreitol. This fraction retains the approximate size and shape of nuclei and resembles the nuclear matrix recently isolated from rat liver nuclei. Ultrastructural and biochemical analyses indicate that this structure consists of nonmembranous elements as well as some membranous elements. Its chemical composition is 87% protein, 12% phospholipid, 1% DNA, and 0.1% RNA by weight. The protein constituents are resolved in SDS- polyacrylamide slab gels into 30-35 distinguishable bands in the apparent molecular weight range of 14,000 - 200,000 with major peptides at 14,000 - 18,000 and 45,000 - 75,000. Analysis of newly synthesized polypeptides by cylindrical gel electrophoresis reveals another cluster in the 90,000-130,000 molecular weight range. Infection with adenovirus results in an altered polypeptide profile. Additional polypeptides with apparent molecular weights of 21,000, 23,000, and 92,000 become major components by 22 h after infection. Concomitantly, some peptides in the 45,000-75,000 mol wt range become less prominent. In synchronized cells the relative staining capacity of the six bands in the 45,000-75,000 mol wt range changes during the cell cycle. Synthesis of at least some matrix polypeptides occures in all phases of the cell cycle, although there is decreased synthesis in late S/G2. In the absence of protein synthesis after cell division, at least some polypeptides in the 45,000- 75,000 mol wt range survive nuclear dispersal and subsequent reformation during mitosis. The possible significance of this subnuclear structure with regard to structure-function relationships within the nucleus during virus replication and during the life cycle of the cell is discussed.     

Isolation of synaptosomal plasma membranes from cholinergic nerve terminals and a comparison of their proteins with those of synaptic vesicles

Plasma membranes were purified from purely cholinergic nerve endings (synaptosomes) isolated from the electric organ of Torpedo marmorata. Synaptosomes were lysed, membranes recovered and further separated by density gradient centrifugation. A fraction was obtained enriched in 5'-nucleotidase, Na+, K+-activated ATPase and acetylcholine esterase. Morphological examination showed abundant membrane fragments of the size range of synaptosomes and few of vesicle size. The fraction has a characteristic protein composition upon gel electrophoresis. Five reproducible major bands with apparent Mr of 100000, 75000, 52000, 42000 and 35000--33000 are found. A gel-electrophoretic comparison with proteins from synaptic vesicles from the same source (major bands Mr 160000, 147000, 34000 and 25000) was made. Comigration of major bands was detected in one-dimensional gel electrophoresis with the 42000-Mr, 35000--33000-Mr and 34000-Mr components. Upon two-dimensional gel electrophoresis the 42000-Mr component comigrates with a similar component in vesicles, recently characterized as actin; the other components are different. The presence of tubulin-like polypeptides is unlikely. Beside actin, all major vesicle proteins are often detected in small amounts in the plasma membrane preparation. It cannot be decided if they result from fused or contaminating vesicle membranes, but since they are essentially absent in some preparations, it seems that the plasma membrane does not contain vesicle proteins.     

Changes in fine structure and polypeptide pattern during development of Holospora obtusa, a bacterium infecting the macronucleus of Paramecium caudatum.

The development of the bacterium Holospora obtusa, which infects the macronucleus of Paramecium caudatum, was investigated in the course of a new infection from the infectious form into the reproductive form and vice versa. In parallel with a complete structural reorganization of the bacterium, the protein pattern changed gradually in this development. During the differentiation of the infectious form into the reproductive form, the voluminous periplasm was gradually reduced and the cytoplasm expanded, until the entire bacterium was filled by the cytoplasm. At this stage the long cell divided into five to seven short cells and thereby established the reproductive form, the main stage of the bacterium being maintained and multiplying in the host nucleus. In parallel with the reduction of the periplasm, some of the main proteins of the infectious form gradually disappeared in the electrophoresis pattern; some proteins disappeared earlier than others. Simultaneously, other proteins appeared and gradually became more prominent in the pattern of the developing reproductive form. In the reverse development, when the reproductive form differentiated into the infectious form, the bacterium grew longer, the cytoplasm was condensed, and electron-dense material was deposited in the extending periplasmic space. In parallel with this morphological development, the polypeptide pattern reverted to that of the infectious form.     

Biochemical characterization of the structural and nonstructural polypeptides of a porcine group C rotavirus.

Purified virions or radiolabeled lysates of infected MA104 cells were used to characterize the structural and nonstructural polypeptides of a porcine group C rotavirus. At least six structural proteins were identified from purified group C rotavirus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of these, two (37,000- and 33,000-molecular-weight polypeptides) were associated with the outer shell, as demonstrated by the ability of EDTA to remove them from the purified virion. The other four polypeptides (molecular weights, 125,000, 93,000, 74,000, and 41,000) were located in the inner shell. The structural or nonstructural nature of a 25,000-molecular-weight protein identified in our studies was unclear. Glycosylation inhibition studies with tunicamycin in infected cells demonstrated that the 37,000- and 25,000-molecular-weight proteins were glycosylated and contained mannose-rich oligosaccharides identified by radiolabeling of the infected cells with [3H]mannose. The 37,000-molecular-weight outer shell glycoprotein was shown by pulse-chase experiments to be posttranslationally processed. The kinetics of viral polypeptide synthesis in infected cells were also studied, and maximal synthesis occurred at 6 to 9 h postinfection. The 41,000-molecular-weight inner capsid polypeptide was the most abundant and was the subunit structure of a 165,000-molecular-weight protein aggregate. Two polypeptides (molecular weights, 39,000 and 35,000) appeared to be nonstructural, as determined by comparison of the protein pattern of radiolabeled infected cell lysates with that of purified virions. Radioimmunoprecipitation was used to examine the serologic cross-reactions between the viral polypeptides of a group C rotavirus with those of a group A rotavirus. No serologic cross-reactivities were detected. The polypeptides of group A and C rotaviruses are compared and discussed.     

Cloning and sequencing of a bile acid-inducible operon from Eubacterium sp. strain VPI 12708.

Two bile acid-inducible polypeptides from Eubacterium sp. strain VPI 12708 with molecular weights of 27,000 and approximately 45,000 have previously been shown to be encoded by genes residing on a 2.9-kb EcoRI fragment. We now report the cloning and sequencing of three additional overlapping DNA fragments upstream from this EcoRI fragment. Together, these four fragments contain a large segment of a bile acid-inducible operon which encodes the 27,000- and 45,000-Mr (now shown to be 47,500-Mr) polypeptides and open reading frames potentially coding for four additional polypeptides with molecular weights of 59,500, 58,000, 19,500, and 9,000 to 11,500. A bile acid-inducible polypeptide with an apparent Mr of 23,500, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was purified to homogeneity, and the N-terminal amino acid sequence that was obtained matched the sequence deduced from the open reading frame coding for the 19,500-Mr polypeptide. A short DNA segment containing the 3' downstream end of the gene coding for the 47,500-Mr polypeptide was not successfully cloned but was directly sequenced from DNA fragments synthesized by polymerase chain reaction. The mRNA initiation site for the bile acid-inducible operon was shown by primer extension to be immediately upstream from the gene encoding the 58,000-Mr polypeptide. A potential promoter region upstream from the mRNA initiation site displayed significant homology with the promoter regions of previously identified bile acid-inducible genes from Eubacterium sp. strain VPI 12708. We hypothesize that this bile acid-inducible operon codes for most of the enzymes involved in the bile acid 7 alpha-dehydroxylation pathway in this bacterium.     

Affinity purification and subunit structures of beta-N-acetylhexosaminidases A and B from boar epididymis.

beta-N-Acetylhexosaminidase from boar epididymis was separated into two forms, A and B, on DEAE-cellulose. Both these forms were excluded from Sepharose S-200 and had apparent Mr values of 510 000 on gradient gel electrophoresis under non-denaturing conditions. Affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylam ine coupled to CNBr-activated Sepharose 4B was used to separate and purify beta-N-acetylhexosaminidases A and B that had specific activities of 115 and 380 mumol/min per mg of protein respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of denatured beta-N-acetylhexosaminidase A gave a single major component of Mr 67 000. beta-N-Acetylhexosaminidase B also had this component, and in addition had polypeptides of Mr 29 000 and 26 000. All these polypeptides were glycosylated. Antiserum to the B form precipitated form A from solution and reacted with the 67 000-Mr component or form A after electrophoretic transfer from sodium dodecyl sulphate/polyacrylamide gels to nitrocellulose sheets. The 67 000-Mr components of forms A and B yielded identical peptide maps when digested with Staphylococcus aureus V8 proteinase, and the 29 000-Mr and 26 000-Mr components in form B may be related to the 67 000-Mr polypeptide.     

Coomassie blue-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for direct visualization of polypeptides during electrophoresis

A method for the direct visualization of Coomassie blue-stained polypeptide bands during electrophoresis with subsequent elution of polypeptides and removal of sodium dodecyl sulfate (SDS) and Coomassie blue is described. Primarily it is intended as a means for easy and--because there is no protein fixation step--nearly quantitative recovery of separated polypeptides for amino acid sequencing. It may also be used to obtain rapid information about the protein patterns during a run. Together with our new high resolution SDS-polyacrylamide gel electrophoresis system for small proteins and polypeptides (H. Sch?gger and G. Von Jagow (1987) Anal. Biochem. 166, 368-379) the method described allows the preparative separation of protein fragments as even protein fragments between 1 and 3.5 kDa are easily detected.     

The gamma-aminobutyrate/benzodiazepine receptor from pig brain. Purification and characterization of the receptor complex from cerebral cortex and cerebellum.

The gamma-aminobutyrate/benzodiazepine-receptor complex has been purified from a Triton X-100 extract of crude synaptic membranes from pig cerebral cortex and cerebellum by a combination of affinity and ion-exchange chromatography. [3H]Flunitrazepam binding activity was purified 2200-fold from cortex with an overall yield of 2%. The dissociation constants for the binding of [3H]muscimol and [3H]flunitrazepam to the receptor complex were 14 +/- 3 nM and 14 +/- 2 nM respectively. The ratio of [3H]muscimol to [3H]flunitrazepam binding sites was in the range 2.2-2.8. There appeared to be no selective inactivation of either binding site during the purification procedure. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed two major polypeptides of Mr 49 000 and 55 000 from both cortex and cerebellum. When the receptor from cortex was photoaffinity labelled with [3H]flunitrazepam, radioactivity was incorporated predominantly into the Mr-49 000 polypeptide, although some radioactivity was detectable in the Mr-55 000 band. The cerebellar receptor was photoaffinity labelled on the 49 000-Mr polypeptide but not on the polypeptide of Mr 55 000. In addition, some radioactivity was detected in a minor polypeptide of Mr 43 000. When purified in the presence of 3-[(3-cholamidopropyl)dimethylammonio]propanesulphonate the same major polypeptide components (Mr 49 000 and 55 000) were isolated, but the receptor now retained its ability to be modulated by secobarbital and by the anaesthetic propanidid.     

Evidence for preferential proteolytic cleavage of one of the two fibronectin subunits and for immunological localization of a site distinguishing them

Purified plasma fibronectin was digested sequentially by thrombin and cathepsin G or by cathepsin G alone and the degradation products and their gelatin-binding and heparin-binding fractions were analyzed in NaDodSO4-polyacrylamide gel electrophoresis followed by immunoblotting with a defined monoclonal anti-fibronectin antibody. In early cathepsin G digests, several gelatin-binding fragments were detected: a few large (Mr greater than or equal to 150 000) polypeptides and fragments of Mr = 85 000, 72 000, 64 000 and 40 000. The 85 000-Mr and 64 000-Mr fragments appeared as closely spaced doublets and reacted with the antibody while the 72 000-Mr and 40 000-Mr fragments did not. Therefore the 64 000-Mr fragments are likely to be derived from the 85 000-Mr fragments. Three large fragments that bound to heparin, but not to gelatin were detected: Mr = 145 000, 135 000 and 120 000. Of these only the 135 000-Mr peptide reacted with the antibody. When fibronectin was digested with thrombin, polypeptides of Mr = 180 000-200 000 and a 30 000-Mr NH2-terminal fragment were produced. Cathepsin G added to this mixture further cleaved the fragments to a digestion pattern resembling that obtained from intact fibronectin except that the 85 000-Mr and 64 000-Mr fragments appeared as single bands and the amount of the 72 000-Mr fragment was reduced. The results suggest that thrombin cleaves the 30 000-Mr fragment preferentially from the NH2-terminal end of one of the two subunits of fibronectin and that the 85 000-Mr, 72 000-Mr and 64 000-Mr fragments obtained by the additional cathepsin G digestion were derived from the other chain. The results are consistent with the model that the antigenic determinant resides 72 000-85 000 Da from the NH2-terminus and is cleaved by cathepsin G alternatively at one of its sides. Thus, the components of the 85 000-Mr and 64 000-Mr doublets are derived from different subunits and the region located by the antibody may be responsible for the difference in their migration in the polyacrylamide gel.     

Structure of the hepatitis A virion: peptide mapping of the capsid region.

Milligram amounts of highly purified hepatitis A virus (HAV) were obtained from persistently infected cell cultures. The HAV polypeptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose for detection by an enzyme-linked immunotransfer blot procedure. The HAV nucleotide-derived amino acid sequence was subjected to computer analysis to identify potential immunogenic regions within the HAV capsid polypeptides. Synthetic peptides corresponding to selected regions of each of the larger putative capsid polypeptides were coupled to keyhole limpet hemocyanin and used to immunize rabbits. Four of six anti-HAV peptide sera were strongly reactive. Antipeptide serum generated against amino acids (a.a.) 75 through 82 reacted with the 27,000-molecular-weight (MW) polypeptide; serum against a.a. 279 through 285 reacted with the 29,000-MW HAV polypeptide; and sera against a.a. 591 through 602 and 606 through 618 reacted with the 33,000-MW HAV polypeptide. These reactions enabled the identification of the gene order of the larger HAV P1 region gene products. Our data indicate the following molecular weights: HAV VP2 or 1B, 27,000; HAV VP3 or 1C, 29,000; and HAV VP1 or 1D, 33,000.