Wt Subunits Research Articles

Virus Maturation Targets the Protein Capsid to Concerted Disassembly and Unfolding

Many animal viruses undergo post-assembly proteolytic cleavage that is required for infectivity. The role of maturation cleavage on Flock House virus was evaluated by comparing wild type (wt) and cleavage-defective mutant (D75N) Flock House virus virus-like particles. A concerted dissociation and unfolding of the mature wt particle was observed under treatment by urea, whereas the cleavage-defective mutant dissociated to folded subunits as determined by steady-state and dynamic fluorescence spectroscopy, circular dichroism, and nuclear magnetic resonance. The folded D75N alpha subunit could reassemble into capsids, whereas the yield of reassembly from unfolded cleaved wt subunits was very low. Overall, our results demonstrate that the maturation/cleavage process targets the particle for an "off pathway" disassembly, because dissociation is coupled to unfolding. The increased motions in the cleaved capsid, revealed by fluorescence and NMR, and the concerted nature of dissociation/unfolding may be crucial to make the mature particle infectious.

Morphological changes related to reconstituted acetylcholine release in a release-deficient cell line

The membrane changes accompanying Ca 2+-dependent acetylcholine release were investigated by comparing release-competent and release-incompetent clones of mouse neuroblastoma N18TG-2 cells. No release could be elicited in native N18 cells or in a N18–choline acetyltransferase clone in which acetylcholine synthesis was induced by transfection with the gene for rat choline acetyltransferase. However, acetylcholine release was operative in a To/9 clone which was co-transfected with complementary DNAs from rat choline acetyltransferase and Torpedo mediatophore 16,000 mol. wt subunit. In thin sections, the aspect of resting N18 and To/9 cells was identical: a very dense cytoplasm with practically no vesicle-like organelles. Cells were chemically fixed at different times during a stimulation using A-23187 and Ca 2+, and examined following both freeze-fracture and thin section. Stimulation of To/9 cells induced a marked change affecting the intramembrane particles. The number of medium-sized particles (9.9–12.38 nm) increased, while that of the small particles decreased. This change was not observed in control, release-incompetent cell lines. In the To/9 clone (but not in control clones), this was followed by occurrence of a large new population of pits which initially had a large diameter, but subsequently became smaller as their number decreased. Coated depressions and invaginations became abundant after stimulation, suggesting an endocytosis process. By considering the succession of events and by comparison with data from experiments performed on synapses in situ, it is proposed that a particle alteration was the counterpart of acetylcholine release in co-transfected To/9 cells; this was followed by a massive endocytosis.

Effects of spinal cord injury on neurofilament immunoreactivity and capsaicin sensitivity in rat dorsal root ganglion neurons innervating the urinary bladder

The effect of chronic spinal cord transection on neurofilament immunoreactivity and capsaicin sensitivity of L6 and S1 dorsal root ganglion neurons innervating the urinary bladder was examined using an antibody (RT97) against 200 000 mol. wt subunit of neurofilament protein and a cobalt uptake assay, respectively. Bladder afferent neurons labelled by axonal transport of a fluorescent dye (Fast Blue) injected into the bladder wall were identified in sections of intact dorsal root ganglia and among dissociated neurons in short-term culture. Approximately two thirds of bladder afferent neurons from spinal intact rats were neurofilament-poor (i.e. C-fibre neurons). These neurons were on average 37% smaller in cross-sectional area than neurofilament-rich neurons (Aδ-fibre neurons). In spinal intact rats, 78% of neurofilament-poor dissociated bladder afferent neurons were sensitive to capsaicin, while only 6.2% of neurofilament-rich neurons were capsaicin-sensitive. Dissociated bladder afferent neurons from spinal transected animals had larger diameters (34.2±1.1μm) than those from spinal intact animals (29.2±1.2μm). In tissue sections from dorsal root ganglia, the mean cross-sectional area of bladder afferent neuron profiles in spinal transected animals was also larger by approximately 35% than in spinal intact animals. Immunoreactivity to neurofilament protein which occurred in 32% of bladder afferent neurons in spinal intact animals was detected in a larger percentage (56% to 62%) of neurons from spinal transected animals. Conversely, the population of capsaicin-sensitive dissociated neurons was reduced from 55% in spinal intact rats to 38% in spinal transected rats.These results indicate that spinal cord injury induces functional and morphological plasticity in C-fibre visceral afferent neurons innervating the urinary bladder.

Cloning and sequencing of a cDNA clone encoding the photosystem I PsaD subunit from Chlamydomonas reinhardtii.

PSI from cyanobacteria, green plants, and algae functions as a plastocyanin: Fd oxidoreductase. It is composed of a heterodimer of the PsaA and PsaB apoproteins (82-83 kD) that hold together in the correct orientation P700, the electron acceptors A, (a Chl molecule), A, (a phylloquinone molecule), and F, (an intermolecular 4Fe-4S center). Besides these two chloroplast-encoded large subunits, PSI contains numerous low mo1 wt subunits of both chloroplast and nuclear origin (for review, see Golbeck and Bryant, 1991). Three of these subunits, PsaC, PsaD, and PsaE, are located on the stromal side of PSI and have been studied extensively. PsaC is the apoprotein of the secondary electron acceptors FA and F,, which are both 4Fe-4S centers (Wynn and Malkin, 1988). PsaE is involved in the cyclic electron transfer around PSI (Yu et al., 1993). PsaD has two major roles on the acceptor side of PSI. First, it is required for the binding of PsaC and PsaE to the complex (Li et al., 1991; Chitnis and Nelson, 1992). Second, PsaD can be cross-linked specifically to Fd and appears therefore to be the Fd docking protein of PSI (Zilber and Malkin, 1988). This subunit is essential for the in vivo function of PSI (Chitnis et al., 1989). The PsaD cDNAs or genes have been cloned in many plant and cyanobacterial species. Because of the importance of this subunit for PSI function, it was of interest to isolate its cDNA from the green alga Chlamydomonas reinhardtii, which is a model system for studying photosynthesis (Rochaix, 1992). Because of the biased codon usage of nuclear genes in this alga, it was not possible to clone the gene by using heterologous probes. Attempts to purify PsaD of C. reinhardtii have failed because of the co-migration of PsaD and PsaF on polyacrylamide gels. An antiPsaF serum was previously described and was found to be contaminated by antibodies recognizing specifically the PsaD subunit from C. reinhardtii (Bassi et al., 1992; J. Farah and J.-D. Rochaix, unpublished data). We have purified these contaminating antibodies by incubating the serum with a filter containing immobilized PSI proteins

Two cDNA Clones Encoding Isoforms of the [beta]-Subunit of the General Mitochondrial Processing Peptidase from Potato

The general MPP removes the amino-terminal extensions of nuclear-encoded mitochondrial proteins. It consists of two nonidentical subunits termed a-MPP and p-MPP, which are supposed to form a heterodimer (Kalousek et al., 1993). Although the enzyme is localized in the matrix space of fungal and mammalian mitochondria, it was shown to reside in the inner mitochondrial membrane in plants (Braun et al., 1992; Glaser et al., 1992). Interestingly, in potato and wheat the complete protease forms part of the Cyt bc, complex (also known as Cyt c reductase or complex 111) of the respiratory chain (Braun et al., 1992; Emmermann et al., 1993a, 1993b; Braun et al., 1995). There are also indications that it forms part of this protein complex in spinach (Eriksson et al., 1994). The complex has been termed Cyt c reductase/processing peptidase complex and is extraordinarily stable (Emmermann et al., 1993a). The complex-integrated processing enzyme exhibits severa1 unique physiological features in comparison with the soluble protease from fungi and mammals (Emmermann and Schmitz, 1993). Here we report the primary structure of two cDNA clones encoding the P-subunit of MPP from potato (Table I). Extensive protein sequencing of a11 subunits of the Cyt bc, complex from potato (Braun et al., 1994) revealed the integration of the processing peptidase into the complex and was the basis for the design of oligonucleotide mixtures for the isolation of cDNA clones. Positively reacting clones were identified and isolated with radiolabeled oligonucleotides. The DNA inserts of two clones designated pp-I MPP and pp-I1 MPP share 71% sequence identity at the nucleotide level and 70% at the protein level. The open reading frame of clone pp-I1 MPP contains a11 61 possible codons. The amino acid sequence of the encoded protein is identical with the peptides sequenced from subunit I1 of the Cyt bc, complex. Similarly, pp-I MPP corresponds to subunit I of this protein complex. Both proteins are more similar to the P-subunits of the general mitochondrial processing peptidase than to subunits I and I1 of fungal and mammalian Cyt bc, complexes. Still, there is around 25% sequence identity with the high mo1 wt subunits of the complex, indi-

Identification of amino acids in the C-terminal region of human follicle-stimulating hormone (FSH) beta-subunit involved in binding to human FSH receptor.

Recent analyses of human FSH (hFSH) using antipeptide antibodies, monoclonal antibodies, and chimeric constructions of hCG/hFSH strongly suggest that the C-terminal region, including residues 81-100 of the hFSH beta-subunit, is involved in subunit association as well as hFSH heterodimer binding and/or activation of receptor. To test this hypothesis, site-directed mutagenesis was used to generate five triple alanine mutants of the C-terminal region of hFSH beta: Q81, H83, G85; K86, D88, S89; D90, S91, T92; D93, T95, V96; and R97, G98, L99. The baculovirus-infected insect cell system was used for expression. High Five cells were infected with virus harboring either delta hFSH beta complementary DNA (cDNA) or wild-type hFSH beta (hFSH beta wt) cDNA and coinfected with virus containing hFSH alpha cDNA. After infections, media were assayed for FSH using a heterodimer-specific enzyme-linked immunosorbent capture assay. All delta hFSH beta subunits formed heterodimers with hFSH alpha wt subunit and were secreted in the medium. These results suggest, for all five mutants, that side chains of amino acids substituted with alanine had no significant role in subunit association. The FSHs delta hFSH and hFSHwt were tested in a RRA, using cell lines that express the hFSH receptor, to determine if there were any changes in binding activity. Similarly, delta hFSH and hFSHwt were compared for receptor activation by measuring the levels of progesterone production in an in vitro FSH bioassay. delta hFSH-(93-96) exhibited minimal binding activity and no detectable steroidogenic activity. delta hFSH-(97-99) showed reduced binding affinity compared with that of hFSHwt, whereas the binding potency and bioactivity of the remaining delta hFSH were comparable to those of hFSHwt. These data demonstrate that within the hFSH beta-(81-99) region, FSH receptor-binding sites are contained within the sequence 93-99.

Development of a monoclonal-based enzyme-linked immunoassay for saxitoxin-induced protein

D. S. Smith and D. D. Kitts. Development of a monoclonal-based enzyme-linked immunoassay for saxitoxin-induced protein. Toxicon 32, 317–323, 1994.—A monoclonal antibody was generated against saxitoxin-induced protein (SIP) from the small shore crab Hemigrapsus oregenesis. SIP was induced by saxitoxin injection and could be detected in the crude crab extracts with both polyclonal and monoclonal antibody preparations. On Western blots, the polyclonal serum reacted against several bands which were induced by saxitoxin in the crude extracts. These bands represented proteins related to SIP. The monoclonal (4G5), however, was specific for the 79,000 mol. wt subunit of SIP. A triple antibody sandwich ELISA was developed in which polyclonal anti-SIP IgG was used as a trapping layer and monoclonal 4G5 was used as the detection layer. This assay was shown to be more specific and more accurate than a direct bind assay which employed the polyclonal antiserum alone. Although the polyclonal serum was more sensitive than the monoclonal on Western blots, the triple antibody sandwich and direct bind ELISAs were of comparable sensitivity.

Identification and characterization of a factor which is essential for assembly of transcarboxylase

Transcarboxylase (TC) from Propionibacterium shermanii is a biotin-containing enzyme which catalyzes the reversible transfer of a carboxyl group from methylmalonyl-CoA to pyruvate. It is composed of a central, hexameric 12S subunit with six outer, dimeric 5S subunits held in a stable 26S complex by twelve 1.3S biotinyl subunits. Each of these subunits has been cloned from the P. shermanii genome and expressed in Escherichia coli. The purified, expressed recombinant proteins are all indistinguishable from their authentic counterparts except for the recombinant 5S subunit (termed 5S WT), which does not form TC complexes or catalyze the overall transcarboxylase reaction. Circular dichroism and isoelectric focusing suggested differences existed between the authentic and E. coli-expressed 5S proteins. HPLC gel filtration was used to separate the authentic 5S dimer from additional components in the preparation. 5S dimer thus purified was unable to form TC complexes or catalyze the overall reaction, behaving identically to the recombinant 5S WT subunit. Fractions from the HPLC gel-filtration purification of authentic 5S were then added to 5S WT or 5S dimer, and one fraction was identified which catalyzed the assembly of TC complexes with either 5S preparation. This assembly activity was shown to be dependent on the concentration of this HPLC fraction. Assembly-promoting factor (APF) is heat-stable and probably a protein, on the basis of its protease susceptibility. Studies with APF and the other TC subunits demonstrate its ability to promote complex formation with 12S and 1.3S subunits. Since the APF was purified from crystals of 26S TC, we believe it to be a novel, previously unidentified subunit of transcarboxylase.

The Conserved Methionines of the 1.3 S Biotinyl Subunit of Transcarboxylase: Effect of Mutations on Conformation and Activity

Transcarboxylase from Propionibacterium shermanii is a biotin-containing enzyme which catalyzes the reversible transfer of a carboxyl group from methylmalonyl-CoA to pyruvate. Transcarboxylase 26 S complexes consist of a central, hexameric 12 S subunit with 6 outer, 5 S subunits attached by 12 1.3 S biotinyl subunits. Each of the subunits has been cloned and expressed in Escherichia coli in active form. We have used the cloned genes in mutagenic studies of the structure-function interactions of these subunits. One particular target of our studies has been the evolutionarily conserved tetrapeptide Ala-Met-Bct-Met which surrounds the biotinyl lysine. We have investigated the properties of subunits containing leucine substitutions at each methionine (1.3 S M88L and 1.3 S M90L) by assaying their activity in the two partial reactions in which this subunit participates. Partial reaction assays demonstrate that leucine substitution at either position has a greater effect on the 12 S partial reaction than on the 5 S reaction and Met 88 is more significant catalytically than Met 90. To determine whether structural alterations in the 1.3 S mutants were responsible for the effects on activity, the conformations of these mutants were investigated. In vitro hydrolysis studies with trypsin and V8 protease demonstrated differences in the susceptibility of 1.3 S M88L relative to 1.3 S WT and 1.3 S M90L. Complexes of avidin with 1.3 S WT or mutant subunits, as monitored by fluorescence properties, indicated that the microenvironment of the biocytin of 1.3 S M88L was different from those of 1.3 S WT and 1.3 S M9OL. By contrast, substrate binding (oxalacetate for 5 S and methylmalonyl-CoA for 12 S) was unaffected by any of the 1.3 S mutants. Taken together, these results indicate that the conserved tetrapeptide of the 1.3 S biotinyl subunit, particularly Met 88, is required to provide an essential conformation and proper binding properties for catalysis of the partial reactions and the overall reaction.

Xanthine dehydrogenase in the fat body of Manduca sexta: Purification, characterization, subcellular localization and levels during the last larval instar

Xanthine dehydrogenase was isolated and purified from the fat body of fifth instar tobacco hornworms, Manduca sexta (L.) by ammonium sulfate precipitation, Sephacryl S-400 gel filtration, QAE-Sephadex ion exchange chromatography and reactive blue-2 Sepharose affinity chromatography. The preparation appeared homogeneous following analysis by polyacrylamide disc gel electrophoresis, sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and rocket and crossed immunoelectrophoresis using rabbit antiserum against purified xanthine dehydrogenase. The molecular weight of native xanthine dehydrogenase was estimated at about 300,000 while SDS-PAGE indicated that the enzyme consisted of two 158,000 mol. wt subunits. Kinetic analyses revealed high affinities for xanthine and hypoxanthine as substrates. Electron acceptors (cofactors) included NAD +, as well as the dyes, p-iodonitrotetrazolium violet and nitro blue tetrazolium. A broad pH optimum was observed between pH 7 and 10, and xanthine dehydrogenase was competitively inhibited by allopurinol and irreversibly inhibited by the sulfhydryl inhibitor, p-hydroxymercuribenzoate. The levels of xanthine dehydrogenase in fat body extracts from fifth instar larvae, as determined by both enzyme assay and immunoelectrophoresis, decreased from days 1 to 5. Ultra-structural analysis of day 3 larval fat body cells by immunocytochemical techniques, using colloidal gold staining, revealed that xanthine dehydrogenase was specifically located either within urate storage vacuoles or near the basal lamina of the cells.

Persistent degenerative state of non-pyramidal neurons in the ca1 region of the gerbil hippocampus following transient forebrain ischemia

Morphological changes in the neurons of the gerbil hippocampus following 5 min of forebrain ischemia were examined using light and electron microscopy. Although non-pyramidal neurons in the CA1 region of the hippocampus survived through the full length of the observation period, up to six weeks after ischemia, they consistently demonstrated degenerative changes distinct from those of the well-known “delayed neuronal death” of CA1 pyramidal cells. When examined with the light microscope, CA1 non-pyramidal neurons were found to be shrunken and their nuclei and cytoplasm were hyperchromatic between seven days and six weeks after ischemia. When examined with the electron microscope, postischemic non-pyramidal neurons were found to have markedly electron-dense profiles; their cytoplasm contained numerous free ribosomes and heterogeneous smaller granular substances, the latter also filling the nuclei. However, there was no loss of ribosomes from the rough endoplasmic reticulum, and mitochondrial cristae were preserved, suggesting that these neurons were viable. CA1 non-pyramidal neurons were studied immunohistochemically using three types of monoclonal antibodies, one each against parvalbumin, a nonphosphorylated epitope on the 168,000 mol. wt and 200,000 mol. wt subunits of neurofilament proteins, and microtubule-associated protein 2. CA1 nonpyramidal neurons lost immunoreactivity to these neuron-specific substances six weeks after ischemia, suggesting that these degenerating cells lacked certain types of normal neuronal activity. We conclude that non-pyramidal neurons in the hippocampal CA1 region survive transient ischemia but undergo degenerative changes following complete loss of CA1 pyramidal cells. These changes may be due to depletion of presumptive target-derived trophic factors within the non-pyramidal neurons.

Evolution of the “titin epitope” in neurofilament proteins

1. 1. The specificity of a monoclonal antibody raised to human titin was characterized. The antibody reacts with an epitope which is common to titin and the high mol. wt subunits NF-H and NF-M of mammalian neurofilaments. 2. 2. Mapping of the epitope indicated that it is located in the carboxyterminal extension of NF-H and NF-M, and that its reactivity does not depend on the phosphorylation state of the molecule. 3. 3. A comparative study on neurofilament protein of lower vertebrates revealed that this epitope has been conserved during vertebrate evolution.

Scatter factor: molecular characteristics and effect on the invasiveness of epithelial cells.

The generation of invasiveness in transformed cells represents an essential step of tumor progression. We have previously shown that MDCK epithelial cells, which are deprived of intracellular adhesion by the addition of anti-Arc-1/uvomorulin antibodies, become invasive for collagen gels and embryonal heart tissue (Behrens, J., M. M. Mareel, F. M. Van Roy, and W. Birchmeier. 1989. J. Cell Biol. 108: 2435-2447.). Here we examined whether invasiveness is also induced by scatter factor, which is known to dissociate epithelial cells (Stoker, M., E. Gherardi, M. Perryman, and J. Gray. 1987. Nature (Lond.). 327:239-242.). Scatter factor was purified to homogeneity from conditioned medium of human fibroblasts by heparin-Sepharose chromatography, followed by cation exchange chromatography, gel filtration, or preparative SDS gel electrophoresis. We found that scatter factor represents a 92,000 mol wt glycoprotein which, apparently, is converted by limited proteolysis into disulfide-linked 62,000 and 34/32,000 mol wt subunits. Reversed phase HPLC and sequence analysis of tryptic peptides confirmed the suggested molecular structure, and revealed further that scatter factor exhibits sequence similarities to hepatocyte growth factor and to plasminogen. Purified scatter factor in fact induces the invasiveness into collagen matrices of MDCK epithelial cells, and induces or promotes the invasiveness of a number of human carcinoma cell lines. Apparently, the effect on the human cells depends on their respective degree of differentiation, i.e., cell lines with a less pronounced epithelial phenotype were more susceptible to the factor. Scatter factor does not seem to influence synthesis, steady-state level, and phosphorylation of the cell adhesion molecule Arc-1/uvomorulin. Thus, scatter factor represents a clearly defined molecular species which induces, in vitro, the progression of epithelial cells to a more motile, i.e., invasive phenotype.

A comparison of cardiac glutathione S-transferases from wild and domestic animals

1. 1. Cardiac glutathione S-transferases from wild animals; hyena, red fox, porcupine, coypu and mountain gazelle were purified and compared with the enzymes from domestic animals; cow, camel, goat and sheep. 2. 2. By using 1-chloro-2,4-dinitrobenzene as a substrate, domestic hearts expressed higher glutathione conjugating activity than wild animals hearts. 3. 3. In all the studied hearts, the bulk of the activity was associated with near neutral and acidic glutathione S-transferase isozymes with pI values ranging from 4 to 7.4. 4. 4. The enzymes from domestic animals displayed homodimeric structure of 25,000 mol. wt subunit while of the wild animals both hyena and coypu displayed homodimers of 26,500 mol. wt subunit and the rest exhibited heterodimers of 25,000 and 28,000 mol. wt subunits.

L-DOPA decarboxylase in Ceratitis capitata white puparia and human: A comparative study

1. 1. l-DOPA decarboxylase (DDC) from Ceratitis capitata and from human kidney have been purified by the same methodology. 2. 2. Both enzymes show mol. wts of 100,000, consisting of two identical mol. wt subunits and solely decarboxylate l-DOPA. 3. 3. In the presence of 5-hydroxytryptophan (5-HTP) only the DDC activity from human kidney is remarkably reduced. 4. 4. Addition of exogenous coenzyme is essential only for human DDC activity. 5. 5. Polyclonal antibodies, raised against DDC purified from insects or humans, cross-react with both antigens.

Human Thyrotropin Receptor Subunits Characterized by Thyrotropin Affinity Purification and Western Blotting*

We studied the subunit structure of the human TSH receptor in thyroid tissue from patients with Graves' disease and multinodular goiter by TSH affinity chromatography, immunoprecipitation with Graves' immunoglobulins (Igs), and a modified technique of Western blotting. Human TSH receptor-binding activity was purified about 1,270-fold by sequential affinity chromatography on wheat germ lectin-agarose and TSH-agarose. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of nonreduced affinity-purified receptors eluted in sodium dodecyl sulfate sample buffer revealed three noncovalently linked subunits of 70,000, 50,000, and 35,000 mol wt. When reduced, a major subunit of 25,000 mol wt was identified. When 3 mol/L NaCl was used to elute affinity-purified receptors only the 50,000 mol wt nonreduced subunit was detected. This subunit bound [125I]bovine TSH and was precipitated by Graves' Igs. Modifications to the conventional Western blotting technique enabled thyroglobulin components (approximately 220,000 mol wt), thyroid microsomal antigen (a doublet of approximately 110,000 mol wt), and putative TSH receptor subunits of 70,000 and 50,000 mol wt to be identified in thyroid particulate membranes by Graves' Igs. Blotting of affinity-purified receptors eluted in sodium dodecyl sulfate sample buffer revealed subunits of either 70,000 or 50,000 mol wt, with a minority of Graves' serum samples. We conclude that the nonreduced human TSH receptor is an oligomeric complex comprising three different subunits of 70,000, 50,000, and 35,000 mol wt. The reduced receptor exists as a single subunit of 25,000 mol wt, which may be disulfide linked to form the higher mol wt forms. The 70,000 and 50,000 mol wt subunits contain epitopes that bind Graves' Igs in modified Western blots, thus directly confirming that the human TSH receptor is a target for Graves' Igs.

Induction of neurofilament phosphorylation in cultured chromaffin cells

The distribution, structural organization and state of phosphorylation of neurofilaments have been examined in chromaffin cells from adult bovine adrenal medulla cultured under various conditions using a series of monoclonal antibodies directed against phosphorylated and nonphosphorylated epitopes of the 200,000 mol. wt subunit. Nonphosphorylated neurofilament epitopes were detected immuno-cytochemically to varying extents in chromaffin cells maintained under standard culture conditions for up to 3 weeks. Staining was usually limited to a perinuclear region from which fine filaments sometimes appeared to radiate around the nucleus. In marked contrast, none of the antibodies directed against phosphorylated neurofilament epitopes stained these structures. When cells were cultured under conditions favouring neurite outgrowth, in conditioned medium derived from intermediate lobe cultures, there was a more extensive expression of the nonphosphorylated neurofilament epitopes. In addition, phosphorylation of neurofilaments was induced. The phosphorylated neurofilament epitopes were restricted to the neurite, whereas the nonphosphorylated neurofilament epitopes were localized in both neurite extensions and perikarya. These results demonstrate that conditioned medium from intermediate lobe cells of the hypophysis not only provokes neurite outgrowth from chromaffin cells, but also supports neuronal maturation as demonstrated by the phosphorylation of neurofilaments in neuntes.

Antigens recognised by the human immune response to infection with Leptospira interrogans serovar hardjo.

Serum samples from patients infected with Leptospira interrogans serovar hardjo were tested by the microscopic agglutination test (MAT), enzyme immunoassay (EIA) and immunoblotting. There was no apparent correlation between MAT titre and EIA optical density (OD) for individual serum samples, but sequential serum samples produced similar profiles in both tests during the course of an infection. Immunoblotting of hardjo sonicate with patients' sera revealed reactions with a number of bands, in the mol. wt (10(3] range 14.4-95. However, all serum samples reacted with the major 28 x 10(3)-mol. wt sub-unit of hardjo lipopolysaccharide (LPS) and most reacted with a (34.5-35) x 10(3)-mol. wt flagella doublet. Examination of sequential serum samples obtained over a period of about 3 months after infection revealed little change in the antigens detected after the second to third week of infection. Absorption of patients' sera with whole viable leptospires revealed that antibodies to several exposed antigens, including LPS, were produced. Sera which reacted with hardjo flagella also reacted with bands of similar mol. wts in preparations from other serovars.

Separation of the Insulin-Like Growth Factor-Binding Proteins in Plasma and Purification of the Larger Molecular Weight Species*

Methods were developed for purification of the high mol wt (150K) insulin-like growth factor (IGF)-binding protein and its acid stable (70K) component from human plasma. High mol wt IGF-binding protein was highly purified by chromatography of Cohn IV-1 fraction of human plasma on Concanavalin A-Sepharose followed by chromatography on IGF-I-Sepharose. The acid-stable component of the high mol wt IGF-binding protein was purified to near homogeneity by chromatography of Cohn IV-1 fraction of human plasma on Con-A Sepharose, followed by chromatography on Sephadex G-50 at pH 2.5 and subsequent chromatography on IGF-I-Sepharose. Both fractions obtained after IGF-I-Sepharose chromatography were capable of binding [125I]IGF-I and gave a single protein band of 79,000 mol wt, when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Coomassie blue staining), suggesting that the large mol wt species may be a dimer of identical or iso-mol wt subunits. Three minor contaminants of less than 5% each were detected upon subsequent silver staining. These methods represent important tools that should aid in furthering our understanding of the role of the IGF carrier proteins in the actions of IGF-I and IGF-II.

Substructure of human von Willebrand factor.

Using electron microscopy, we have visualized the substructure of human von Willebrand factor (vWf) purified by two different approaches. vWf multimers, which appear as flexible strands varying in length up to 2 micron, consist of dimeric units (protomers) polymerized linearly in an end-to-end fashion through disulfide bonds. Examination of small multimers (e.g., one-mers, two-mers, and three-mers) suggests that each protomer consists of two large globular end domains (22 X 6.5 nm) connected to a small central node (6.4 X 3.4 nm) by two flexible rod domains each approximately 34 nm long and approximately 2 nm in diameter. The protomer is 120 nm in length when fully extended. These same structural features are seen both in vWf molecules that were rapidly purified from fresh plasma by a new two-step procedure and in those purified from lyophilized intermediate-purity Factor VIII/vWf concentrates. The 240,000-mol wt subunit observed by gel electrophoresis upon complete reduction of vWf apparently contains both a rod domain and a globular domain and corresponds to one half of the protomer. Two subunits are disulfide-linked, probably near their carboxyl termini, to form the protomer; disulfide bonds in the amino-terminal globular ends link promoters to form vWf multimers. The vWf multimer strands have at least two morphologically distinct types of ends, which may result from proteolytic cleavage in the globular domains after formation of large linear polymers. In addition to releasing fragments that were similar in size and shape to the repeating protomeric unit, plasmic degradation of either preparation of vWf reduced the size of multimers, but had no detectable effect on the substructure of internal protomers.