The three dimensional structure of the type I insulin-like growth factor receptor.

Ever since the discovery of insulin and its role in the regulation of glucose metabolism, there has been great interest in the molecule itself, the insulin-like growth factors (IGFs), and their receptors (IR and IGF-R). These receptors form a subfamily of tyrosine kinase receptors which are large, transmembrane proteins consisting of several structural domains. Their ectodomains have a similar arrangement of two homologous domains (L1 and L2) separated by a Cys rich region. The C-terminal half of their ectodomains consists of three fibronectin type 3 repeats, and an insert domain that contains the alpha-beta cleavage site. This review summarises the key developments in the understanding of the structure of this family of receptors and their relation to other multidomain proteins. Data presented will include multiple sequence analyses, single molecule electron microscope images of the IGF-1R, insulin receptor (IR), and IR-Fab complexes, and the three dimensional structure of the first three domains of the IGF-1R determined to 2.6 A resolution by x ray crystallography. The L domains each adopt a compact shape consisting of a single stranded, right handed beta-helix. The Cys rich region is composed of eight disulphide bonded modules, seven of which form a rod shaped domain with modules associated in an unusual manner.

Expression and purification of antigenically active soluble derivatives of the heterodimeric and homodimeric forms of the mouse CD8 lymphocyte membrane glycoprotein.

The T lymphocyte membrane glycoprotein CD8 enhances antigen recognition by class I-restricted T cells. There are two naturally occurring forms of CD8, an alphabeta heterodimer expressed by the majority of CD8(+) T cells, and a less abundant alphaalpha homodimer found on specialised T cell subsets. An expression strategy was developed for production of soluble CD8alphaalpha and CD8alphabeta extracellular domains for use in ligand binding studies. Mouse CD8alpha was expressed autonomously as a homodimer at 10 mg/l in mammalian fibroblasts, but CD8beta was not expressed at significant levels in the absence of CD8alpha. Co-expression with CD8alpha led to significant enhancement in the level of CD8beta expression, which was secreted as a non-covalent heterodimer at 3 mg/l with CD8alpha. Despite the marked increase of CD8beta expression in the presence of CD8alpha, an excess of soluble CD8alphaalpha homodimer was also present in the supernatant of co-expressing cell clones. In order to resolve the CD8alphaalpha homodimer from the CD8alphabeta heterodimer, affinity chromatographic techniques specific for the CD8beta subunit were employed. Purification procedures requiring elution from affinity matrices at low pH led to substantial losses in the total antigenic activity and partial subunit dissociation of the soluble CD8alphabeta heterodimer. The inclusion of a hexahistidine tag at the C-terminus of CD8beta enabled affinity purification of soluble CD8alphabeta (and sCD8alphaalpha) under neutral conditions, yielding recombinant protein with the correct stoichiometry and full antigenic activity. This method may prove useful for production of other soluble recombinant heterodimeric receptor proteins whose antigenicity is affected by denaturation during immunoaffinity purification.

Textilinins from Pseudonaja textilis textilis. Characterization of two plasmin inhibitors that reduce bleeding in an animal model.

The incidence of vein-graft occlusion associated with myocardial infarction and thrombosis following the use of the plasmin inhibitor, aprotinin, to reduce blood loss during vascular surgery has prompted the isolation of an alternative kinetically distinct inhibitor of plasmin from the venom of Pseudonaja textilis. This inhibitor has been called textilinin (Txln) and two distinct forms have been isolated from the Brown-snake venom (molecular weight, 6688 and 6692). A comparison of plasmin inhibitor constants for aprotinin and the Txlns 1 and 2 indicated that the former bound very tightly (inhibitor constant, Ki approximately 10(-11) mol/l), while both of the latter bound less tightly (Ki approximately 10(-9) mol/l). Homogeneity of Txlns 1 and 2 was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry. A sequence difference of six amino acids was observed between the two forms of Txln. Txln 1 and 2 showed, respectively, 45 and 43% homology with aprotinin, while there was 58 and 55% homology, respectively, with a plasmin inhibitor from the venom of eastern Taipan, Oxyuranus scutellatus. Both Txlns have six cysteines, like other inhibitors of this group, and homology was determined by alignment of these cysteines. Both have been shown to reduce blood loss by about 60% in a murine tail vein bleeding model. It is proposed that the kinetic profiles of Txln 1 and 2 for plasmin allow the arrest of haemorrhage without the possible threat of thrombosis.

The disulfide bonds in the C-terminal domains of the human insulin receptor ectodomain.

The human insulin receptor is a homodimer consisting of two monomers linked by disulfide bonds. Each monomer comprises an alpha-chain that is entirely extracellular and a beta-chain that spans the cell membrane. The alpha-chain has a total of 37 cysteine residues, most of which form intrachain disulfide bonds, whereas the beta-chain contains 10 cysteine residues, four of which are in the extracellular region. There are two classes of disulfide bonds in the insulin receptor, those that can be reduced under mild reducing conditions to give alpha-beta monomers (class I) and those that require stronger reducing conditions (class II). The number of class I disulfides is small and includes the alpha-alpha dimer bond Cys524. In this report we describe the use of cyanogen bromide and protease digestion of the exon 11 plus form of the receptor ectodomain to identify disulfide linkages between the beta-chain residues Cys798 and Cys807 and between the alpha-chain Cys647 and the beta-chain Cys872. The latter bond is the sole alpha-beta link in the molecule and implies a side-by-side alignment of the two fibronectin III domains of the receptor. Also presented is evidence for additional alpha-alpha dimer bond(s) involving at least one of the cysteine residues of the triplet at positions 682, 683, and 685. Evidence is also presented to show that Cys884 exists as a buried thiol in the soluble ectodomain.

Type II intermediate-filament proteins from wool. The amino acid sequence of component 5 and comparison with component 7c.

Component 5 is one of the four type II intermediate-filament proteins found in the hard keratin wool. It was isolated as the S-carboxymethyl derivative from Merino wool and its amino acid sequence was determined by manual and automatic sequencing of peptides produced by chemical and enzymic cleavage. Component 5 is an N-terminally blocked molecule of 503 residues and Mr (not including the blocking group) of 56,600. The blocking group has not been identified. The amino acid sequence of component 5 shows 77% sequence identity with that of component 7c, another type II wool intermediate-filament protein [Sparrow, Robinson, McMahon & Rubira (1989) Biochem. J. 261, 1015-1022]. The sequence similarity extends from the N-termini of the two molecules to residue 459 (component 5 sequence); however, there is no recognizable sequence similarity in the remaining C-terminal 43 amino acid residues. Details of procedures used in determining the sequence of component 5 have been deposited as a Supplementary Publication SUP 50168 (80 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire, LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1992) 281, 5. The information comprises: (1) details of chemical and enzymic methods used for cleavage of component 5, peptide CN1, the peptide mixture CN2/3 and various other peptides, (2) details of the procedures used for the fractionation and purification of peptides from (1), including Figures showing the elution profiles from the chromatographic steps used, and (3) details of the method used to determine the C-terminal sequence of component 5.

Nondenaturing protein electrotransfer of the esterase activity of lipolytic preparations.

Lipolytic enzymes subjected to nondenaturing polyacrylamide gel electrophoresis were electrophoretically transferred under nondenaturing conditions onto a solid-state matrix. Electrotransfer permitted the visualization of hydrolytic activity to the long-chain water insoluble substrate alpha-naphthyl palmitate. Four commercial preparations: a lipase from Candida cylindracea, an esterase from porcine liver, a lipase from Pseudomonas sp., and a cholesterol esterase from Pseudomonas fluorescens were examined.

Interactions of intermediate filament proteins from wool.

Filaments of wool are heteropolymers formed by interaction of type I and type II intermediate filament (IF) proteins. There are four proteins in each of these two classes. Interaction of the reduced wool IF proteins was studied by two-dimensional electrophoresis which showed that complexes between type I and type II proteins were formed in solution at urea concentrations below 6 M. Complex formation between the carboxymethyl derivatives of wool IF proteins was studied using a filter binding assay in which radio-labelled individual components were allowed to react under various conditions with SDS-PAGE separated components after transfer to nitrocellulose. The results suggested that (i) absolute type specificity of interaction was maintained, (ii) fine specificity, i.e. preferential reaction between specific components is observed, (iii) wool IF proteins (hard keratins) also react, with the same type specificity, with soft keratins isolated from cow snout, (iv) the initial step in the polymerization sequence that leads to filament formation yields heterodimers.

Sequences of wool keratin proteins: the CSIRO connection.

Wool, a dead tissue of epithelial origin, derives many of its properties as a textile fibre from the structure and arrangement of the proteins from which it is comprised. Much of the progress in the elucidation of wool protein structures, as a step towards understanding this relationship between structure and properties, has been made in the Division of Protein Chemistry of Australia's Commonwealth Scientific and Industrial Research Organization.

The esterase profile of a lipase from Candida cylindracea.

A commercial preparation of a lipase produced by Candida cylindracea catalysed the hydrolysis of both long- and short-chain esters of p-nitrophenol. Six major bands of hydrolytic activity to alpha-naphthyl acetate were detected on polyacrylamide gel electrophoresis and two on isoelectric focusing. The esterase activity fractionated into two major peaks of activity on ion-exchange chromatography and into several peaks of activity on hydrophobic interaction chromatography. These esterase activities showed different substrate specificities to p-nitrophenyl esters, tributyrin and cetyl palmitate.

The amino acid sequence of component 7c, a type II intermediate-filament protein from wool.

Component 7c is one of the four homologous type II intermediate-filament proteins that, by association with the complementary type I proteins, form the microfibrils or intermediate filaments in wool. Component 7c was isolated as the S-carboxymethyl derivative from Merino wool and its amino acid sequence was determined by manual and automatic sequencing of peptides produced by chemical and enzymic cleavage reactions. It is an N-terminally blocked molecule of 491 residues and Mr (not including the blocking group) of 55,600; the nature of the blocking group has not been determined. The predicted secondary structure shows that component 7c conforms to the now accepted pattern for intermediate-filament proteins in having a central rod-like region of approximately 310 residues of coiled-coil alpha-helix flanked by non-helical N-and C-terminal regions. The central region is divided by three non-coiled-coil linking segments into four helical segments 1A, 1B, 2A and 2B. The N-and C-terminal non-helical segments are 109 and 71 residues respectively and are rich in cysteine. Details of procedures use in determining the sequence of component 7c have been deposited as a Supplementary Publication SUP 50152 (65 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1989) 257,5. The information comprises: (1) details of chemical and enzymic methods used for cleavage of component 7c, peptides CN1, CN2 and CN3, and various other peptides, (2) details of the procedures used for the fractionation and purification of peptides from (1), including Figures showing the elution profiles from the chromatographic steps used, (3) details of methods used to determine the C-terminal sequence of peptide CN3, and (4) detailed evidence to justify a number of corrections to the previously published sequence.

Lipolytic enzymes of the digestive organs of the crown-of-thorns starfish (Acanthaster planci): comparison of the stomach and pyloric caeca.

1. Stomach and pyloric caeca homogenates from the crown-of-thorns starfish hydrolysed p-nitrophenyl esters, alpha-naphthyl esters, cholesteryl oleate and tributyrin. The pyloric caeca contained the highest activities. 2. The p-nitrophenyl acetate hydrolytic activity eluted at 0.23 M NaCl on ion exchange chromatography while the p-nitrophenyl palmitate hydrolytic activity eluted between 0.2 and 1.0 M NaCl. 3. Polyacrylamide gel zymograms for alpha-naphthyl acetate hydrolytic activity revealed one major band and several minor bands of activity for both tissues. 4. Isoelectric focusing zymograms revealed one major band with a pI = 4.2 for both tissues, with an additional band at pI = 3.5 for pyloric caeca. 5. The pyloric caeca contained twice as much lipid as the stomach. Lipid extracts contained mixtures of steroids and steroid-esters; a cholesterol-like sterol was tentatively identified.

Granulocyte/macrophage colony-stimulating factor from mouse lung conditioned medium. Purification of multiple forms and radioiodination.

Four forms of mouse granulocyte/macrophage colony-stimulating factor (GM-CSF) were purified 100,000-fold from mouse lung conditioned medium. Each of the CSF species stimulated the formation of both granulocyte and macrophage colonies, and half-maximal stimulation in the semi-solid mouse bone-marrow colony assay occurred at 1 pm. The four GM-CSF species exhibited similar charge microheterogeneity, focusing between pH 4.2 and pH 5.2. On SDS/polyacrylamide gels two of the GM-CSF sub-species had apparent Mr values of 23,000, and the other two, 21, 000. Treatment with neuraminidase decreased the Mr values of these two sets to 21,000 and 19,000 respectively. Incubation with endoglucosidase F decreased the charge heterogeneity and the Mr of all species to 16,500. A gas-phase radioiodination procedure was used to incorporate 2-3 atoms of 125I/molecule into purified GM-CSF without any loss of biological activity. The 125I-labelled GM-CSF was analysed on a microbore reversed-phase h.p.l.c. column to determine its specific radioactivity directly. This 125I-labelled GM-CSF molecule is suitable for cell-surface receptor-binding studies.

Purification and partial amino acid sequence of asialo murine granulocyte-macrophage colony stimulating factor.

A procedure utilizing reversed-phase high-performance liquid chromatography is described for the purification of asialo granulocyte-macrophage colony stimulating factor (asialo-GM-CSF) from mouse lung-conditioned medium. In the purification, the partially purified factor was treated with neuraminidase to reduce charge heterogeneity due to variable degrees of sialation. Three active forms of the asialo factor were separated by the final reversed-phase liquid chromatography step. These each gave a single major band and several minor bands on polyacrylamide gel electrophoresis and had similar amino acid compositions. The specific activity of purified murine asialo-GM-CSF was approximately 8 X 10(9) colonies per mg of protein. Amino acid sequence determination of the major form gave a single amino-terminal sequence, which has been used to develop oligonucleotide probes for the isolation of two cDNA clones encoding GM-CSF. The nucleotide sequence of these two clones gave a deduced amino acid sequence almost identical with that determined for the amino terminus of asialo-GM-CSF and an amino acid composition very similar to that for asialo-GM-CSF.

Monoclonal antibody studies of alpha-keratin low-sulfur proteins.

Monoclonal antibodies to the two families of low-sulfur proteins from wool were produced. Selection was applied to identify those hybridomas secreting antibody that was effective in the Electro-blot system. The specificities of eight different monoclonal antibodies were investigated by their binding to alpha-keratin low-sulfur proteins which had been subjected to electrophoresis from wool, goat hair, porcupine quill, rat hair and echidna quill, using the Electro-blot procedure. Considerable cross-reactivity was found both within the low-sulfur protein components of individual keratins from a particular species, and also between the keratins of the different species. Some antibodies were found to bind selectively to components of one family of low-sulfur proteins in wool, while others recognized determinants in both families, indicating some homology between the two families.

Structural homology between hard alpha-keratin and the intermediate filament proteins desmin and vimentin.

Although it has been assumed that the microfibrils in hard -keratin are members of the class of structures known as intermediate filaments (IF), no firm chemical evidence relating the low-sulfur proteins in hard -keratin to other IF proteins has yet been published. We now present primary sequence data for two components from wool keratin which show striking similarities with two IF proteins, desmin and vimentin. The sequences show marked homology, a heptad repeat and a 9.5-residue periodicity in the linear disposition of the acidic and the basic residues. These data thus provide the first evidence that the low-sulfur proteins in hard -keratin and the other IF proteins do indeed have both a similar structure and a common evolutionary origin.

Two forms of murine epidermal growth factor: rapid separation by using reverse-phase HPLC.

Epidermal growth factor (EGF) has been isolated from acid extracts of C57BL6/J mouse submaxillary glands by using hydrophobic chromatography. High yields of EGF in large amounts (10 mg) can be isolated reliably from the acid extract of the glands in less than 4 hr. The reverse-phase HPLC techniques used to purify the EGF initially yielded what appeared to be a single homogeneous EGF molecule. However, ion pairing reagents (e.g., heptafluorobutyric acid) altered the chromatographic properties, revealing two distinct species: EGF-alpha and EGF-beta. The apparent molecular weights, isoelectric points, and antigenic properties of EGF-alpha and EGF-beta were identical, and both forms stimulated a mitogenic response in 3T3 cells. Analysis of different preparations of purified EGF (commercial and experimental) indicated the presence of EGF-alpha and EGF-beta in constant proportion. Previous EGF binding studies must have used mixtures of 125I-labeled EGF-alpha and 125I-labeled EGF-beta. The two molecules appear to compete for an identical receptor on the cell surface.

Partial purification and characterization of two Peptide hydrolases from pea seeds.

Two peptide hydrolases have been found in pea seeds (Pisum sativum var. Greenfeast) and extensively purified by ion exchange chromatography using benzoyl-dl-arginine-p-nitroanilide as substrate. The enzymes which both have molecular weights of 65,000 can be separated by anion exchange chromatography but are otherwise virtually identical in the properties tested. They did not hydrolyze several common protease substrates but readily hydrolyzed small peptides containing basic amino acids on the carboxyl side of these residues. They are completely inhibited by diisopropylfluorophosphate and are inhibited to varying extents by thiol reagents.