Molecular cloning and analysis of DNA complementary to three mouse Mr = 68,000 heat shock protein mRNAs

The construction and isolation of three recombinant DNAs complementary to different mouse L-cell Mr = 68,000 heat shock protein (hsp68) mRNAs is described. cDNA libraries derived from heat-shocked mouse L-cell poly(A)+ RNA by the vector-linked primer strategy of cDNA synthesis and cloning of Okayama and Berg (Okayama, H., and Berg, P. (1982) Mol. Cell. Biol. 2, 161-170) were screened first with a Drosophila hsp70 heterologous probe and subsequently with a cDNA probe isolated from the first screening. Positive clones were assigned to one of three sets based on their restriction map, and the largest member of each group was chosen for further analysis. All three cDNAs hybrid-select mRNA for the mouse major heat shock protein (hsp68) as assayed by in vitro translation and hybridize preferentially to two heat shock-induced hsp68 mRNAs on Northern blots. The coding regions of the cDNAs are almost identical and closely resemble other HSP70 genes but the 3' untranslated regions diverge considerably. Differences in the lengths of the untranslated regions are responsible for the two different sized induced hsp68 mRNAs in mouse L-cells. The physical maps of these cDNA clones and the limited number of mouse genomic DNA fragments detected on Southern blots suggest that there are at least three closely related heat shock-inducible members of the mouse HSP70 gene family. None of the cloned cDNAs are derived from the two related cognate genes known to be present in the mouse genome.     

Chicken liver basic fatty acid-binding protein (pI = 9.0). Purification, crystallization and preliminary X-ray data

Chicken liver basic fatty acid-binding protein (pI = 9.0) has been purified with a high yield by a modification of a method originally applied to rat liver. The final product is highly homogeneous and can be used to grow crystals that belong to two different space groups. The crystals are either tetragonal, space group P4₂2₁2 with a = b = 60.2 Å and c = 138.1 Å or orthorhombic, space group P2₁2₁2₁ with a = 60.7 Å, b = 40.1 Å and c = 66.7 Å. The second form appears to be more suitable for X-ray diffraction studies, it diffracts to at least 2.8 Å resolution and it is believed to contain one protein molecule in the crystallographic asymmetric unit.     

Fluorescence and circular-dichroism properties of pig intestinal calcium-binding protein (Mr=9000), a protein with a single tyrosine residue.

Spectral properties of pig intestinal Ca2+-binding protein (CaBP) and its apoprotein have been examined by fluorescence, absorption and c.d. Direct fluorescence from some of the five phenylalanine residues is observed and excitation spectra show that there is also energy transfer from some phenylalanine residues to the tyrosine. Absorption and c.d. spectra show that the tyrosine hydroxy group does not ionize significantly below pH 12. Tyrosine fluorescence is reversibly quenched by a lysine residue with a pK of 10.05 in the Ca2+ form. At low pH the tyrosine fluorescence is enhanced with transitions with pK values of approx. 4.2. The c.d. spectrum of the Ca2+ form shows a decrease of the ellipticity band at 276nm with a transition similar to that of the fluorescence titration. The apoprotein, however, shows an additional transition with a pK of about 6. The results are interpreted in terms of the recently published structure of the cow intestinal CaBP [Szebenyi, Obendorf & Moffat (1981) Nature (London) 294, 327-332]. The single tyrosine has a very high pK, although it apparently lies on the surface of the protein molecule.     

A Mr 80K hepatocyte surface protein(s) interacts with basement membrane components

We have identified a Mr 80K cell surface protein(s) from adult rat hepatocytes that binds basement membrane components, including collagen IV, heparan sulfate proteoglycan, and laminin. Freshly isolated hepatocytes were cell surface-labeled with 125I using the lactoperoxidase-catalyzed method, and detergent-solubilized membrane proteins were chromatographed on affinity columns prepared with purified basement membrane components. A Mr 80K protein was eluted with 0.15-1 M NaCl from a collagen IV column. Two proteins (Mr 80K and 38K) were eluted from a heparan sulfate proteoglycan column. The larger protein was also eluted from a column made with heparan sulfate side chains. Several proteins (Mr 80K, 67K, 45K, and 32K) bound to an affinity chromatography column made with the laminin A chain-derived synthetic peptide PA22-2, which is active for promoting cell attachment. When fractions eluted from these columns were analyzed by two-dimensional gel electrophoresis, the Mr 80K proteins showed similar patterns with a pI ranging from 8 to 9. The Mr 80K protein(s) may have an important role in the interaction of hepatocytes with basement membrane.     

New gene assignments in the rabbit (Oryctolagus cuniculus). Comparison with other species

Nineteen cell hybrids were obtained by fusing rabbit (Oryctolagus cuniculus, OCU) fibroblasts and a Chinese hamster cell line HGPRT-. Eleven enzymatic markers were previously investigated (Soulié and Grouchy 1982); seven of these could be assigned (LDHA, LDHB, TPI, PEPB, NP, ITP, and G6PD). Two assignments were uncertain (MDH2 and GUK). Two markers could not be assigned (MDH1 and PGD). Seven further markers were investigated and are the subject of this report. Six could be assigned: GALT to chromosome OCU1, GAPD to OCU4, GPX and ACY to OCU9, PGM1 to OCU13, and GSR to OCU19. One could not be assigned (GPI). MDH2 and GUK were previously considered uncertain. Now MDH2 was found impossible to assign and GUK was mapped on OCU15. These assignments were compared with those known in man, Cebus capucinus, Microcebus murinus, cat, and mouse. It was impossible to assign any enzymatic marker belonging to the ten linkage groups known in the rabbit. The esterase locus could not be investigated since the rabbit enzyme migrates in the same position as the hamster enzyme.     

Comparison of copper binding components in dog serum with those in other species.

The copper content of dog serum and its distribution to copper binding proteins was compared with that of rat and mouse. Total serum Cu concentrations of dogs and mice were one third those of the rat. Plasma ceruloplasmin, determined by azide-inhibitable oxidase activity with two substrates, was 8-fold less in the dog and 9- to 20-fold less in the mouse than in the rat, and, in both dogs and mice, there was 70-75% less ceruloplasmin Cu, determined by atomic absorption after gel filtration. In the dog, the largest proportion of total and exchangeable serum Cu was with the transcuprein fraction. Only one third as much Cu was with albumin in the dog (and mouse) versus the rat, and this was released much more readily through dialysis. In dogs and mice, the exchangeable (nonceruloplasmin) serum copper pool was half the size of that in rats and humans. Especially in the mouse (but also in rats and dogs), a small proportion of the exchangeable pool appeared bound to ferroxidase II. We conclude that the dog may rely more on transcuprein and low molecular weight complexes and less on albumin and ceruloplasmin for transport of copper to cells.     

Eosinophil cationic protein cDNA. Comparison with other toxic cationic proteins and ribonucleases

Human eosinophil granules contain several basic proteins including eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN) and major basic protein (MBP). ECP and MBP are potent helminthotoxins while EDN is less so. Both ECP and EDN possess neurotoxic and ribonuclease activities. A clone representing ECP mRNA was isolated from an eosinophil lambda ZAP cDNA library. The cDNA sequence codes for a preprotein of 160 amino acids and a protein of 133 amino acids, the amino terminus of which is identical to the known partial amino acid sequence of ECP. The ECP nucleotide sequence shows similarity to EDN, rat pancreatic ribonuclease, and human angiogenin; all are members of the ribonuclease gene superfamily. Although the deduced amino acid sequence of ECP shares identical active site and substrate binding site residues with EDN, angiogenin, and human pancreatic ribonuclease, the ribonuclease activity of ECP is 50 to 100 times less than that of EDN possibly because of the lack of a positively charged residue at human pancreatic ribonuclease position 122. The calculated isoelectric point (10.8), electronic charge (14.5), and cationic charge distribution of ECP are different from those of EDN but similar to those of MBP, which may account in part for the greater helminthotoxic activity of ECP when compared to EDN. These data suggest that ECP and EDN are derived from a common ancestral ribonuclease gene and that ECP has evolved into a potent helminthotoxin similar in some respects to MBP, while losing much of its ribonuclease activity.     

Fine structure of muscle and other components of the trunk of Sagitta setosa (Chaetognath).

The histology of the Chaetognath's trunk is largely based upon works of Hertwig (1880) and Burfield (1927) and is revised here essentially on ultrastructural basis, in a study on Sagitta setosa. The trunk is composed of a thick multilayered epidermis, without cuticle, and contains an abundant nervous system. It is separated from the underlying general musculature by a "basement membrane" which is not crossed by nerves fibres in the region of the ventral ganglion. We have been mainly interested in the muscular tissue. It has no close contact with the body cavity but is surrounded by a thin monolayer epithelium. The general musculature is composed of two types of muscles. The first one, forming the primary musculature, is divided by mesenteries and lateral fields into four quadrants. It contains groups of fibres (C then A) alternating with other groups of fibres (B). The second one, constituting the secondary musculature, is divided into four bands. Both types of fibres are readily identifiable by their myofibrils. The fibres of primary musculature, in close contact with their membrane level, have no basement lamina except at the myoepidermic junctions.     

Chicken riboflavin-binding protein. cDNA sequence and homology with milk folate-binding protein

The Rd gene is expressed in the livers and oviducts of laying hens and codes for the riboflavin-binding protein (RfBP) of egg yolk and egg white. A lambda gt11 cDNA library derived from chicken oviduct poly(A)+ RNA was screened with polyclonal rabbit antiserum to chicken RfBP. Positive clones were isolated and rescreened with a mixed oligonucleotide probe corresponding to residues 20-25 of the mature protein. The largest cDNA clone (969 base pairs) was subcloned into plasmid pIBI21, and the nucleotide sequence was determined by the dideoxynucleotide method. This clone contained the entire coding region for RfBP. The published amino acid sequence of the mature protein was confirmed. In addition, the following 17-residue signal peptide was deduced: Met-Leu-Arg-Phe-Ala-Ile-Thr-Leu-Phe-Ala-Val-Ile-Thr-Ser-Ser-Thr-Cys. Unexpectedly, the nucleotide sequence codes for 2 adjacent arginine residues at the carboxyl terminus that are not observed in the mature protein. The amino acid sequence of RfBP is homologous with bovine milk folate-binding protein. Eight of the nine pairs of cysteines involved in disulfide bonds in RfBP are conserved in folate-binding protein, as are all of the tryptophan residues. Sequence identity between homologous regions of these two vitamin-binding proteins is more than 30%.     

The human ubiquitin carrier protein E2(Mr = 17,000) is homologous to the yeast DNA repair gene RAD6.

Components of the ubiquitin conjugating system were purified from human placenta by covalent affinity chromatography on ubiquitin sepharose. In contrast to E2 preparations obtained from rabbit reticulocytes and erythrocytes or Saccharomyces cerevisiae, the placental E2 preparation lacks E2(Mr = 14,000) and E2(Mr = 20,000) which are both unique in catalysing the ligase-independent transfer of ubiquitin to histones. A novel technique was employed to detect ubiquitin carrier function of the E2 proteins after SDS-electrophoresis and blotting to nitrocellulose. A cDNA of E2(Mr = 17,000) was isolated from a human cDNA library by screening with a degenerate oligonucleotide whose sequence was based on a partial amino acid sequence obtained from an E2(Mr = 17,000) peptide. Sequence analysis demonstrated an identity of 69% in the primary sequence of human E2(Mr = 17,000) and the protein encoded by the yeast DNA repair gene RAD6, which was recently shown to be an E2 species in yeast. Such a high degree of similarity between the human E2(Mr = 17,000) and the yeast DNA repair enzyme is suggestive of important common structural constraints or roles in addition to ubiquitin carrier activity, since in yeast this function itself is not necessarily dependent on high conservation of primary structure.     

Association of the Mr 58,000 postsynaptic protein of electric tissue with Torpedo dystrophin and the Mr 87,000 postsynaptic protein.

Dystrophin was purified by immunoaffinity chromatography from detergent-solubilized Torpedo electric organ postsynaptic membranes using monoclonal antibodies. A major doublet of proteins at Mr 58,000 and minor proteins at Mr 87,000, Mr 45,000, and Mr 30,000 reproducibly copurified with dystrophin. The Mr 58,000 and Mr 87,000 proteins were identical to previously described peripheral membrane proteins (Mr 58,000 protein and 87,000 protein) whose muscle homologs are associated with the sarcolemma (Froehner, S. C., Murnane, A. A., Tobler, M., Peng, H. B., and Sealock, R. (1987) J. Cell Biol. 104, 1633-1646; Carr, C., Fischbach, G. D., and Cohen, J. B. (1989) J. Cell Biol. 109, 1753-1764). The copurification of dystrophin and Mr 58,000 protein was shown to be specific, since dystrophin was also captured with a monoclonal antibody against the Mr 58,000 protein but not by several control antibodies. The Mr 87,000 protein was a major component (along with the Mr 58,000 protein) in material purified on anti-58,000 columns, suggesting that the Mr 58,000 protein forms a distinct complex with the Mr 87,000 protein, as well as with dystrophin. Immunofluorescence staining of skeletal and cardiac muscle from the dystrophin-minus mdx mouse with the anti-58,000 antibody was confined to the sarcolemma as in normal muscle but was much reduced in intensity, even though immunoblotting demonstrated that the contents of Mr 58,000 protein in normal and mdx muscle were comparable. Thus, the Mr 58,000 protein appears to associate inefficiently with the sarcolemmal membrane in the absence of dystrophin. This deficiency may contribute to the membrane abnormalities that lead to muscle necrosis in dystrophic muscle.     

Monoclonal antibodies to the glucose transporter from human erythrocytes. Identification of the transporter as a Mr = 55,000 protein

Using the preparation of purified glucose transporter from human erythrocytes as antigen, we have prepared and characterized six monoclonal antibodies. Three of these antibodies have been shown to be to the glucose transporter by several criteria: they immunoprecipitate the transport activity, the cytochalasin B binding activity, and 75% of the protein from the solubilized purified preparation. The remaining three antibodies were shown to recognize the same polypeptide by a Western blot procedure. All of the antibodies reacted with the deglycosylated transporter and are thus against peptide determinants; most bound to the cytoplasmic domain of the transporter. The antibodies exhibited a range of effects on cytochalasin B binding, from slight enhancement to modest inhibition to strong inhibition; for this reason they must bind to at least three different epitopes. Western blot analysis of erythrocyte membranes prepared in the presence of protease inhibitors showed that all six antibodies bound to a polypeptide of average Mr = 55,000. Moreover, by immunological assay this polypeptide accounted for 5.3% of the membranes protein, a value similar to that given by cytochalasin B binding. Thus, the proposal that the native transporter is a Mr = 100,000 polypeptide is highly unlikely. The antibodies also react with the glucose transporter in other human cell types, but not with that in rodent or avian cells.     

Comparison of mitochondrial and nucleolar RNase MRP reveals identical RNA components with distinct enzymatic activities and protein components

RNase MRP is a ribonucleoprotein endoribonuclease found in three cellular locations where distinct substrates are processed: the mitochondria, the nucleolus, and the cytoplasm. Cytoplasmic RNase MRP is the nucleolar enzyme that is transiently relocalized during mitosis. Nucleolar RNase MRP (NuMRP) was purified to homogeneity, and we extensively purified the mitochondrial RNase MRP (MtMRP) to a single RNA component identical to the NuMRP RNA. Although the protein components of the NuMRP were identified by mass spectrometry successfully, none of the known NuMRP proteins were found in the MtMRP preparation. Only trace amounts of the core NuMRP protein, Pop4, were detected in MtMRP by Western blot. In vitro activity of the two enzymes was compared. MtMRP cleaved only mitochondrial ORI5 substrate, while NuMRP cleaved all three substrates. However, the NuMRP enzyme cleaved the ORI5 substrate at sites different than the MtMRP enzyme. In addition, enzymatic differences in preferred ionic strength confirm these enzymes as distinct entities. Magnesium was found to be essential to both enzymes. We tested a number of reported inhibitors including puromycin, pentamidine, lithium, and pAp. Puromycin inhibition suggested that it binds directly to the MRP RNA, reaffirming the role of the RNA component in catalysis. In conclusion, our study confirms that the NuMRP and MtMRP enzymes are distinct entities with differing activities and protein components but a common RNA subunit, suggesting that the RNA must be playing a crucial role in catalytic activity.     

Properties and distribution of the protein inhibitor (Mr 17,000) of protein kinase C.

Ca2+-dependent hydrophobic-interaction chromatography is a powerful tool for the identification and isolation of a variety of Ca2+-binding proteins which expose a hydrophobic site(s) in the presence of Ca2+ [Gopalakrishna & Anderson (1982) Biochem. Biophys. Res. Commun. 104, 830-836; Walsh, Valentine, Ngai, Carruthers & Hollenberg (1984) Biochem. J. 224, 117-127; McDonald & Walsh (1985) Biochem. J. 232, 559-567]. Using this approach, we isolated two potent and specific protein inhibitors of protein kinase C, of 17 kDa [McDonald & Walsh (1985) Biochem. J. 232, 559-567] and 12 kDa [McDonald & Walsh (1986) Biochem. Soc. Trans. 14, 585-586]. Although these inhibitors were purified by Ca2+-dependent hydrophobic-interaction chromatography and exhibit properties similar to those of calmodulin and related Ca2+-binding proteins, we were unable to demonstrate high-affinity Ca2+ binding to these inhibitors, using equilibrium dialysis. Protein kinase C exhibited half-maximal activity at 0.6 microM-Ca2+ in the presence of phospholipid and diacylglycerol, and complete inhibition by both inhibitors was observed over the range of Ca2+ concentrations examined (10 nM-10 microM). These observations suggest that the inhibitory action of these proteins does not require Ca2+. The inclusion of proteinase inhibitors during isolation of the kinase C inhibitors, as well as two-dimensional peptide mapping and amino acid analysis of the isolated proteins, suggested that the 12 kDa inhibitor is a proteolytic fragment of the 17 kDa protein which is generated during purification. Antibodies raised in rabbits against the bovine brain 17 kDa inhibitor were shown to be specific by Western immunoblotting and the competitive enzyme-linked immunosorbent assay method and were used to study the tissue and species distribution of this protein. The inhibitor was found to be present in several bovine, murine, avian and human tissues, consistent with a role in the regulation of a variety of physiological functions involving the widely distributed protein kinase C.     

Topology of the hydrophobic membrane-bound components of the histidine periplasmic permease. Comparison with other members of the family.

The membrane-bound complex of periplasmic permeases comprises two hydrophobic proteins which have been hypothesized to be integral membrane-spaninning proteins. We have investigated the topological organization of the hydrophobic components of the Salmonella typhimurium histidine permease, HisQ and HisM. Both proteins are digested by trypsin and proteinase K when either inside-out or right-side-out membrane vesicles are used. Therefore, these proteins are exposed to both surfaces of the membrane. Digestion with carboxypeptidase and binding studies with antibodies directed against the carboxyl terminus of HisQ and HisM have localized their carboxyl termini to the inside surface of the cytoplasmic membrane. Aminopeptidase digestion suggests periplasmic localization of their amino termini. Alkaline phosphatase fusions to HisQ and HisM indicate the existence of five spanners in both proteins. The periodicity and orientation of spanners and loops in HisQ and HisM match those of the five carboxyl-terminal spanners of MalF, the only other hydrophobic component of the periplasmic permeases for which topological information is available. An alignment of the sequences of all known hydrophobic components of periplasmic permeases is presented which indicates clear conservation of secondary structure and some conservation of primary sequence. The structural conservation of the components is discussed, and a role for a hydrophilic loop containing a conserved sequence (the EAA loop) is proposed.     

Phosphorylase phosphatase regulatory subunit. "Western" blotting with immunoglobulins against inhibitor-2 reveals a protein of Mr = 60,000

Rabbit muscle phosphorylase phosphatase has been isolated in different laboratories as an inactive complex of Mr = 70,000, composed of separate catalytic (Mr = 38,000) and regulatory (Mr = 31,000) proteins. The regulatory protein is identical to one of two heatstable inhibitors called inhibitor-2 (I2). Antiserum raised in sheep against I2 by repeated immunization potently blocked inhibitory activity, whereas preimmune serum did not. Immunoglobulins which blocked inhibitory activity were purified by affinity chromatography with I2 as the immobilized ligand. Using a "Western" immunoblotting procedure, as little as 1-5 ng of pure I2, obtained by electroelution of the Mr = 31,000 band of I2 from a polyacrylamide gel segment, were detected. Immunoblotting of the immunogen revealed only a band at Mr = 31,000, indicating the absence of contaminating antigenic proteins. When extracts of skeletal muscle and other rabbit tissues were denatured directly in dodecyl sulfate for immunoblotting the most intensely stained band was present at Mr = 60,000, rather than at Mr = 31,000 as expected. A small amount of I2 and other bands were detected, in particular at Mr = 36,000 and 25,000. Subsequent to heat treatment of the tissue extracts, there was an enrichment of I2 content relative to the Mr = 60,000 band. The results indicate the existence of a Mr = 60,000 protein related to I2. Activation of phosphorylase phosphatase in a muscle extract by treatment with Co2+ plus trypsin exactly coincided with digestion of the Mr = 60,000 immunoreactive protein. Available data indicate that this protein may function as a regulatory subunit of phosphorylase phosphatase.     

EPR spectra of photosystem I and other iron protein components in intact cells of cyanobacteria.

Electron paramagnetic resonance (EPR) spectra were recorded of whole filaments of the cyanobacteria Nostoc muscorum and Anabaena cylindrica. Signals due to manganese were removed by freezing and thawing the cells in EDTA. EPR spectra were assigned on the basis of their g values, linewidths, temperature dependence and response to dithionite and light treatments. The principal components identified were: (i) rhombic Fe3+ (signal at g = 4.3), probably a soluble storage form of iron; (ii) iron-sulfur centers A and B of Photosystem I; (iii) the photochemical electron acceptor 'X' of Photosystem I; this component was also observed for the first time in isolated heterocysts; (iv) soluble ferredoxin which was present at a concentration of 1 molecule per 140 +/- 20 chlorophyll molecules; (v) a membrane-bound iron-sulfur protein (g = 1.92). A signal g = 6 in the oxidized state was probably due to an unidentified heme compound. During deprivation of iron the rhombic Fe3+, centers A, B and X of Photosystem I, and soluble ferredoxin were all observed to decrease.