Isolation and characterization of mannan-binding proteins from chicken liver

Two binding proteins which recognize and bind mannose and N-acetylglucosamine (mannan-binding proteins, MBP) have been isolated from chicken liver to near homogeneity mainly by affinity chromatography on a column of Sepharose 4B-mannan. The neutral binding protein (pI 7.0), which has a high glycine content, is an analog of mammalian liver MBP (F-I). F-I consists of a series of proteins composed of two subunits of 28,000 (A) and 32,000 (B) Da. The proteins have molecular weights ranging from 280,000 to 740,000 and subunit compositions ranging from 6A + 4B to 5A + 19B. With increasing molecular weight the specific activity of mannan binding increases gradually, accompanied by a slight change in specificity to a preference for mannose rather than N-acetylglucosamine. The acidic binding protein (pI 5.1) is a glycoprotein with a high glutamic acid content (F-II). The molecular weight of F-II was estimated to be 640,000, and it is composed of single subunits of 41,000 Da. The two MBPs isolated in this study are distinct from the liver lectin specific for N-acetylglucosamine-terminated glycoproteins isolated from the same source [T. Kawasaki and G. Ashwell (1977) J. Biol. Chem. 252, 6536-6543] in chemical properties and binding specificities.     

Isolation and characterization of a mannan-binding protein from rabbit serum

A binding protein which recognizes mannose and N-acetylglucosamine has been isolated from rabbit serum to apparent homogeneity. The serum binding protein was nearly identical to the mannan-binding protein isolated previously from rabbit liver [Kawasaki, T., Etoh, R. and Yamashina, I. (1978) Biochem. Biophys. Res. Commun. $\text{81}$, 1018–1024] in respect of immunochemical properties and subunit profiles, but could be differentiated from the liver protein in its larger molecular size and inferior sensitivity to monosaccharides as haptenic inhibitors of the binding to ¹²⁵I-mannan. A postulation was made that the plasma was, comparable with the liver, a major locus of mannan-binding protein in the rabbit.     

Purification and characterization of two mannan-binding lectins from mouse serum

Mannan-binding lectin (MBL) is a serum protein that activates the complement system after binding to glycoconjugates found on the surface of microorganisms. By molecular cloning two forms of MBL have been identified in the mouse (mMBL-A and mMBL-C), but only mMBL-A has been purified and characterized at the protein level. MBL-C has been termed the liver form of MBL. The present report describes the purification and characterization of mMBL-A and mMBL-C from serum. The two forms of mMBL could be separated both by ion-exchange and carbohydrate-affinity chromatography. The initial identification by immunochemical technique was confirmed by N-terminal amino-acid sequencing. Both proteins give bands corresponding to polypeptide chains of 28 kDa on SDS-PAGE in the reduced state, but mMBL-A migrated more rapidly than mMBL-C in acid/urea-PAGE, in accordance with the calculated pIs. Both forms mediated activation of complement component C4 in mannan-coated microtiter wells. MBL-A showed a higher affinity for d -glucose and alpha-methyl-d -glucose then did MBL-C. Serum concentrations of mMBL-A in laboratory strains and wild mice were found to vary from 5 to 80 microg/ml, with wild mice tending to show higher levels than laboratory strains.     

Isolation and characterization of a mannan-binding protein from rabbit liver

A membrane protein which binds mannan has been isolated from rabbit liver by affinity chromatography. Upon polyacrylamide gel electrophoresis, a single major band was recovered with an estimated molecular weight of 31,000. When assayed as inhibitors, N-acetylmannosamine, N-acetylglucosamine, and mannose were potent inhibitors of mannan binding; N-acetylgalactosamine and mannose-6-phosphate were inert. Glycoproteins with terminal N-acetylglucosamine and/or mannose residues which are cleared rapidly from the circulation by the liver were the most potent inhibitors. On the basis of these results, it is proposed that the mannan-binding protein is the principle mediating the hepatic uptake of glycoproteins with terminal N-acetylglucosamine and/or mannose residues.     

Isolation and chemical characterization of two distinct "link proteins" from bovine nasal cartilage proteoglycan complex

Bovine nasal cartilage proteoglycan aggregates have been dissociated and separated by dissociative density gradient centrifugation into proteoglycan sub-units and "link fraction". The latter contained mainly the two "link proteins" as shown by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two "link proteins" were then separated by preparative gel electrophoresis under dissociative conditions. Molecular weight and amino acid composition of both proteins are presented.     

Isolation and characterization of rat cDNA clones for two distinct thyroid hormone receptors

Two distinct v-erbA-related cDNA clones representing the products of different genes were isolated from a rat liver cDNA library. The first, rc-erbA-alpha, was 82% identical to v-erbA and encoded a polypeptide with a calculated molecular mass of 45,000 daltons. This cDNA clone arises from the same gene product as a v-erbA-related cDNA isolated from rat brain by Thompson et al. (Thompson, C. C., Weinberger, C., Lebo, R., and Evans, R. (1987) Science 237, 1610-1614). The second cDNA clone, rc-erbA-beta, was 76% identical to v-erbA and encoded a polypeptide with a calculated molecular mass of 52,000 daltons. Both rc-erbA-alpha and rc-erbA-beta translational products bound 3,5,3'-triiodo-L-thyronine with affinities equal to each other (Kd approximately equal to 0.4 nM) and comparable to the nuclear thyroid hormone receptor extracted from rat liver. The relative affinities of a series of thyroid hormone analogs for both translational products were also identical. In various tissues and cell lines, the relative levels of rc-erbA-beta RNA, but not rc-erbA-alpha RNA, correlated with measurements of nuclear 3,5,3'-triiodo-L-thyronine binding sites. Based on this correlation, we suggest that rc-erbA-beta may encode the "classical" nuclear thyroid hormone receptor, whereas rc-erbA-alpha may encode an isoreceptor species with differing functional properties.     

Isolation and partial characterization of two antifungal proteins from barley

We have developed a simple assay for detecting antifungal compounds utilizing impregnated paper discs on agar to inhibit mycelial spread of an indicator organism, Trichoderma reesei. Using this assay we have isolated and purified to apparent homogeneity two antifungal proteins from dehusked barley grain. Both proteins are present at high concentrations: over 10 mg of each protein can be isolated per 100 g of grain. The first protein has a molecular weight of 30 000 and is identical to the 30 kDa ribosome-inactivating protein previously isolated from barley. This protein very effectively inactivates fungal ribosomes and this may explain its antifungal activity and biological role. The second antifungal protein has a molecular weight of 28 000 and is 20-fold more potent than the 30 kDa protein in inhibiting growth of Trichoderma. In addition to Trichoderma, the 28 kDa protein also efficiently inhibits growth of Phycomyces blakesleeanus, Alternaria alternaria and a protoplast-forming mutant of Neurospora crassa. The 28 kDa protein does not inactivate fungal ribosomes and we are currently investigating other possible enzymatic activities of this protein.     

Isolation and characterization of two cDNAs from atlantic cod encoding two distinct psychrophilic elastases

The cDNAs encoding two different Atlantic cod elastases have been isolated and sequenced. The predicted amino acid sequences revealed two preproelastases, consisting of a signal peptide, an activation peptide and a mature enzyme of 242 and 239 amino acids. Amino acid sequence identity between the two cod elastases was 60.1% and identity with mammalian elastases ranged from 50–64%. The two cod elastases contain all the major structural features common to serine proteases, such as the catalytic triad His57, Asp102 and Ser195. Both cod elastases have a high content of methionine, consistent with previous findings in psychrophilic fish enzymes.     

Isolation and partial characterization of two distinct DNA topoisomerases from cauliflower inflorescence

DNA topoisomerases which remove superhelical turns in closed circular DNA have been isolated from cauliflower inflorescences using polyethylene glycol fractionation, ammonium sulfate precipitation, and column chromatography on CM-Sephadex or CM-cellulose and DNA-cellulose. Two distinct enzymes, topoisomerase-I and ATP-dependent topoisomerase, were separated clearly by CM-Sephadex or CM-cellulose, and partially characterized using agarose gel electrophoresis with plasmid pBR322 DNA. Topoisomerase-I acts like other eucaryotic DNA topoisomerases in the absence of ATP, is stimulated by spermidine and inhibited by EDTA. The ATP-dependent topoisomerase acts like topoisomerase-I only in the presence of ATP in the reaction medium, is inhibited by spermidine and EDTA, and does not introduce supertwists into closed duplex DNA or produce catenate aggregates under the present reaction conditions.     

Isolation and characterization of three distinct 34 kDa EDTA-extractable proteins from bovine lens

EDTA-extractable protein (EEP) is known to be a major lens membrane protein with a molecular mass in the range 32 kDa to 38 kDa, and is also known to bind to the lens membrane and phospholipid-containing liposomes in a calcium-dependent manner. Recent results (Russell, P., Zelenka, P., Martensen, J., and Reid, T.W. (1977) Curr. Eye Res. 6, 533-538) on antibody cross-reactivity have demonstrated that a 34-35 kDa component of EEP is identical to calpactin I (lipocortin II). In this study, we have identified and purified three distinct 34 kDa components of EEP (designated as EEP-34A1, EEP-34A2 and EEP-34B) from bovine lens that inhibit phospholipase A2 activity. These proteins bind to phospholipid-containing liposome and F-actin in a calcium-dependent fashion. Two-dimensional electrophoresis demonstrates that the three proteins were distinct from one another. However, immunochemical studies and one-dimensional peptide mapping indicate that EEP-34A1 and EEP-34B are very similar. Our results also indicate that EEP-34A1 is very similar to calpactin II and that EEP-34A2 corresponds to calpactin I. The bovine lens 34-35 kDa component of EEP is a mixture of proteins rather than a single protein.     

Isolation of two distinct activator proteins for lipoprotein lipase from ovine plasma

Two distinct activator proteins for lipoprotein lipase (LPL) have been isolated in approximately equal amounts from ovine plasma. These low molecular weight proteins were readily separated from each other on the basis of size and charge. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated proteins of Mr about 8000 and 5000, with pI in urea-containing gels of about 5.1 and 4.8 respectively. Each of the ovine activator proteins was as effective as human apolipoprotein C-II (apo C-II) in activating LPL, with 1 microgram/ml giving near to maximum activation, and in lowering the apparent Km of LPL for triolein substrate. As the ratio of activator to triolein increased from 0.16 to 5.2 (micrograms/mg) the apparent Km fell from about 0.5 to 0.18 mM. Whereas human apo C-II and the two ovine activators were equally effective in stimulating the hydrolysis of triolein, differences were observed between the human and ovine activators when p-nitrophenylbutyrate was used as substrate. Unlike human apo C-II, which produced significant inhibition of p-nitrophenylbutyrate hydrolysis, the ovine activators were without effect. This suggests that the interaction between the ovine activators and LPL is different from that of human apo C-II.     

Isolation and characterization of two alkaline ribonucleases from calf serum.

Treatment of calf serum at 60 degrees C and pH 3.5 followed by chromatography on carboxymethyl (CM) cellulose resulted in the separation of two major peaks of alkaline RNAse activity. One was eluted from CM-cellulose at 0.075 M KCl with an overall purification of 5400-fold and the other was eluted at 0.25 M KCl with a 6700-fold purification. The RNAse eluted from CM-cellulose at 0.075 M KCl was almost completely inhibited by anti-RNAse A serum and by the endogenous RNAse inhibitor and a 33% inhibition was observed in the presence of 5 mM MgCl2. This enzyme seems to be similar or identical to RNAse A. The other RNAse, eluted from CM-cellulose at 0.25 M KCl was not inhibited by anti-RNAse A or 5 mM MgCl2 and was much less sensitive to the endogenous inhibitor. Both enzymes degraded RNA endonucleolytically and the nucleoside monophosphates obtained after partial hydrolysis of RNA by the two serum RNAases were primarily 2'- or 3' -CMP and 2'- or 3' -UMP. Poly(A), native DNA and denatured DNA were degraded slowly or not at all. The RNAase A-like enzyme degraded poly(C) at a significantly faster rate, and poly(U) at a slower rate, than RNA. However, the other serum RNAase was more active with poly(U) than with RNA and almost inactive with poly(C) as the substrate.     

Isolation and characterization of two novel calcium-dependent phospholipid-binding proteins from bovine lung

Two calcium-dependent proteins of apparent Mr 32,000 and 34,000 were isolated from bovine lung. Approx. 70 mg/kg of each was obtained. Two-dimensional gel electrophoresis in the presence of 8 M urea showed their apparent p/values to be 5.1 and 5.0, respectively. Both proteins are related immunologically to calelectrin from Torpedo marmorata. They also have very similar amino acid compositions to calelectrin. Partial sequence information shows that both proteins contain the highly conserved sequence described for the annexins, a new family of calcium-dependent membrane-binding proteins. In common with other members of this family, the new proteins bind to acidic phospholipids in a calcium-dependent manner.     

Isolation and characterization of two distinct forms of protein kinase C

Protein kinase C (Ca2+- and phospholipid-dependent protein kinase) has been purified from rat brain by a three-step, 18-h procedure resulting in the isolation of milligram quantities of enzyme. Unlike previous preparations from published protocols, which yield a single polypeptide, this procedure yields a protein which consists of a 78/80-kDa doublet upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two polypeptides have been characterized with respect to structure and function and are very similar in both regards. However, the two forms can be distinguished immunologically by polyclonal antisera generated against purified protein kinase C. The 78- and 80-kDa proteins do not appear to be related to one another by proteolytic cleavage or by differential phosphorylation, although the two purified proteins do contain stoichiometric amounts of phosphate. The 78- and 80-kDa polypeptides therefore appear to represent two distinct forms of protein kinase C, thus providing evidence for the existence of multiple isozymes of this key regulatory protein.     

Isolation and characterization of endogenous ligands for liver mannan-binding protein

Endogenous ligands for the hepatic lectin which is specific for mannose and N-acetylglucosamine (mannan-binding protein, MBP) were isolated from rat liver rough microsomes and primary cultured hepatocytes by affinity chromatography on an immobilized MBP column. Western blotting using specific antisera revealed that serum glycoproteins, alpha 1-macroglobulin, alpha 1-antitrypsin, and alpha 1-acid glycoprotein, and a lysosomal enzyme, beta-glucuronidase were the major constituents of the endogenous ligands. These endogenous ligands consisted of high mannose-type oligosaccharides of Man9GlcNAc2 and Man8GlcNAc2, and had rapid turnover rates with an average half-life of 45 min, indicating that they were mainly composed of biosynthetic intermediates of glycoproteins. In view of the identification of the endogenous ligands as the biosynthetic intermediates of glycoproteins, the possible functions of the intracellular lectin are discussed in relation to the intracellular transport of glycoproteins.     

Isolation of zymogen granules from rat pancreas and characterization of their membrane proteins

A zymogen granule fraction has been isolated from rat pancreas, and its purity has been assessed by biochemical and morphological criteria. Specific activities of two marker enzymes, amylase and chymotrypsin, are increased by 4.6 and 5.4-fold, respectively, as compared to the homogenate. The purified fraction is devoid of detectable RNA, DNA and 5'-nucleotidase, glucose-6-phosphatase, and cytochrome c oxidase activities. Electron micrographs confirm the absence of mitochondria, lysosomes, and rough endoplasmic reticulum fragments. Zymogen granule membranes were isolated from this fraction on a sucrose gradient following lysis in alkaline buffer. Secretory contaminants were efficiently removed from the membranes as indicated by experiments in which labeled secretory proteins were added during the isolation procedure and secondly by measuring residual levels of amylase and chymotrypsin. Three enzyme activities were found in the membranes: thiamine pyrophosphatase, ATP-diphosphohydrolase, and low levels of acid phosphatase. Membrane proteins were solubilized by urea-Triton X-100 and separated in double-dimension (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Isoelectric point and molecular weight of each protein band were determined.     

Isolation and characterization of two distinct myo-inositol transporter genes of Saccharomyces cerevisiae

By the complementation of a yeast mutant defective in myo-inositol transport (Nikawa, J., Nagumo, T., and Yamashita, S. (1982) J. Bacteriol. 150, 441-446), we isolated two myo-inositol transporter genes, ITR1 and ITR2, from a yeast gene library. The ITR1 and ITR2 genes contained long open reading frames capable of encoding 584 and 612 amino acids with calculated relative molecular masses of 63,605 and 67,041, respectively. The sequence similarity between the ITR1 and ITR2 products was extremely high, suggesting that the two genes arose from a common ancestor. Both gene products show significant sequence homology with a superfamily of sugar transporters, including human HepG2 hepatoma/erythrocyte glucose transporter and Escherichia coli xylose transporter. Hydropathy analysis indicated that the ITR1 and ITR2 products are both hydrophobic and contain 12 putative membrane-spanning regions. Thus, yeast myo-inositol transporters could be classified into the sugar transporter superfamily. Gene disruption and tetrad analysis showed that yeast cells contain two separate myoinositol transporters. The ITR1 product was the major transporter and the ITR2 product the minor one in cells grown in minimum medium containing glucose. Northern blot analysis showed that ITR1 mRNA was much more abundant than ITR2 mRNA. The previously isolated myo-inositol transport mutant was determined to be defective in ITR1.     

Isolation of two ATPase inhibitor proteins from mitochondria of rat skeletal muscle

An ATPase inhibitor protein was isolated from mitochondria of rat skeletal muscle by alkaline extraction and then was purified, It differed in definitive ways from the ATPase inhibitor protein isolated previously by Ca²⁺-stripping of submitochondrial particles of rat skeletal muscle. The two ATPase inhibitor proteins were shown to be present together in intact mitochondria.