Purification and characterization of novel calmodulin-binding protein from cardiac muscle

A novel protein which represents the most abundant calmodulin-binding protein in bovine heart cytosolic fraction was purified to apparent homogeneity. The purification procedure involved DEAE-Sepharose CL-6B (to remove calmodulin), calmodulin-Sepharose 4B affinity, and Sepharose 6B column chromatographies. This purified calmodulin-binding protein is a highly asymmetric protein with a sedimentation coefficient of approximately 5.0 S and a Stokes radius of about 83.0 A. The molecular weight of the calmodulin-binding protein was determined to be 175,000 from the sedimentation constant and Stokes radius of the protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein showed a single protein band with an apparent molecular weight of 140,000. The result suggests that the protein is monomeric. Although this molecular weight is similar to that of caldesmon, a known ubiquitous calmodulin-binding protein, the protein did not react with caldesmon-specific antibodies, nor did it display a proteolytic fragmentation pattern similar to that of the former. In addition, caldesmon was found almost exclusively in the particulate fraction in low ionic strength cardiac muscle extract, whereas this protein is purified the soluble fraction.     

Purification and characterization of glycolactin, a novel glycoprotein from bovine milk

A novel glycoprotein designated glycolactin, with a molecular weight of 64 kDa, a sequence hitherto unknown in the literature and capable of inhibiting the hemagglutinating activities of soybean lectin and Ricinus communis agglutinin 120, was isolated from bovine milk. Its lectin-inhibiting activity differed from that of lactoferrin, another milk protein. Like other milk proteins, glycolactin inhibited superoxide formation in vitro. Glycolactin inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC50 of about 31 nM. It exhibited ribonucleolytic (RNase) activity towards yeast transfer RNA with a pH optimum of 7.5, and specific RNase activity towards poly C. The purification protocol of glycolactin involved removal of globulin from the acid whey fraction of bovine milk by precipitation with 1.8 M (NH4)2SO4, and adsorption on the ion exchangers CM-Sepharose and Mono S. Deglycosylation of glycolactin using glycopeptidase F produced only a slight decrease of 4 kDa in the molecular weight of glycolactin.     

A novel kinesin-like protein with a calmodulin-binding domain

Calcium regulates diverse developmental processes in plants through the action of calmodulin. A cDNA expression library from developing anthers of tobacco was screened with ³⁵S-labeled calmodulin to isolate cDNAs encoding calmodulin-binding proteins. Among several clones isolated, a kinesin-like gene (TCK1) that encodes a calmodulin-binding kinesin-like protein was obtained. The TCK1 cDNA encodes a protein with 1265 amino acid residues. Its structural features are very similar to those of known kinesin heavy chains and kinesin-like proteins from plants and animals, with one distinct exception. Unlike other known kinesin-like proteins, TCK1 contains a calmodulin-binding domain which distinguishes it from all other known kinesin genes. Escherichia coli-expressed TCK1 binds calmodulin in a Ca²⁺-dependent manner. In addition to the presence of a calmodulin-binding domain at the carboxyl terminal, it also has a leucine zipper motif in the stalk region. The amino acid sequence at the carboxyl terminal of TCK1 has striking homology with the mechanochemical motor domain of kinesins. The motor domain has ATPase activity that is stimulated by microtubules. Southern blot analysis revealed that TCK1 is coded by a single gene. Expression studies indicated that TCK1 is expressed in all of the tissues tested. Its expression is highest in the stigma and anther, especially during the early stages of anther development. Our results suggest that Ca²⁺/calmodulin may play an important role in the function of this microtubule-associated motor protein and may be involved in the regulation of microtubule-based intracellular transport.     

A novel guinea pig macrophage-specific polymorphic molecule. I. Biochemistry, genetics, and tissue distribution

In the course of studying Ia molecules from strain 2 and strain 13 guinea pig macrophages, with the intent of comparing them to B cell Ia molecules, it was observed that guinea pig alloserum prepared by cross-immunization of guinea pig lymphocyte Ag non-identical inbred guinea pigs immunoprecipitated not only conventional class I and class II molecules, but also a 98,000-Da molecule, termed gp98. Two different forms of the molecule were detected, indicating it is polymorphic. The genes encoding gp98 were shown not to be linked to the guinea pig lymphocyte Ag complex. The molecule gp98 was found on macrophages within populations of peritoneal exudate cells, resident peritoneal cells, bone marrow cells, and spleen. All gp98-bearing macrophages were also Ia-positive. However, only a subpopulation of macrophages bore gp98. The gp98 was not found on Ly-1 or Ig-bearing cells, indicating that B and T cells do not bear Ia. Thus, gp98 appears to be a highly immunogenic polymorphic macrophage-specific molecule that allows the characterization of guinea pig macrophage subsets.     

Isolation and characterization of a novel calmodulin-binding protein from potato.

Tuberization in potato is controlled by hormonal and environmental signals. Ca(2+), an important intracellular messenger, and calmodulin (CaM), one of the primary Ca(2+) sensors, have been implicated in controlling diverse cellular processes in plants including tuberization. The regulation of cellular processes by CaM involves its interaction with other proteins. To understand the role of Ca(2+)/CaM in tuberization, we have screened an expression library prepared from developing tubers with biotinylated CaM. This screening resulted in isolation of a cDNA encoding a novel CaM-binding protein (potato calmodulin-binding protein (PCBP)). Ca(2+)-dependent binding of the cDNA-encoded protein to CaM is confirmed by (35)S-labeled CaM. The full-length cDNA is 5 kb long and encodes a protein of 1309 amino acids. The deduced amino acid sequence showed significant similarity with a hypothetical protein from another plant, Arabidopsis. However, no homologs of PCBP are found in nonplant systems, suggesting that it is likely to be specific to plants. Using truncated versions of the protein and a synthetic peptide in CaM binding assays we mapped the CaM-binding region to a 20-amino acid stretch (residues 1216-1237). The bacterially expressed protein containing the CaM-binding domain interacted with three CaM isoforms (CaM2, CaM4, and CaM6). PCBP is encoded by a single gene and is expressed differentially in the tissues tested. The expression of CaM, PCBP, and another CaM-binding protein is similar in different tissues and organs. The predicted protein contained seven putative nuclear localization signals and several strong PEST motifs. Fusion of the N-terminal region of the protein containing six of the seven nuclear localization signals to the reporter gene beta-glucuronidase targeted the reporter gene to the nucleus, suggesting a nuclear role for PCBP.     

Purification and characterization of a bovine pregnancy-associated glycoprotein

A 67000 Mr bovine pregnancy-associated glycoprotein (bPAG) has been isolated from fetal cotyledons and purified to homogeneity by HPLC. The purification was monitored by a double immunodiffusion test and by RIA in conjunction with an antiserum raised against a crude fraction of placenta-specific antigens. The molecular weight of bPAG was estimated to be 67000 by SDS-PAGE. The isoelectric points (pI) of the four isoforms, determined by high-resolution analytical electrofocusing in polyacrylamide gel, were 4.4, 4.6, 5.2, and 5.4. The carbohydrate content of the bPAG consisted of approximately 10.02 +/- 1.09% neutral sugar and variant amounts of sialic acid (from 0.29 +/- 0.06% in the most basic isoform to 2.1 +/- 0.31% in the most acidic isoform). A specific antiserum was raised against the purified bPAG. A specific RIA showed that the bPAG was antigenically unrelated to BSA, alphafetoprotein (AFP), and human schwangerschafts-spezifischen (pregnancy-specific) beta 1 glycoprotein (SP1). According to some characteristics (e.g. the molecular weight), the purified bPAG may correspond to a form of the pregnancy-specific protein B previously described by Sasser and colleagues (Biol Reprod 1986; 35:936-942).     

Purification and partial characterization of a novel hemagglutinating glycoprotein from the cultured mycelia of Hericium erinaceus

HEG-5, a novel glycoprotein with hemagglutinating activity, was firstly isolated and purified from the cultured mycelia of Hericium erinaceus CZ-2. SDS–PAGE, Native-PAGE and MALDI-TOF-MS proved that HEG-5 was a single band with the molecular weight of approximately 14.4 kDa. HEG-5 had the protein: polysaccharide ratio of approximately 10:1 (%/%) and contained d-glucose, l-rhamnose, d-galactose and d-mannose with a molar ratio of 1.00:1.09:2.45:7.14 in polysaccharide fraction. HEG-5 was an acidic glycoprotein with a PI value of 6.3 and the higher content of acidic amino acids (Asp, 12.42 ± 0.25% and Glu, 12.24 ± 0.26%) in protein fraction. FT-IR and NMR spectra revealed that HEG-5 contained the protein and carbohydrate portions with (1→4)-linked β-galactose residues and β-linked glucose residues. Circular dichroism (CD) demonstrated that HEG-5 was a β-sheet predominant glycoprotein. Hemagglutination assay proved it was a thermo-unstable glycoprotein. The HEG-5 structural novelty was finally presented by protein sequencing and modeling by using MALDI-TOF-MS, NCBI blast search and online SWISS-MODEL Workspace service.     

Purification and partial characterization of a malignancy-associated glycoprotein

The isolation from cancer patient serum of a glycoprotein (Cc) associated with the presence of a variety of malignancies was previously reported. Although preliminary chemical and physical data indicated that Cc was different from identified circulating glycoproteins, subsequent immunological studies suggested that it was closely related to alpha 1-acid glycoprotein. Further analysis revealed the presence of two components in some Cc preparations and prompted a re-examination of the isolation and characterization data. In the present study, Cc was purified by a modified protocol which involved the use of pleural fluid obtained from individuals with cancer, and an alpha 1-acid glycoprotein antibody column to remove contaminating alpha 1-acid glycoprotein. Typically, the material not retained by the antibody column gave a single band with Mr 53,000 when subjected to sodium dodecyl sulfate-polyacrylamide electrophoresis. Amino terminal analysis revealed that the protein contained a blocked amino terminus, and carbohydrate analysis indicated that complex, asparagine-linked saccharide units were present. The product could be distinguished from alpha 1-acid glycoprotein and other previously described circulating glycoproteins by several criteria, including molecular weight, isoelectric point, and amino acid and carbohydrate composition. One of three preparations isolated had an apparent Mr of 59,000. Carbohydrate analysis as well as deglycosylation studies showed that the change in molecular weight was due to increased glycosylation.     

Purification and chemical study of a Collocalia glycoprotein]

A glycoprotein was purified from the aqueous extract of "edible bird's nest" (Collocalia) using free flow preparative electrophoresis and represented the main fraction of Collocalia glycoproteins. This glycoprotein is homogeneous upon agarose electrophoresis and slightly polydisperse upon ultracentrifugation (S So 20w = 3,0). The carbohydrate moiety contains galactose, mannose, glucosamine, galactosamine and sialic acid, which is completely released by Clostridium perfringens or Diplococcus pneumoniae neuraminidases and has the same chromatographic behaviour as N-acetyl-neuraminic acid. The peptide part of the glycoprotein is rich in serine, threonine and proline. About 40 p. cent of the hydroxyaminoacids are involved in carbohydrate-peptide linkages.     

Purification and characterization of a soybean leaf storage glycoprotein

Removing the pods from soybean (Glycine max [L.] Merr. cv Wye) plants induces a change in leaf function which is characterized by a change in the leaf soluble protein pattern. The synthesis of at least four polypeptides (~27, 29, 54, and 80 kilodaltons) is enhanced, and these polypeptides accumulate to levels comprising over 50% of the soluble protein. Heat girdling the petiole also causes the accumulation of these polypeptides, suggesting that the signal for changing leaf function may be associated with inhibition of phloem transport. The 27 and 29 kilodalton polypeptides are glycosylated and have been purified to greater than 90% by (NH₄)₂SO₄ fractionation, concanavilin A affinity, and gel filtration chromatography. These peptides appear to comprise a single protein. Mouse antiserum has been prepared against this glycoprotein and has been used to check for cross-reactivity with seed proteins and to quantitate changes with leaf development. No cross-reactivity was observed with seed soluble proteins from several stages of development. Quantitation showed the highest content in podded plants at, and shortly following, flowering, with levels subsequently declining in conjunction with seed growth. In depodded plants, the level of glycoprotein continued to increase following flowering and accounted for 45% of the soluble leaf protein by 4 weeks after depodding.     

Purification and characterization of catalytic fragments of phosphorylase kinase gamma subunit missing a calmodulin-binding domain

A catalytic fragment preparation of rabbit muscle phosphorylase kinase produced by limited chymotryptic digestion was isolated and identified as the NH2-terminal region of the gamma subunit by Edman degradation. Mass spectral analysis, gas phase sequence analysis, and amino acid analysis of the active fragment carboxyl-terminal peptides revealed multiple COOH termini generated at residues Tyr290, Arg296, and Phe298 in the gamma subunit sequence. These active fragment species are about 24% smaller than the gamma subunit (Mr 44,673) and range in size from Mr 33,279 to Mr 34,275. The active fragment preparation exhibits a specific activity about 6-fold higher than that of the gamma subunit-calmodulin complex. Calmodulin confers calcium sensitivity to the gamma subunit but has no effect on the enzymatic properties of active fragment. Affinity measurements demonstrated a dissociation constant of 0.7 microM for active fragment binding to dansylcalmodulin, a value about 28-fold weaker than reported for the gamma subunit. These data support the presence of a calmodulin binding domain in the COOH-terminal region of the gamma subunit.     

Maize cap1 encodes a novel SERCA-type calcium-ATPase with a calmodulin-binding domain

A cDNA (CAP1) isolated from maize roots shares sequence identity with genes encoding P-type Ca(2+)-ATPases and restores the growth phenotype of yeast mutants defective in Ca(2+)-pumps. CAP1 was transcribed and translated in the yeast mutant. Furthermore, the membrane-integrated product formed a Ca(2+)-dependent phosphorylated intermediate and supported Ca(2+) transport. Although CAP1 shares greater sequence identity with mammalian "endoplasmic reticulum-type" Ca(2+)-pumps, it differs from these genes by having features of calmodulin (CaM)-regulated Ca(2+)-pumps. CAP1 from yeast microsomes bound CaM, and the CAP1-dependent Ca(2+) transport in yeast was stimulated by CaM. Peptides from the C terminus of CAP1 bound CaM. Anti-CAP1 antibodies specifically recognized a maize microsomal polypeptide that also bound CaM. A similar polypeptide also formed a Ca(2+)-dependent phosphoenzyme. Our results suggest that cap1 encodes a novel form of CaM-regulated Ca(2+)-ATPase in maize. CAP1 appears to be encoded by one or two genes in maize. CAP1 RNA is induced only during early anoxia, indicating that the Ca(2+)-pump may play an important role in O(2)-deprived maize cells.     

Tissue distribution of high molecular weight calmodulin-binding protein

Polyclonal antibodies raised against bovine heart high molecular weight calmodulin-binding protein were used to study the distribution of this protein in diverse bovine tissues. The high molecular weight calmodulin-binding protein, in addition to bovine heart, is also present in lung and brain at much lower levels, but not in skeletal muscle, spleen, kidney or uterus.     

Purification of a 47-kDa calmodulin-binding polypeptide as an actin-binding protein from Neurospora crassa

We have enriched a 47-kDa polypeptide (p47) from Neurospora crassa on the basis of its affinity to calmodulin. The p47 was purified to homogeneity by chromatography on a Mono S cation exchange column and evidence is presented that the polypeptide co-sediments specifically with F-actin. The intracellular distribution of p47 and actin was also examined using indirect double immunofluorescence staining of cells at different stages of development. Our results suggest that by altering the conformation binding site of actin to p47, calmodulin could play a regulatory role in the polarized hyphal growth of N. crassa.     

Purification and characterization of a glycoprotein elicitor from Alternaria tenuissima

Glycoprotein elicitor can induce plant resistance and become a potential agent for biological control of plant diseases. Here, a new glycoprotein elicitor was purified with the method of cold alcohol precipitation and anion exchange chromatography from the mycelium of Alternaria tenuissima strain JH505, which was identified on the basis of morphological features and sequence analysis of rDNA internal transcribed spacer. The protein showed a single band on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) stained with silver and appeared one main protein peak in HPLC. The apparent molecular weight of the purified protein was 66 kDa and isoelectric point was about 4.27. This protein was identified as glycoprotein by glycoprotein staining Kit. Anthrone-colorimetric assay and Coomassie blue G-250 staining showed that carbohydrate and protein content was in a ratio of 1.75. After deglycosylation by trifluoromethane-sulfonic acid, this glycoprotein showed two bands on the SDS–PAGE, and which means the glycoprotein may have at least two glycosylation sites. The glycoprotein induced tobacco resistance against tobacco mosaic virus and enhanced wheat seedling growth at 15°C. The glycoprotein elicitor provided an effective way of alternative strategies for plant disease control.     

Subcellular distribution of calmodulin and calmodulin-binding sites in Tetrahymena pyriformis

The subcellular distribution of calmodulin and particulate calmodulin-binding activity was studied in a eukaryotic protozoan, Tetrahymena pyriformis NT-1. The particulate calmodulin-binding activity was found to be localized principally in microsomes and to some extent in cilia and surface membranes called pellicles. Nearly all (93%) of the total amount of calmodulin was recovered in two soluble compartments, the ciliary and postmicrosomal supernatant fractions.     

Molecular cloning sequence and distribution of rat calspermin, a high affinity calmodulin-binding protein

Calspermin is a heat-stable, acidic calmodulin-binding protein predominantly found in mammalian testis. The cDNA representing the rat form of this protein has been cloned from a rat testis lambda gt11 library. Sequence analysis of two overlapping clones revealed a 232-nucleotide 5'-nontranslated region, 510 nucleotides of open reading frame, a 148-nucleotide 3'-untranslated region, and a poly(A) tail. Authenticity of the clones was confirmed by comparison of a portion of the deduced amino acid sequence with the sequence of a tryptic peptide obtained from the rat testis protein. The lambda gt11 fusion protein was recognized by affinity purified antibodies to pig testis calspermin and bound 125I-calmodulin in a Ca2+-dependent manner. Calspermin cDNA encodes a 169-residue protein with a calculated Mr of 18,735. The putative calmodulin-binding domain is very close to the amino terminus of the protein. This region shows 46% identity with the calmodulin-binding region of rat brain Ca2+/calmodulin-dependent protein kinase II and 32% identity with the equivalent region of chicken smooth muscle myosin light chain kinase. The 5'-nontranslated region reveals significant homology with a portion of the catalytic region of the calmodulin-dependent protein kinase family. Calspermin contains a stretch of 17 contiguous glutamic acid residues in the central region of the molecule. Computer analysis predicts calspermin to be 81% alpha-helix and 14% random coil. Analysis of genomic DNA indicates calspermin to be the product of a unique gene. Northern blot analysis of rat testis RNA reveals a 1.1-kilobase mRNA. This RNA is restricted to testis among several rat tissues examined and could not be identified in total RNA isolated from testes of other mammals. Analysis of cells isolated from rat testis reveals calspermin mRNA to be predominantly expressed in postmeiotic cells indicating that it may be specific to haploid cells.