Identification of Macrophage Migration Inhibitory Factor Isoforms in Bovine Brain

In the course of the study of the primary structures and molecular mechanisms of action of immunologically active compounds of the nervous system we have isolated from the soluble fraction of total bovine brain two heat-stable proteins. The purification procedure was mainly based on DEAE-Servacel ion-exchange chromatography and reversed-phase HPLC. The proteins were identified by the N-terminal Edman microsequence analysis and database searching as macrophage migration inhibitory factor (MIF). The N-terminal sequences for MIF1 and MIF2 were found to be identical. According to mass spectral analysis, the molecular masses for MIF1 and MIF2 were determined respectively as 12,369.21 and 12,299.7 Da. In addition, we have also isolated a third peptide having the same N-terminal sequence and Mr 9,496.2 that seems to be a proteolytic fragment of MIF. Using p-hydroxyphenylpyruvate as a substrate, we have not revealed tautomerase activity of either MIF1 or MIF2. As both the immunologic and enzymatic activities were reported to be expressed by the oligomeric structure of MIF, we suggest that the present study may give additional information on MIF in terms of structural properties of this protein. A comparatively simple purification procedure is presented that may be widely used for simultaneous isolation in one run of MIF isoforms.     

Charge Heterogeneity of Bovine Brain Macrophage Migration Inhibitory Factor

Macrophage migration inhibitory factor (MIF) is known as a ubiquitous pluripotent cytokine originally identified for its capacity to inhibit the random migration of macrophages in vitro. It is recognized as an important regulator of the immunological, neuroendocrine and enzymatic processes. MIF is widely expressed in brain, but its role in the nervous system is not yet understood. In the course of the study of the primary structure of bovine brain MIF we have previously identified a number of MIF-related proteins having identical N-terminal sequences. In this paper we report the results of isoelectric focusing of MIF isolated to a homogeneous state from bovine brain that revealed MIF charge heterogeneity. We have detected isoelectric forms of MIF with pI values of 6.9, 7.0, 7.3, and 7.8. The diverse actions of MIF within the immuno-neuroendocrine system is suggested to be a result of its occurrence in different isoforms and oligomerization states.     

Macrophage migration inhibitory factor: Isolation from bovine brain

The purification of macrophage migration inhibitory factor (MIF) from bovine brain cytosol and its partial characterization are reported. A rapid and relatively simple method for MIF isolation was developed based mainly on size-exclusion chromatography on Toyopearl TSK polymer having a tendency to adsorb MIF as compared to elution of other proteins with similar molecular weights. The method gives a high yield of MIF (0.1 mg homogenous protein per g wet tissue). The retardation is conveniently utilized to achieve good separations of MIF from other proteins of similar molecular weights. The isolated protein was identified as MIF by SDS-electrophoresis, immunoblotting, sequencing of the N-terminal amino acid residues, and also by determination of keto-enol tautomerase activity that is characteristic of MIF with p-hydroxyphenylpyruvic acid as a substrate.     

Isolation and sequence of cDNA clones encoding rat phosphatidylinositol transfer protein

Phosphatidylinositol (PtdIns) transfer protein is a cytosolic protein that catalyzes the transfer of PtdIns between membranes. It is expressed in organisms from yeast to man, and activity has been found in all animal tissues examined. Using antibodies prepared against bovine brain PtdIns transfer protein, lambda gt11 rat brain cDNA libraries were screened and several clones isolated. DNA sequence analysis showed that the cDNAs encoded a polypeptide of 271 amino acids with a mass of 31,911 Da. Comparison of the deduced amino acid sequence with N-terminal sequence data obtained for the intact purified bovine brain protein and rat lung phospholipid transfer protein verified that the cDNAs were PtdIns transfer protein clones. The predicted protein shows no significant sequence similarity to other known (phospholipid)-binding proteins. DNA blot hybridization suggests that the rat genome may contain more than one gene encoding PtdIns transfer protein. RNA blot hybridization reveals that the PtdIns transfer protein gene is expressed at low levels in a wide variety of rat tissues; all tissues examined showed a major mRNA component of 1.9 kilobases and a minor component of 3.4 kilobases. The isolation of clones encoding rat PtdIns transfer protein will greatly facilitate studies of the structure and function of PtdIns transfer proteins and their role in lipid metabolism.     

Similarities of physico-chimical and antigenic properties of neurocupreins and extremely acidic copper proteins from chromaffin granules

Extremely acidic copper-containing proteins, neurocupreins, were isolated from brains of various mammals (bovine, rabbit, pig and sheep). Neurocupreins from all these sources were found to have similar physico-chemical and antigenic properties. Using the immunological approach, it was shown that neurocuprein is located only in brain cytosol and synaptosomal fractions. Extremely acidic copper-containing proteins were also isolated from soluble and membranous fractions of chromaffin granules from bovine adrenal medulla. The soluble form of the protein from the granules has practically the same physico-chemical and antigenic properties as neurocupreins. The copper protein isolated from membranes of granules has slightly higher molecular weight and somewhat different amino acid composition, although their EPR spectra are identical. However, both copper proteins from chromaffin granules are immunoprecipitated with antibodies to neurocuprein. It is suggested that the membranous form differs from the soluble one in possessing a peptide which prolongs the protein chain without changes in its antigenic properties.     

Similarities of physico-chemical and antigenic properties of neurocupreins and extremely acidic copper proteins from chromaffin granules

Extremely acidic copper-containing proteins, neurocupreins, were isolated from brains of various mammals (bovine, rabbit, pig and sheep). Neurocupreins from all these sources were found to have similar physico-chemical and antigenic properties. Using the immunological approach, it was shown that neurocuprein is located only in brain cytosol and synaptosomal fractions. Extremely acidic copper-containing proteins were also isolated from soluble and membranous fractions of chromaffin granules from bovine adrenal medulla. The soluble form of the protein from the granules has practically the same physico-chemical and antigenic properties as neurocupreins. The copper protein isolated from membranes of granules has slightly higher molecular weight and somewhat different amino acid composition, although their EPR spectra are identical. However, both copper proteins from chromaffin granules are immunoprecipitated with antibodies to neurocuprein. It is suggested the the membranous form differs from the soluble one in possessing a peptide which prolongs the protein chain without changes in its antigenic properties.     

Physicochemical Properties and Biological Activity of the Peptide–Protein Complex from Bovine Scleral Tissue

The peptide–protein complex from bovine sclera was studied. It is shown that it contained a protein with a molecular weight of 66387 Da with the partial N-terminal amino acid sequence DTHKSEIAHRFKDLG-, which is homologous to the mature molecule of bovine serum albumin, and polypeptides with molecular weights of 1300–5080 Da. With a model of the organotypic cultivation of posterior eye tissues of the newt Pleurodeles waltl in vitro, it was shown that the effect of this peptide–protein complex in low doses increased the viability of scleral fibroblasts.     

Isolation and characterization of fatty acid-binding protein from rat brain

A fatty acid-binding protein has been identified and isolated from the cytosol fraction of rat brain. The fatty acid-binding protein was purified to homogeneity by gel filtration and preparative isoelectric focusing. The binding protein was different from Z protein from rat liver in its isoelectric point and immunological reactivity, in spite of its similar molecular weight of 12,000. Rabbit antibodies against rat liver Z protein were used to demonstrate that the fatty acid-binding proteins from rat liver and brain are immunologically unrelated, and that no Z protein is present in rat brain cytosol.     

Isolation of a cDNA clone encoding a high molecular weight precursor to a 6-kDa pulmonary surfactant-associated protein

Mammalian surfactant is an incompletely defined mixture of lipids and associated proteins of molecular mass 35,000 Da and approximately 6,000 Da. Surfactant preparations which are highly effective in treating respiratory distress syndrome in premature infants lack the 35-kDa proteins, but contain the 6-kDa proteins. We isolated and partially sequenced one of these low molecular weight proteins from the lung lavage material of an alveolar proteinosis patient. Oligonucleotides deduced from the sequence were used as probes to isolate a human cDNA clone. The clone codes for a 42-kDa protein which contains the sequence of the 6-kDa protein. Messenger RNA coding for the 42-kDa protein was identified in human lung RNA by in vitro translation and immunoprecipitation of the translation products with an antiserum against purified bovine surfactant 6-kDa proteins. Immunoprecipitation of the 42-kDa primary translation product is inhibited by the presence of the bovine 6-kDa protein. These observations suggest a precursor-product relationship of the 42-kDa protein to one of the 6-kDa proteins.     

Putative Synaptic Vesicle Nucleotide Transporter Identified as Glyceraldehyde-3-Phosphate Dehydrogenase

Abstract: Synaptic vesicles isolated from electric ray electric organ have been shown previously to contain a 34-kDa protein that binds azido-ATP, azido-AMP, and N -ethylmaleimide. The protein was found to share similarities with the mitochondrial ADP/ATP carrier and assumed to represent the synaptic vesicle nucleotide transporter. Synaptic vesicles were purified by sucrose density gradient centrifugation and subsequent chromatography on Sephacryl S-1000 from both Torpedo electric organ and bovine brain cerebral cortex. They contained ATP-binding proteins of 35 kDa and 34 kDa, respectively. ATP binding was inhibited by AMP. Both proteins were highly enriched after column chromatography of vesicle proteins of AMP-Sepharose. Antibodies were obtained against both proteins. Antibodies against the bovine brain synaptic vesicle protein of 34 kDa bound specifically to the 35-kDa protein of Torpedo vesicles. An N-terminal sequence obtained against the 34-kDa protein of bovine brain synaptic vesicles identified it as glyceraldehyde-3-phosphate dehydrogenase. The previously observed molecular characteristics of the putative vesicular nucleotide transporter in Torpedo fit those of glyceraldehyde-3-phosphate dehydrogenase. We, therefore, suggest that the protein previously identified as putative nucleotide transporter is, in fact, glyceraldehyde-3-phosphate dehydrogenase.     

Immunological evidence that the nonhormone binding component of avian steroid receptors exists in a wide range of tissues and species

A monoclonal antibody to a fungal protein has been used to demonstrate the presence of the nonhormone binding component of molybdate-stabilized steroid receptors in a variety of vertebrate tissues. We recently identified a steroid receptor in the aquatic fungus Achlya ambisexualis where sexual morphogenesis of the male is directed by the steroid antheridiol. This receptor resembles receptors of higher organisms in exhibiting an 8S, molybdate-stabilized form. In the chick oviduct, a 90 000 molecular weight protein has previously been shown to be associated with the molybdate-stabilized complex of the progesterone receptor. We have isolated a similar protein of molecular weight about 88 000 from A. ambisexualis and have obtained a hybridomal-derived monoclonal antibody directed against it. This mouse anti-Achlya immunoglobulin G1 (IgG1) cross-reacts with the 90 000 molecular weight protein in chick oviduct cytosol and was used to detect analogous 90 000 molecular weight proteins in mammalian tissues. Tissue cytosols were incubated with antibody, and the complexes were isolated onto protein A-Sepharose. The resin-bound proteins were then analyzed by gel electrophoresis. This procedure revealed the presence of 90 000 molecular weight proteins in several mammalian tissues including rat liver, mouse liver and uterus, pig ovarian granulosa cells, human endometrium, and HeLa cells. These results demonstrate that the 90 000 molecular weight protein is not peculiar to the chick oviduct but is present in several different tissues from a variety of animals. This antibody should be a useful probe for further studies on the biological role of these proteins.     

Isolation and identification of antimicrobial components from the epidermal mucus of Atlantic cod (Gadus morhua)

The epidermal mucus of fish species has been found to contain antimicrobial proteins and peptides, which is of interest in regard to fish immunity. An acidic extract from the epidermal mucus of the Atlantic cod (Gadus morhua) was found to exhibit antimicrobial activity against Bacillus megaterium, Escherichia coli and Candida albicans. This activity varied significantly when salt was added to the antimicrobial assay, and was eliminated by pepsin digestion. No lysozyme activity was detected in the extract. By using weak cationic exchange chromatography together with reversed-phase chromatography, and monitoring the antimicrobial activity, we have isolated four cationic proteins from the mucus extract. Using N-terminal and C-terminal amino acid sequence analysis, together with MS, the antimicrobial proteins were identified as histone H2B (13 565 Da), ribosomal protein L40 (6397 Da), ribosomal protein L36A (12 340 Da) and ribosomal protein L35 (14 215 Da). The broad spectra of antimicrobial activities in the cod mucus and the characterization of four antimicrobial polypeptides suggest that mucus compounds contribute to the innate host defence of cod.     

Tissue and subcellular distributions of an inhibitory GDP/GTP exchange protein (GDI) for the rho proteins by use of its specific antibody

We have recently purified from bovine brain cytosol to near homogeneity a GDP/GTP exchange protein for the rho proteins, named rho GDI, that inhibits the dissociation of GDP from and the subsequent binding of GTP to the rho proteins. In the present study, we made a monoclonal antibody against rho GDI and studied its tissue distribution in rat and its subcellular distribution in rat cerebrum by use of this antibody. rho GDI was found in most rat tissues as described for the rho proteins. In rat cerebrum, rho GDI was mostly found in the cytosol of neuron body and synaptosome. In synaptosome, it was mainly found in the synaptic cytosol.     

多头绒泡菌中动蛋白的免疫化学鉴定

动蛋白(kinesin)是一种微管系统的运动蛋白(motor protein),它能通过水解ATP将化学能转化为机械能,推动微管产生运动.微管系统作为一种主要的细胞骨架存在于所有真核细胞中,它们对于维持细胞形态,细胞的分裂,染色体的运动及细胞内的物质运输起着重要作用.细胞质力蛋白(dynein)和动蛋白是公认的推动这类运动的运动蛋白.自从1985年Vole首次在鱿鱼大轴突(squidgiant axon)中发现动蛋白以来,人们先后在许多种动物细胞中发现有动蛋白存在,甚至在低等真核生物棘状变形虫,盘基网柄茵和高等植物烟草花粉管中发现有动蛋白的存在.研究结果表明,动蛋白参与了真核细胞中的许多重要生命活动,如细胞中的细胞器及囊泡的运动,染色体排裂和分离等运动.动蛋白很可能是普通存在于所有真核细胞中的一种运动蛋白.多头绒泡菌(Physarum poly-cephalum)属于粘菌纲(Myxomycetes)的一种低等真核生物,它表现出许多显著的细胞运动特征如原生质团迁移,细胞质的穿梭运动(shuttle streaming)等,是研究非肌细胞运动和收缩蛋白的经典材料.在多头绒泡菌胞质中也具有微管系统,它们构成其纺锤丝等,参与染色体的运动及其它胞质运动,但至今国内外尚无人证明其中有与微管作用的运动蛋白——动蛋白的存在,作者利用抗牛脑动蛋白的单克隆抗体,     

Isolation and primary structure of an amphibian neurotensin.

Using a radioimmunoassay system employing an antiserum which recognises the common C-terminal tripeptide (YIL) of neurotensin (NT) and neuromedin N (NN), immunoreactivity was identified in extracts of brain (65.8 pmol/g), small intestine (44.2 pmol/g) and rectum (13.2 pmol/g) of the European common frog (Rana temporaria). No immunoreactivity was detected in extracts of stomach and skin. Reverse-phase HPLC analysis of each tissue extract resolved a single immunoreactive peptide with identical retention time in each case. The immunoreactive peptide was isolated by reverse-phase HPLC from brain extracts and an N-terminal pyroglutamyl residue was successfully removed enzymatically. The molecular mass of des(pyroglutamyl) frog NT, determined by plasma desorption mass spectroscopy, was 1440 Da. The primary structure of this peptide was determined by gas-phase sequencing and the calculated molecular mass, 1440.7 Da, was in close agreement with that derived by mass spectroscopy. The full primary structure of frog NT was established as: QSHISKARRPYIL. When compared with bovine NT, frog NT exhibits five amino acid substitutions in the N-terminal region, whereas the C-terminal hexapeptide sequence (RRPYIL), which mediates the classical biological effects of NT, is completely conserved. Amphibia thus possess a tridecapeptide NT which is analogous to that of higher vertebrates and considerable constraints on the primary structure of the C-terminal biologically-active core have existed for a vast evolutionary time span.     

Brain neurite-stimulating protein

A protein possessing the neurite-stimulating activity in organotypic cultures of chicken embryo spinal ganglia was isolated and purified from bovine brain tissue. The isolation and purification procedures included acid extraction, ultrafiltration, preparative polyacrylamide gel electrophoresis and chromatography on heparin-Sepharose. The molecular weight of the protein is about 15000 Da. The neurite-stimulating activity of the purified protein manifests itself at a protein concentration of about 10(-9) M.     

Molecular cloning of the cDNA for stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like small GTP-binding proteins) and characterization of stimulatory GDP/GTP exchange protein.

We have recently purified to near homogeneity the stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like GTP-binding proteins) from bovine brain cytosol. This regulatory protein, named GDP dissociation stimulator (GDS), stimulates the GDP/GTP exchange reaction of smg p21s by stimulating the dissociation of GDP from and the subsequent binding of GTP to them. In this study, we have isolated and sequenced the cDNA of smg p21 GDS from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of the purified smg p21 GDS. The cDNA has an open reading frame encoding a protein of 558 amino acids with a calculated Mr value of 61,066, similar to the Mr of 53,000 estimated for the purified smg p21 GDS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits smg p21 GDS activity. smg p21 GDS is overall hydrophilic, but there are several short hydrophobic regions. The smg p21 GDS mRNA is present in bovine brain and various rat tissues. smg p21 GDS has low amino acid sequence homology with the yeast CDC25 and SCD25 proteins, which may regulate the GDP/GTP exchange reaction of the yeast RAS2 protein, but not with ras p21 GTPase-activating protein, the inhibitory GDP/GTP exchange proteins (GDP dissociation inhibitor) for smg p25A and rho p21s, and the beta gamma subunits of heterotrimeric GTP-binding proteins such as Gs and Gi.     

Tissue distribution, purification and characterization of rat phosphatidylinositol transfer protein

Phosphatidylinositol transfer activity is measured in cytosol fractions prepared from 13 rat tissues; specific activity is highest in brain and lowest in adipose and skeletal muscle. Based upon electrophoretic analysis phosphatidylinositol transfer protein is purified to homogeneity from whole rat brain. The protein has a molecular weight of 36,000 and exists as a mixture of species having isoelectric points of 4.9 and 5.3. In a vesicle-vesicle assay system, the intermembrane transfer rate is greatest for phosphatidylinositol and less by a factor of 2 for phosphatidylcholine; transfer of phosphatidylethanolamine, phosphatidylserine or sphingomyelin is not observed. Using a polyclonal rabbit antibody against bovine phosphatidylinositol transfer protein, immunologic cross-reactivity is noted between the rat protein and other mammalian phosphatidylinositol transfer proteins. A strong correlation is established between a tissue's capacity for phosphatidylinositol transfer and the amount of immunoreactive transfer protein seen in that tissue. Purified phosphatidylinositol transfer protein is capable of transporting newly synthesized phosphatidylinositol molecules from rat brain microsomes to small unilamellar phospholipid vesicles. The results are discussed within the context of cellular phosphoinositide metabolism and the maintenance of the metabolically responsive pool of phosphatidylinositol in the plasma membrane.     

Purification and characterization of the 27,000 Da calcium-binding protein of bovine brain.

A Ca2+-binding protein named CAB-27 was purified from bovine brain 100,000 g supernatant. The protein has a molecular mass of 27,000 Da as determined by SDS/polyacrylamide-gel electrophoresis and 35,500 Da by sedimentation-coefficient and Stokes-radius analysis. The protein contains about 26% Glx and Asx and 13% basic residues. The acidic nature of the molecule is confirmed by its pI of 4.80. In the presence of 3 mM-MgCl2 and 150 mM-KCl, CAB-27 binds 2.0 mol of Ca2+/mol of protein, with an apparent Kd of 0.2 microM. Ca2+-binding is unaffected by prior incubation of the protein at 80 degrees C for 2 min. Brain contains about 130 mg of CAB-27/kg. Immunoblotting identified CAB-27 in several bovine tissues; it appears to be particularly rich in brain and kidney. In addition, CAB-27 is identified as an inhibitor of bovine pancreas phospholipase A2 in vitro. The inhibitory activity of CAB-27 was 20-fold less potent than lipocortin. On the basis of the Ca2+-binding properties, intracellular concentration and tissue distribution of this protein, we suggest that CAB-27 may be an important intracellular Ca2+ receptor.     

Purification and characterization of the major whey proteins from the milks of the bottlenose dolphin (Tursiops truncatus), the Florida manatee (Trichechus manatus latirostris), and the beagle (Canis familiaris)

The major whey proteins of the milks of the dolphin, manatee, and beagle were purified by gel filtration and ion exchange chromatography and characterized and identified by molecular weight determination, amino acid analysis, N-terminal sequencing, and activity measurements. The major whey protein components from all three species were found to be monomeric beta-lactoglobulins. These proteins were all active in binding retinol. Dolphin milk contained two beta-lactoglobulins (designated 1 and 2) which showed a slight difference in molecular weight and considerably divergent N-terminal sequences, whereas the other milks only contained a single form of beta-lactoglobulin. alpha-Lactalbumins were purified from dolphin and dog milks and were active in promoting lactose synthesis by bovine galactosyltransferase. The dolphin protein had an N-terminal sequence more similar to ruminant alpha-lactalbumins than to those known from other species. Although alpha-lactalbumin activity has been detected in manatee milk at low levels, the corresponding protein was not isolated. In addition, dog milk was found to contain high levels of lysozyme (greater than 1.0 mg/ml), which were identified by activity and sequencing. The functional and evolutionary implications of these results are discussed.