Heat-stable Protein Research Articles

Purification and Identification of a 28-kDa Calcium-regulated Heat-stable Protein: A NOVEL SECRETAGOGUE-REGULATED PHOSPHOPROTEIN IN EXOCRINE PANCREAS

This study reports the purification and identification of a novel 28 kDa phosphoprotein from rat pancreatic acini, previously described as being highly regulated by calcium mobilizing secretagogues, which we have designated calcium-regulated heat-stable protein 28 (CRHSP-28). Internal amino acid sequences of purified CRHSP-28 were obtained following trypsin digestion and found to match with >95% identity the predicted amino acid sequence of a novel cDNA recently identified as being highly expressed in human breast carcinomas. Verification that this cDNA codes for human CRHSP-28 was demonstrated by the ability of antiserum raised against purified rat CRHSP-28 to recognize the recombinant human protein when expressed in bacteria. Furthermore, this antibody was found to specifically react with CRHSP-28 in rat acini following one- and two-dimensional electrophoresis and underwent a marked acidic shift in mobility after cholecystokinin stimulation, a phenomenon indicative of an increase in its phosphorylation. CRHSP-28 is predicted to be extremely hydrophilic, is phosphorylated entirely on serine residues, and bears little homology to any known proteins. Finally, the distribution of the CRHSP-28 protein in various rat tissues revealed that although it was present at low levels in almost all tissues, it was most highly expressed in pancreas, followed by the gastric, intestinal, and colonic mucosa. In view of its relative abundance throughout the digestive system and its apparent regulation by calcium-mobilizing agents, this protein may provide valuable insight into the mechanism(s) of calcium signaling in these tissues.

Cloning and sequencing of a cDNA encoding a heat-stable sweet protein, mabinlin II

A cDNA clone encoding a heat-stable sweet protein, mabinlin II (MAB), was isolated and sequenced. The encoded precursor to MAB was composed of 155 amino acid (aa) residues, including a signal sequence of 20 aa, an N-terminal extension peptide of 15 aa, a linker peptide of 14 aa and one residue of C-terminal extension. Comparison of the proteolytic cleavage sites during posttranslational processing of MAB precursor with those of like 2S seed-storage proteins of Arabidopsis thaliana, Brassica napus and Bertholletia excelsa shows that the three individual cleavage sites between respective species are conserved.

Identification of the first major allergen of a squid (Todarodes pacificus)

Background: In Japan, squid is an important seafood, and some patients with food allergies are sensitive to squid. There has been no report, however, describing the major allergens of squid. Objective: To characterize squid allergens, we isolated a major allergen from the Pacific flying squid (Todarodes pacificus) and compared it with a major allergen from a shrimp (Penaeus orientalis). Methods: The major squid and shrimp allergens were isolated by column chromatography on diethylaminoethyl-Sepharose (Pharmacia, Uppsala, Sweden), hydroxylapatite, and Sephacryl S-300 (Pharmacia). The IgE reactivity of the isolated allergens was assessed by immunoblotting. The cross-reactivity between the squid and shrimp allergens was examined by use of mouse polyclonal and monoclonal antibodies to the major allergens. Amino acid sequence analyses of the isolated allergens were done. Results: The isolated squid allergen is a 38 kd, heat-stable protein. IgE antibody binding to the purified squid allergen was demonstrated by immunoblotting. Cross-reactivity between major squid and shrimp allergens was demonstrated with sera from patients allergic to squid or shrimp or with allergen-specific monoclonal antibodies. The amino acid sequence analysis of the major squid allergen showed a marked homology with tropomyosin from blood fluke planorbid (Biomphalaria glabrata), which is a common vector snail of Schistosoma mansoni. Conclusion: This 38 kd protein is a major allergen of the squid, Todarodes pacificus, and is believed to be squid muscle protein tropomyosin. We named it Tod p 1 according to International Union of Immunological Societies allergen nomenclature regulation. (J A LLERGY C LIN I MMUNOL 1996;98:948-53.)

Cyclic 3,5 adenoise monophosphate and cyclosporin A inhibit cellular proliferation and serine/threonine protein phosphatase activity in pituitary cells.

cAMP and cyclosporin A exert antiproliferative effects in many different cell types. In cultured pituitary cells, the antiproliferative effects of both agents correlate with the inhibition of a serine/threonine protein phosphatase activity. This inhibition is mediated by the heat-stable protein, inhibitor-1. The increase of cAMP levels, through the activation of the protein kinase A, induces inhibitor-1 phosphorylation and activation. On the other hand, cyclosporin A, inhibiting the calcium-dependent serine/threonine phosphatase calcineurin, prevents the dephosphorylation and inactivation of inhibitor-1. This dual regulation by cAMP and calcium on the inhibitor-1 activity parallel the effects of these agents on DNA synthesis and serine/threonine phosphatase activity. Because inhibitor-1 is the main regulator of protein phosphatase 1 activity, these results suggest that protein phosphatase 1 may be the common target of cAMP and cyclosporin A in regulating cell proliferation. We propose that protein-phosphatase 1 stimulates growth in these cells and that cAMP and cyclosporin A block this effect through their actions on inhibitor-1.

An abscisic acid analog inhibits abscisic acid-induced freezing tolerance and protein accumulation, but not abscisic acid-induced sucrose uptake in a bromegrass (Bromus inermis Leyss) cell culture

The application of abscisic acid (ABA), either as a racemic mixture or as optically resolved isomers, increases freezing tolerance in a bromegrass (Bromus inermis Leyss) cell culture and induces the accumulation of several heat-stable proteins. Two stereoisomers of an ABA analog, 2′3′ dihydroacetylenic abscisyl alcohol (DHA), were used to study the role of ABA-induced processes in the acquisition of freezing tolerance in these cells. Freezing tolerance was unchanged in the presence of (−) DHA (LT50 -9°C), and no increase in heat-stable protein accumulation was detected; however, the (+) enantiomer increased the freezing tolerance (LT50 -13°C) and induced the accumulation of these polypeptides. All three forms of ABA increased freezing tolerance in the bromegrass cells, although (−) ABA was less effective than either (+) or (±) ABA when added at equal concentrations. Cells pretreated with 20 or 50 μM (−) DHA displayed lower levels of freezing tolerance following the addition of 2.5, 7.5 or 25 μM (±) ABA. Full freezing tolerance could be restored by increasing the concentration of (±) ABA to > 25 μM. Pretreatment of cells with (−) DHA (20 or 50 μM) had no effect on freezing tolerance when 25 μM (+) ABA was added. The induction of freezing tolerance by 25 μM (−) ABA was completely inhibited by the presence of 20 μM (−) DHA. The accumulation of ABA-responsive heat-stable proteins was inhibited by pretreatment with 20 μM (−) DHA in cells treated with 2.5 or 7.5μM (+) ABA, and in cells treated with 25 μM (−) ABA. The accumulation of these polypeptides was restored when (±) or (+) ABA was added at a concentration of 25 μM. The analysis of proteins which cross-reacted with a dehydrin antibody revealed a similar inhibitory pattern as seen with the other ABA-responsive proteins. The effects of the various isomers of ABA and DHA on cell osmolarity and sucrose uptake was also investigated. In both cases, (±) and (+) ABA had pronounced effects on the parameters measured, whereas (−) ABA treated cells gave substantially different results. In both sucrose uptake and cell osmolarity, DHA had no significant effect on the results obtained following (±) or (+) ABA treatment. Maximum freezing tolerance was only observed in cells when both heat-stable protein accumulation and sucrose uptake were observed.

Import of RNA into Leishmania mitochondria occurs through direct interaction with membrane-bound receptors.

Cytoplasmic tRNAs are imported into the kinetoplast mitochondrion of Leishmania, but the mechanism of import is unknown, particularly whether RNA is transferred as a ribonucleoprotein complex through the protein import pathway or by a distinct receptor-mediated mechanism. Using isolated mitochondria, it was shown that a small, importable RNA, which is structurally homologous to tRNA, binds rapidly, specifically, and with high affinity to the mitochondrial surface in the absence of soluble protein factors to form an import intermediate. Two classes of binding site of apparent Kd 0.3 and 10 n, respectively, were distinguished. tRNA from Leishmania, but not yeast, competitively inhibited the binding. Northwestern blot analysis revealed the presence of a 15-kDa RNA binding protein on the mitochondrial surface. Whereas receptor binding was resistant to heparin and KCl, internalization was sensitive to both reagents. These results are consistent with the presence of a direct mechanism of receptor-mediated RNA import on Leishmania mitochondria.

Strategy for large scale solubilization of coal — Characterization of Neurospora protein and gene

Low grade coal placed on mycelial mat of Neurospora crassa growing on Petri plate was found to be solubilized by this fungus. A heat stable protein has been purified to near homogeneity which can solubilize low grade coal in in vitro. The biochemical properties of the Neurospora protein will be presented. The nature of the product obtained after solubilization of coal by Neurospora protein in vivo and in vitro will also be presented. The N-terminus sequence of the amino acids of this protein will be used to design primer for possible cloning of gene for Neurospora protein capable of solubilization of coal in order to develop methodology for coal solubilization on a large scale.

Some effect of CuSO4on carp

Copper treatment, muscle and liver accumulate a great amount of copper ions. The higher concentration of copper ions in the organism caused an elevated level of metallothionein. Copper induced the synthesis of metallothionein, mostly in the livers. In gel chromatography heat stable low molecular weight protein certain coming from the destruction of high molecular weight proteins could be observed. On the effect of higher copper ion concentration in the liver, the level of the metallothionein bound to copper (as related to total protein bounded to copper) was decreased. This phenomenon shows the weaker affinity of apothionein to copper, compared to other proteins. Follows copper sulphate treatment, every measured blood serum parameter was changed GOT activity rose to 130% and the GPT to 300% of control. LDH activity increased, too According to this, the anaerobic way became preferred in glucose metabolism. On the effect of copper sulphate stress the blood serum glucose concentration was higher than the control value.

The Myeloid Leukemia-associated Protein SET Is a Potent Inhibitor of Protein Phosphatase 2A

Two potent heat-stable protein phosphatase 2A (PP2A) inhibitor proteins designated I1PP2A and I2PP2A have been purified to apparent homogeneity from extracts of bovine kidney (Li, M., Guo, H., and Damuni, Z. (1995) Biochemistry 34, 1988-1996). N-terminal and internal amino acid sequencing indicated that I2PP2A was a truncated form of SET, a largely nuclear protein that is fused to nucleoporin Nup214 in acute non-lymphocytic myeloid leukemia. Experiments using purified preparations of recombinant human SET confirmed that this protein inhibited PP2A. Half-maximal inhibition of the phosphatase occurred at about 2 nM SET. By contrast, SET (up to 20 nM) did not affect the activities of purified preparations of protein phosphatases 1, 2B, and 2C. The results indicate that SET is a potent and specific inhibitor of PP2A and suggest that impaired regulation of PP2A may contribute to acute myeloid leukemogenesis.

Binding of endogenous copper and zinc to cadmium-induced metal-binding proteins in various tissues of Perna viridis

Green lipped mussels, Perna viridis, were exposed to cadmium chloride (CdCl2; 0.52 and 1 μg/ml) in water for 4 days. The concentrations of cadmium (Cd), copper (Cu) and zinc (Zn) were measured in the viscera, gill, gonads, mantle, and muscle. There was a significant increase (p<0.05) in Cd concentration in all tissues studied. Results from Sephadex G-75 chromatography indicated that most Cd was bound to a fraction of heat-stable proteins similar to the metal-binding protein (MBP) metallothionein. After exposure to Cd, there was no significant change in Cu concentration in total tissue proteins or in total cytosolic proteins. A significant increase (p<0.05) in Cu, however, was detected in heat-stable proteins bound to the Cd-induced MBP in both viscera and gill. Copper bound to MBP also occurred in the gonad, mantle, and muscle, but to a much lesser extent. These results showed that Cd-induced MBP can also bind endogenous Cu. Zinc concentration in total heat-stable protein was increased only in gill and the muscle. Unlike Cu, a small amount of Zn binding to Cd-induced MBP was detected only in these tissues. Considering that both Cu and Zn exist intracellularly in dynamic equilibrium, the binding of Cu, but not Zn, to MBP may be explained by the kinetic reactivity of the two different metals to protein. The results of this study support the thesis that induction of intracellular MBP may also bind endogenous Cu and Zn.

Identification of a Protein That Interacts with Tubulin Dimers and Increases the Catastrophe Rate of Microtubules

Using a polymerization inhibition assay, we have purified a small, heat stable protein that physically interacts with tubulin dimers and increases the catastrophe rate of microtubules. Sequence analysis identified this protein as oncoprotein 18 (Op18)/stathmin, a conserved phosphoprotein that is highly expressed in leukemia cells. Immunodepletion experiments in Xenopus egg extracts showed that Op18/stathmin is involved in physiological regulation of mitotic microtubule dynamics. Op18/stathmin is a microtubule regulator that preferentially interacts with unpolymerized subunits. It is a candidate for increasing the microtubule catastrophe rate in mitosis and might also regulate microtubule dynamics in response to external signals.

Demonstration of a heat-stable 120-kilodalton protein of Rickettsia japonica as a spotted fever group-common antigen.

Genomic libraries of Rickettsia japonica were cloned into an expression vector lambda gt11. A clone expressing a protein reactive with antiserum against 120-kilodalton (kDa) proteins, a mixture of heat-modifiable and heat-stable polypeptides, was selected and designated as lambda Rj120-1. The expressed protein has a molecular mass of 180 kDa. Western immunoblotting demonstrated that the expressed protein was a fusion protein with beta-galactosidase. The antiserum against 120-kDa proteins was absorbed by the induced lysogen, resulting in the removal of reactivity to the heat-stable 120-kDa polypeptide. The antiserum against the expressed protein reacted with heat-stable 120- to 130-kDa polypeptides of spotted fever group (SFG) rickettsiae in addition to R. japonica. The findings indicated that the protein expressed from the cloned gene of R. japonica possessed the antigenicity group-common to SFG rickettsiae. Primers designed from the gene coding for R. conorii heat-stable 120-kDa protein (Schuenke, K.W., and Walker, D.H., Infect. Immun. 62: 904-909, 1994) and lambda gt11 lacZ gene amplified the lambda Rj120-1 DNA by the polymerase chain reaction (PCR). Analysis of restriction fragment length polymorphism (RFLP) of the PCR-amplified products revealed that the cloned DNA corresponds to a portion of the gene coding for the heat-stable 120-kDa protein of R. conorii with 2,519 nucleotides beginning at nucleotide 190 of the open reading frame. RFLP demonstrated that the cloned gene was highly homologous to the corresponding gene of R. conorii.

Biological properties of the colony-promoting activity in extracts prepared from murine kidney

Aqueous extracts prepared from the murine kidney (MKE) promoted colony formation derived from murine hematopoietic progenitor cells in serum-free cultures stimulated by interleukin-3 (IL-3) and erythropoietin (Epo). MKE itself did not stimulate any colony formation. MKE preferentially enhanced granulocyte-macrophage colony forming units (CFU-GM), but did not promote any erythroid colony formation. The CFU-GM colony promotion by MKE was observed at day 6 after the culture started, and the colony-promoting activity (CPA) was maintained at the same level until day 16. MKE showed no CPA in the cultures using cells obtained from 5-FU-injected mice and from c-kit(+)-enriched treatment. Furthermore, MKE acted synergistically with granulocyte-colony-stimulating factor (CSF), macrophage-CSF, IL-6 and IL-11 on colony formation, but did not act with GM-CSF, stem cell factor and Epo. From the results of various experiments and gel-filtration chromatography, it is estimated that the colony-promoting factor detected in MKE is a heat stable protein with about 20 KDa molecular weight. These results suggest that MKE promotes colony formation by murine myeloid progenitor cells, and that the target cell populations of MKE are relatively mature in the hematopoietic differentiation pathway.

Molecular identification of I1PP2A, a novel potent heat-stable inhibitor protein of protein phosphatase 2A.

The amino acid sequences of two tryptic peptides derived from purified preparations of I1PP2A indicated that this potent heat-stable protein inhibitor of protein phosphatase 2A (PP2A) may be equivalent to putative histocompatibility leukocyte antigens class II-associated protein I (PHAP-I). Experiments using purified preparations of recombinant human PHAP-I confirmed that this protein inhibited PP2A. Half-maximal inhibition of the phosphatase occurred at about 4 nM PHAP-I, similar to the half-maximal inhibition obtained with purified preparations of bovine kidney I1PP2A. In addition, PHAP-I did not affect the activities of protein phosphatase 1, 2B, and 2C in a manner analogous to that of I1PP2A. Together, the results establish the identity of I1PP2A on a firm basis.

A cGMP kinase mutant with increased sensitivity to the protein kinase inhibitor peptide PKI(5-24).

Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent protein kinase. In this study, we tried to confer PKI sensitivity to cGMP kinase by site-directed mutagenesis. The molecular requirements for high affinity inhibition by PKI were deduced from the crystal structure of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr235 and Phe239 in the catalytic subunit, which from a sandwich-like structure with Phe10 of the PKI(5-24) peptide. To increase the sensitivity for PKI, the cGMP kinase codons at the corresponding sites, Ser555 and Ser559, were changed to Tyr and Phe. The mutant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM). Wild type and mutant cGMP kinase did not differ significantly in their Km and Vmax for three different substrate peptides. The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher potency than that of wild type, with Ki values of 42 +/- .3 microM and 160 +/- .7 microM, respectively. The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide. Mutation of the essential Phe10 in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with Ki values of 160 +/- 11 and 169 +/- 27 microM, respectively. These results suggest that the mutations Ser555Tyr and Ser559Phe are required, but not sufficient, for high affinity inhibition of cGMP kinase by PKI.

Effects of Mercury, Chromium and Nickel on Glycogen Reserves and Protein Levels in Tissues of Cyprinus carpio

The carps Cyprinus carpio were exposed to sublethal concentrations of mercury (0.01 and 0.1 mg/1), chromium (2 and 20 mg/1) and nickel (2 and 20 mg/1) for seven days. Following the metal exposures, total and heat-stable protein levels in the liver, muscle and gill and glycogen contents in the liver and muscle of fish were measured. Results were statistically compared with a control group which was kept in the same conditions without any metal addition. Glycogen levels in the tissues of all metal-exposed carps significantly decreased. When compared to the control levels, mercury caused highest depletions of glycogen in the tissues (up to 96 %), and was followed by nickel (up to 86 %) and chromium (up to 75 %). Total protein contents in the tissues of metal-exposed animals decreased significantly over control values, except in the liver and gill of chromium exposed animals. In the heat-treated homogenates, percent protein loss differed between control and metal-exposed fish. Highest protein loss after the heat-treatment occurred in chromium exposed fish (up to 84 %), and it was followed by nickel (up to 43 %), mercury (up to 41 %) and control (up to 33 %) fish. Except in the chromium experiment, highest percentages of protein loss were found in the muscle, and were followed by the liver and gill.

Identification of a Rapidly Dephosphorylating 95-kDa Protein as Elongation Factor 2 during 8-Br-cAMP Treatment of N1E115 Neuroblastoma Cells

Treatment of 8-Br-cAMP promotes neurite outgrowth and neuronal differentiation in N1E115 mouse neuroblastoma cells. Prior or simultaneous treatment of PMA blocks 8-Br-cAMP-mediated neurite outgrowth. Phosphorylation of cellular proteins during these treatments was examined in a permeabilized cell system. While PMA promotes phosphorylation of the heat-stable protein kinase C substrates MARCKS and neuromodulin, 8-Br-cAMP hastens the dephosphorylation of a protein of Mr 95k (p95). Extensively purified, N-terminal sequenced, and judged from its phosphorylation properties, p95 was identified as the eukaryotic elongation factor-2 (eEF-2), whose dephosphorylation has been reported to be related to an increase in protein synthesis. It is likely 8-Br-cAMP stimulates dephosphorylation of eEf-2, promotes protein synthesis that eventually leads to neuronal differentiation in N1E115 cells.

The multifunctional calcium/calmodulin-dependent protein kinase: from form to function.

The regulation of cellular function by hormones and extracellular molecules, which bind to cell surface receptors, is initiated by signal transduction mech­ anisms at the plasma membrane that lead to the generation of intracellular signals, or second messengers. These second messengers interact with specific target molecules to initiate a cascade of biochemical events leading to a change in cellular function. A variety of intracellular second messengers, including divalent metals, phospholipid metabolites, and cyclic nucleotides, are known to be generated in response to extracellular stimuli. Calcium acts as an intra­ cellular second messenger in species as diverse as yeast to humans and is involved in cellular processes ranging from contraction to secretion to gene expression. Elevation of cytosolic calcium can occur by influx via regulated ion channels and transporters, or by release from intracellular stores via recep­ tor-generated second messengers (Figure 1). Cells contain a number of intra­ cellular calcium-binding proteins, and in most cell types, the major calcium­ binding protein is calmodulin (44, 91), an -17 kDa, heat-stable protein that binds four calcium ions with an overall high affinity of -1 11 M. This complex of 4(Ca)-calmodulin activates downstream targets. Although the importance of calcium as an intracellular messenger has been long recognized, identifica-

Cloning and structural analysis of the gene for cAMP-dependent protein kinase catalytic subunit from Plasmodium yoelii

We isolated the gene encoding the cAMP-dependent protein kinase catalytic subunit (cAPK[C]) from Plasmodium yoelii by screening a genomic library for the DNA fragment as produced by the polymerase chain reaction. The deduced protein of 341 amino acids conserves residues that are important for the function of serine/threonine protein kinases and shows the highest homology to cAPK[C]s of other organisms. However, P. yoelii cAPK[C] has 8 residues, which are perfectly conserved in cAPK[C]s of other organisms, radically replaced with residues having different side-chain properties. It is stressed that two radical replacements occur in regions for the binding with a regulatory subunit and/or a heat-stable inhibitor protein.

A novel heart derived inhibitor of vascular cell proliferation. Purification and biological activity

Recently, growth factors with mitogenic properties for vascular wall cells have been isolated from adult heart tissue. Since angiogenesis in the heart typically does not occur under normal physiological conditions, despite the presence of many growth factors, we hypothesized the existence of growth inhibitors. To test this hypothesis, we subjected whole bovine heart extracts to a series of protein purification steps in search of such an inhibitor. The purification procedure consisted of ammonium sulfate precipitation followed by cation exchange chromatography, hydroxylapatite chromatography, ultrafiltration and gelfiltration. We isolated a small protein, which is an inhibitor of cell proliferation from the bovine heart. The inhibitor reversibly suppressed [3H]-thymidine incorporation into nuclei of bovine aortic endothelial and smooth muscle cells. The moiety responsible for the inhibitory activity was identified biochemically (SDS Page, isoelectric focusing, HPEC) as an 11 kD protein with an isoelectric point of 7. The substance is a heat and acetic acid stable protein which does not bind to reversed phase columns because of its hydrophilic character. The inhibitor has no affinity to heparin sepharose. The inhibitory activity was destroyed by hydrolysis. No homology to any hitherto structurally investigated growth inhibitor was observed using the chemical determination of the amino acid sequence by microsequencing after previous trypsin digestion. We conclude that the described growth inhibitor may counteract the activity of mitogens that are abundantly present in normal heart. Vascular cell proliferation may be regulated by inhibition or production of the inhibitor.