cDNA cloning and nucleotide sequence of lipocortin-like 33 kDa protein in guinea pig neutrophils

cDNA clones of guinea pig neutrophil 33 kDa protein, a lipocortin like-protein, were isolated from two lambda gt10 libraries and one primer-extended lambda gt10 library of guinea pig neutrophils using synthetic oligonucleotide probes or cDNA fragment probe. The cDNA consists of 1389 nucleotides, and contains 1038 nucleotides encoding 346 amino acids of 33 kDa protein and a poly(A) tail. The deduced amino acid sequence shows high homology with those of lipocortin 1 from human U937 cells (89% homology) and rat lung (86%).     

Translocation of a small cytosolic calcium-binding protein (MRP-8) to plasma membrane correlates with human neutrophil activation.

To further understand the mechanisms involved in phagocyte activation in general and in NADPH oxidase activation in particular, a polyclonal antibody was raised in rabbit against a partially purified oxidase preparation. The enzyme was solubilized from zymosan-activated human neutrophils and resting cells and separated by preparative isoelectric focusing electrophoresis. A polyclonal antibody was raised in rabbit against the pI 5.0 fraction, which had the maximum superoxide-producing capacity. Analysis of the polyclonal antibody revealed marked differences between activated and resting neutrophils. The antibody recognized in particular an 8-kDa protein (p8) in resting human neutrophil cytosol and in the membrane of zymosan-activated cells. A polyclonal antibody (anti-p8) was raised against the pure cytosolic p8 protein. This anti-p8 reacted not only with p8, but also with cytosolic proteins of 14 kDa and 6 kDa. N-terminal amino acid sequence analysis of p8 revealed homology with the calcium-binding myeloid related protein (MRP-8). Upon neutrophil activation, translocation of the 8- and 14-kDa proteins to the membrane was observed with stimuli known to depend on extracellular calcium. In calcium-depleted medium, the absence of translocation correlated with a depression of superoxide production, supporting a role for the calcium-binding protein in cellular activation.     

The structure and function of the 33 kDa extrinsic protein of Photosystem II: A critical assessment

In this review the structure and function of the 33 kDa protein of Photosystem II is examined. Significant controversies exist concerning the solution secondary structure of the protein, the location of its binding site(s) within Photosystem II, the amino acid residues of the 33 kDa protein required for binding and its stoichiometry within the photosystem. The studies which examine these topics are considered from a critical perspective. A hypothetical model of the folding of the 33 kDa extrinsic protein which is supported by site-specific labeling studies and site-directed mutagenesis experiments is presented. Additionally, the function of the protein within the photosystem is unclear. We present a hypothesis that the 33 kDa protein is involved in maintaining the chloride associated with photosynthetic oxygen evolution in close proximity to the oxygen-evolving site.     

The extrinsic 33 kDa polypeptide of the oxygen-evolving complex of photosystem II is a putative calcium-binding protein and is encoded by a multi-gene family in pea

The extrinsic 33 kDa polypeptide of the water-oxidizing complex has been extracted from pea photosystem II particles by washing with alkaline-Tris and purified by ion-exchange chromatography. The N-terminal amino acid sequence has been determined, and specific antisera have been raised in rabbits and used to screen a pea leaf cDNA library in gt11. Determination of the nucleotide sequence of positive clones revealed an essentially full-length cDNA for the 33 kDa polypeptide, the deduced amino acid sequence showing it to code for a mature protein of 248 amino acids with an N-terminal transit peptide of 81 amino acids. The protein showed a high degree of conservation with previously reported sequences for the 33 kDa protein from other species and the sequence contained a putative Ca²⁺-binding site with homology to mammalian intestinal calcium-binding proteins. Northern analysis of total pea RNA indicated a message of approximately 1.4 kb, in good agreement with the size of the cDNA obtained at 1.3 kbp. Southern blots of genomic DNA probed with the labelled cDNA give rise to several bands suggesting that the 33 kDa polypeptide is coded by a multi-gene family.Abbreviations ATZ - anilinothiazolinone - DITC - p-phenylenediisothiocyanate - PTH - phenylthiohydantoin - TFA - trifluoroacetic acid - Tris - tris (hydroxymethyl) aminomethane - bis-Tris - bis (2-hydroxyethyl) imino-tris (hydroxymethyl)-methane - p.f.u. - plaque-forming units     

Assocation of the 33 kDa extrinsic polypeptide (water-splitting) with PS II particles: immunochemical quantification of residual polypeptide after membrane extraction

Various washing procedures were tested on Triton-prepared PS II particles for their ability to remove the 33 kDa extrinsic polypeptide (33 kDa EP) associated with the water-splitting complex. Residual 33 kDa EP was evaluated by Coomassie blue staining of SDS gels of washed particles and by Western blotting with an antibody specific for the 33 kDa EP. A wash with 16 mM Tris buffer, pH 8.3, inhibited water-splitting activity but did not remove all the 33 kDa EP. Sequential washes with 30 mM octyl glucoside (pH 8.0 and 6.8), and a single wash with 0.8 M Tris were also ineffective in removing all the 33 kDa EP. Washing with 1 M CaCl₂ was more effective in removing 33 kDa EP; while only a faint trace of protein was detectable by Coomassie-staining, immunoblotting revealed a considerable remainder. The treated particles retained some water-splitting activity. The two step procedure of Miyao and Murata (1984) involving 1 M NaCl and 2.3 M urea was most effective, removing all but a trace of antibody positive protein. Our finding suggests that (1) the degree of depletion of the 33 kDa EP cannot be judged on the basis of Coomassie stain alone, and (2) this extrinsic protein is very tightly associated with the membrane, perhaps via a hydrophilic portion of this otherwise hydrophilic protein. The results also suggest that the presence or absence of the 33 kDa protein per se is not the primary determinant of residual water splitting activity.Abbreviations Chl chlorophyll - DCPIP dichlorophenolindophenol - DPC diphenolcarbazide - DTT dithiothreitol - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2(N-morpholino)ethanesulfonic acid - SDS sodium dodecyl sulfate - Tris Tris(hydroxymethyl)aminomethane     

The status of cysteine residues in the extrinsic 33 kDa protein of spinach photosystem II complexes

Two cysteine residues of the extrinsic 33 kDa protein in the oxygen-evolving photosystemII (PS II) complexes were found to exist as cystine residues in situ. The 33 kDa protein, when reduced by 2-mercaptoethanol in either the presence or the absence of 6 M guanidine-HCl (Gdn-HCl), could not rebind with the CaCl₂-treated PS II complexes, from which the 33 kDa protein was removed, and evolve any oxygen. Two sulfhydryl (SH) groups of the 33 kDa protein were easily reoxidized to a disulfide (S-S) bond by stirring under aerobic conditions with the concomitant regaining of both the binding ability to the CaCl₂-treated PS II complexes and the oxygen-evolving activity.The molecular conformation of the 33 kDa protein was examined by circular dichroic (CD) spectrometry in the UV regions to reveal that the conformation in the reduced state was completely different from those of the untreated and reoxidized states. The disulfide (S-S) bond of the 33 kDa protein is thus essential to maintain the molecular conformation required to function.Abbreviations CD circular dichroism - Chl chlorophyll - DMQ 2,5-dimethyl-p-benzoquinone - DTNB 5,5-dithio-bis (2-nitrobenzoic acid) - EDTA ethylendiamine-tetraacetic acid - Gdn-HCl guanidine-hydrochloric acid - PS II photosystem II - SDS sodium dodecylsulfate This paper was presented at the U.S.-Japan Binational Seminar on Solar Energy Conversion, Okazaki, Japan, March 17–21, 1987     

Identification of domains on the extrinsic 23 kDa protein possibly involved in electrostatic interaction with the extrinsic 33 kDa protein in spinach photosystem II.

To elucidate the domains on the extrinsic 23 kDa protein involved in electrostatic interaction with the extrinsic 33 kDa protein in spinach photosystem II, we modified amino or carboxyl groups of the 23 kDa protein to uncharged methyl ester groups with N-succinimidyl propionate or glycine methyl ester in the presence of a water-soluble carbodiimide, respectively. The N-succinimidyl propionate-modified 23 kDa protein did not bind to the 33 kDa protein associated with PSII membranes, whereas the glycine methyl ester-modified 23 kDa protein completely bound. This indicates that positive charges on the 23 kDa protein are important for electrostatic interaction with the 33 kDa protein associated with the PSII membranes. Mapping of the N-succinimidyl propionate-modified sites of the 23 kDa protein was performed using Staphylococcus V8 protease digestion of the modified protein followed by determination of the mass of the resultant peptide fragments with MALDI-TOF MS. The results showed that six domains (Lys11-Lys14, Lys27-Lys38, Lys40, Lys90-Lys96, Lys143-Lys152, Lys166-Lys174) were modified with N-succinimidyl propionate. In these domains, Lys11, Lys13, Lys33, Lys38, Lys143, Lys166, Lys170 and Lys174 were wholly conserved in the 23 kDa protein from 12 species of higher plants. These positively charged lysyl residues on the 23 kDa protein may be involved in electrostatic interactions with the negatively charged carboxyl groups on the 33 kDa protein, the latter has been suggested to be important for the 23 kDa binding [Bricker, T.M. & Frankel, L.K. (2003) Biochemistry42, 2056-2061].     

Purification and characterization of a lipocortin-like 33 kDa protein from guinea pig neutrophils

A lipocortin-like, phospholipase A2 inhibitory 33 kDa protein was purified from guinea pig neutrophils. From amino acid composition and sequence data, this protein was found to have a high degree of homology to human lipocortin I. This protein inhibited porcine pancreatic phospholipase A2 activity in the presence of [3H]oleic acid-labeled Escherichia coli membranes as substrate. Maximal inhibition amounted to 65% whereas 50% inhibition occurred at 83.5 nM. This protein showed F-actin-binding ability in a Ca2+-dependent manner.     

Properties of a peripheral 34 kDa protein in Synechococcus vulcanus Photosystem II particles. Its exchangeability with spinach 33 kDa protein in reconstitution of O2 evolution

Photosystem II (PS II) particles retaining a high rate of O₂ evolution were prepared from a thermophilic cyanobacterium, Synechococcus vulcanus Copeland, and the composition and properties of their peripheral proteins were investigated. The following results were obtained. (1) The O₂-evolving PS II particles of S. vulcanus contained only one peripheral protein with a molecular mass of 34000 which corresponded to the 33 kDa protein in higher plant PS II particles, but no other peripheral proteins corresponding to the 24 and 16 kDa proteins of higher plant PS II particles. (2) The cyanobacterial peripheral 34 kDa protein was removed from the particles by 1 M CaCl₂-washing concomitant with total inactivation of O₂ evolution, and the inactivated O₂ evolution was reconstituted to 75% of the original activity by rebinding of this protein back to the washed particles. (3) The cyanobacterial peripheral 34 kDa protein rebound to CaCl₂-washed spinach PS II particles and restored O₂ evolution to an appreciable extent (28%). (4) The spinach peripheral 33 kDa protein rebound to CaCl₂-washed PS II particles of S. vulcanus and partially restored O₂ evolution (60%). These results suggested that the peripheral 34 kDa protein of S. vulcanus possesses the determinants for both binding and activity reconstitution identical with those of the peripheral 33 kDa protein of spinach.     

The 33 kDa protein can be removed without affecting the association of the 23 and 17 kDa proteins with the luminal side of PS II of spinach

An earlier study shows that a 30 min incubation of spinach PS II submembrane fragments at pH 6.3 in the presence of 10 microM HgCl(2) induces a 40% depletion of the 33 kDa protein without the apparent release of the 17 and 23 kDa proteins [Bernier, M., and Carpentier, R. (1995) FEBS Lett. 360, 251-254]. Here we report that the photosystem II 33 kDa extrinsic protein is fully removed by HgCl(2) added at micromolar and higher concentrations (0.25, 20, and 50 microM), with the 17 and 23 kDa extrinsic proteins and other intrinsic proteins remaining bound to the reaction center. The data presented here put in doubt the "regulatory cap" model of PS II, which follows the OEC-33 kDa-23 kDa-17 kDa binding order, as these results directly demonstrate that the 33 kDa protein can be removed without affecting the binding of the 23 and 17 kDa proteins to the intrinsic subunits of PS II. This suggests that each extrinsic protein may possess its own binding site on PS II. A possible mechanism for HgCl(2) upon the release of the 33 kDa protein is discussed.     

Origin of 33 kDa protein of vitelline membrane of quail egg: Immunological studies

The inner layer of vitelline membrane is an investment of avian ovum at the time of ovulation, but its formation is poorly understood. In order to elucidate the origin of the inner layer of vitelline membrane, a 33 kDa protein, one of the components of the inner layer, was purified from quail eggs and polyclonal antibody was raised against this protein. The tissue distribution of protein interacted with the antibody was studied by Western blotting technique. No immunoreactive component could be observed in extracts of liver, kidney, heart, lung, small intestine, brain, infundibulum, albumen-secreting region of oviduct, uterus, and wall of small white follicles. The intensive band was detected in the granulosa layer, which was isolated from the large preovulatory follicles as a monolayer of granulosa cells sandwiched between the inner layer of vitelline membrane and the basal lamina. The granulosa cells isolated from the granulosa layer also reacted with this antibody. Theca layer had no immunoreactive components. The position of the band of the 33 kDa protein on SDS-PAGE was sifted to higher molecular weight in follicular tissues as compared with that in the laid eggs, indicating that the structural change of the protein occurs after ovulation. These studies indicate that the material reactive to the antibody raised against a 33 kDa protein of quail vitelline membrane is synthesized by the granulosa cells.     

The 33 kDa protein of photosystem II is a low-affinity calcium- and lanthanide-binding protein

We have shown that the isolated 33 kDa protein of photosystem II contains one calcium and one lanthanide low-affinity binding site with binding constants (K(D)) on the order of 10(-5) M. Binding of calcium or lanthanides to this site induces conformational changes in the protein that manifest in fluorescence emission spectra of the protein, circular dichroism spectra, and calorimetric thermograms where the phase transitions are shifted to lower temperatures. The role of calcium binding to the 33 kDa protein in the attainment of its native structure and the significance of this interaction for the oxygen evolution process are discussed.     

Improved isolation of a 50 kDa anorexigenic protein from rat urine

In prior work, a 50 kDa protein was purified to homogeneity from rat urine. This protein reduces food intake when injected into rats and is the only natural substance other than satietin known to be effective for long (24 hour) time periods and which does not make animals ill. However, when attempts were made to repeat the purification, contamination appeared in the 50 kDa fraction. The present contribution documents successful reisolation of the 50 kDa anorexigen by an improved method. Reisolation involved Cibacron blue-Sepharose, DEAE-Sephacel and Sephacryl S-200 chromatography, and SDS disc preparatory electrophoresis. The reisolated 50 kDa anorexigen contains no detectable carbohydrate. Partially purified preparations of the 50 kDa anorexigen were fragmented with trypsin and proteinase K without loss of anorexigenic activity. It is concluded that the 50 kDa anorexigen may be reproducibly purified to homogeneity and may contain within its amino acid sequence a peptide which is the basis of its anorexigenic activity.     

Effect of linolenic acid on release of the 43 kDa polypeptide and manganese from photosystem II membranes depleted of the 33 kDa extrinsic polypeptide

The effect of linolenic acid (18:3) on release of the 43 kDa polypeptide and manganese from photosystem II ( PS II ) membranes depleted of extrinsic polypeptides was studied. In both control and NaCl-washed particles which were depleted of the extrinsic 23 and 16 kDa polypeptides, the 18:3 treatment caused a 20% release of the 33 and 43 kDa polypeptides. In CaCl₂, (or urea + NaCl)-washed particles, which were depleted of the 33 kDa polypeptide in addition to the 23 and 16 kDa polypeptides, the release of the 43 kDa polypeptide increased to 70%, whereas only 25% of the 47 kDa polypeptide was removed. These findings suggest (i) that the 33 and the 43 kDa polypeptides are neighbows in the photosynthetic membrane and (ii) that the 33 kDa polypeptide shields the 43 kDa polypeptide against the action of 18:3. Incubation of CaCl₂, or (urea + NaCI)-treated PSII particles in the presence or absence of 18:3 resulted in the loss of only 2 of the 4 Mn atoms present per reaction center. this indicates that the 2 Mn atoms more firmly associated with PSII are not affected by the removal of the extrinsic 16, 23 and 33 kDa polypeptides, and the intrinsic 43 kDa polypeptide. nor by the treatment with linolenic acid.     

Isolation and some properties of bovine brain 100 kDa heat shock protein

1. The 100 kDa protein was purified from bovine brains. 2. The antibody against the 100 kDa brain protein was prepared and was monospecific to the antigen. 3. The antibody cross-reacted with HeLa cell HSP100 (100 kDa heat shock protein). 4. The physicochemical, immunochemical properties and a partially amino acid sequence indicated that the 100 kDa protein was HSP100. 5. Peptide mapping using Staphylococcus aureus V8 protease showed a core peptide with 10 kDa molecular mass common to both HSP100 and HSP90. 6. The amino acid sequence of the 10 kDa fragment of the 100 kDa protein showed a high homology with that of human HSP90 (38-60); the difference was only two of 23 amino acid residues determined.     

Comparison of the structure of the extrinsic 33 kDa protein from different organisms

The psbO gene encoding the extrinsic 33 kDa protein of oxygen-evolving photosystem II (PSII) complex was cloned and sequenced from a red alga, Cyanidium caldarium. The gene encodes a polypeptide of 333 residues, of which the first 76 residues served as transit peptides for transfer across the chloroplast envelope and thylakoid membrane. The mature protein consists of 257 amino acids with a calculated molecular mass of 28,290 Da. The sequence homology of the mature 33 kDa protein was 42.9-50.8% between the red alga and cyanobacteria, and 44.7-48.6% between the red alga and higher plants. The cloned gene was expressed in Escherichia coli, and the recombinant protein was purified, subjected to protease-treatments. The cleavage sites of the 33 kDa protein by chymotrypsin or V8 protease were determined and compared among a cyanobacterium (Synechococcus elongatus), a euglena (Euglena gracilis), a green alga (Chlamydomonas reinhardtii) and two higher plants (Spinacia oleracea and Oryza sativa). The cleavage sites by chymotrypsin were at 156F and 190F for the cyanobacterium, 159M, 160F and 192L for red alga, 11Y and 151F for euglena, 10Yand 150F for green alga, and 16Y for spinach, respectively. The cleavage sites by V8 protease were at 181E (cyanobacterium), 182E and 195E (red alga), 13E, 67E, 69E, 153D and 181E (euglena), 176E and 180E (green alga), and 18E or 19E (higher plants). Since most of the residues at these cleavage sites were conserved among the six organisms, the results indicate that the structure of the 33 kDa protein, at least the structure based on the accessibility by proteases, is different among these organisms. In terms of the cleavage sites, the structure of the 33 kDa protein can be divided into three major groups: cyanobacterial and red algal-type has cleavage sites at residues around 156-195, higher plant-type at residues 16-19, and euglena and green algal-type at residues of both cyanobacterial and higher plant-types.     

On the role of the N-terminus of the extrinsic 33 kDa protein of Photosystem II

The role of the N-terminus of the extrinsic 33 kDa protein of Photosystem II has been investigated by means of site-directed mutagenesis and cross-linking. Replacement of Asp-9 resulted in a dramatic increase in proteolytic sensitivity leading to the degradation of the protein forming a 31 kDa fragment with an undefined N-terminus. This fragment was unable to restore oxygen evolution. However, the variants of the 33 kDa protein which remained intact could reconstitute oxygen evolution as effectively as the wild-type protein. Cross-linking experiments with a water-soluble carbodiimide revealed that mutagenesis of residue D9 led to the disruption of an intramolecular salt bridge. Therefore we suggest that the N-terminus of the 33 kDa protein is necessary for maintaining the binding ability of the protein to Photosystem II but might not be involved in binding itself.     

Nuclear translocation of mouse polycomb m33 protein in regenerating liver

Immunoblots probed with an antibody to M33 protein, a homolog of Drosophila Polycomb, revealed that most M33 in adult mouse liver had a higher electrophoretic mobility than that in F9 embryonal carcinoma cells. High-mobility 60-kDa M33 localized in the cytoplasmic fraction of liver homogenates, and two less abundant 66- and 70-kDa species were detected in the nuclear fraction. Immunocytochemistry of freeze-substituted tissues showed a punctate pattern of immunofluorescence in the cytoplasm of hepatic parenchymal cells. Nuclear M33 isoforms treated with alkaline phosphatase had increased mobilities corresponding to cytoplasmic M33. In partially hepatectomized mice, nuclear M33 isoforms appeared after 48 h, near the time of maximum DNA synthesis as measured by bromodeoxyuridine incorporation. By 60 h, most M33 was in the form of these low-mobility species, and the pattern of immunofluorescence suggested the existence of chromatin-bound and free states of the protein in the nucleus. Thereafter, high-mobility 60-kDa M33 reappeared. The data are consistent with a phosphorylation-associated translocation mechanism that is a cell cycle-dependent.     

Cloning and sequencing of the 23 kDa mouse photoreceptor cell-specific protein.

The 23 kDa protein was localized by immunocytochemistry to photoreceptor cells of the mouse retina, and bovine and mouse cDNA clones were isolated and sequenced. The deduced amino acid sequences showed that the mouse 23 kDa protein is 91% identical to the bovine protein, and is the same as S-modulin, the CAR (cancer-associated retinopathy) protein and recoverin, the Ca(2+)-dependent activator of photoreceptor guanylate cyclase. The amino acid sequence reveals two Ca2+ binding sites, no internal repeats, 59% homology to the chicken visinin protein and 40% homology to calmodulin while Northern analysis demonstrated a single 1.0 kb mRNA species in bovine and mouse retina.     

棉铃虫核型多角体病毒ORF33基因的原核表达和在宿主细胞中亚细胞定位

对棉铃虫Helicoverpa ar migera核型多角体病毒HearSNPV的ORF33基因(ha33)进行克隆和原核表达,hass在E.coli中表达不完全,表达产物的大小为17kDa,小于预测的分子量28.4kDa。用纯化的原核表达产物免疫家兔,制备了多克隆抗体,应用多克隆抗体检测了HearSNPV感染的宿主细胞(HzAMI)中ORF33基因的表达,表达产物的分子量为31kDa。并通过共聚焦荧光显微镜方法,用多克隆抗体检测编码的蛋白在宿主细胞(HzAM1)中的亚细胞定位,发现ha33编码的蛋白存在于宿主细胞的细胞质中,并持续到感染后期。