Subtilisin Protein Inhibitor from Potato Tubers

A protein with molecular weight of 21 kD denoted as PKSI has been isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii). The isolation procedure includes precipitation with (NH4)2SO4, gel chromatography on Sephadex G-75, and ion-exchange chromatography on CM-Sepharose CL-6B. The protein effectively inhibits the activity of subtilisin Carlsberg (Ki = 1.67 +/- 0.2 nM) by stoichiometric complexing with the enzyme at the molar ratio of 1 : 1. The inhibitor has no effect on trypsin, chymotrypsin, and the cysteine proteinase papain. The N-terminal sequence of the protein consists of 19 amino acid residues and is highly homologous to sequences of the known inhibitors from group C of the subfamily of potato Kunitz-type proteinase inhibitors (PKPIs-C). By cloning PCR products from the genomic DNA of potato, a gene denoted as PKPI-C2 was isolated and sequenced. The N-terminal sequence (residues from 15 to 33) of the protein encoded by the PKPI-C2 gene is identical to the N-terminal sequence (residues from 1 to 19) of the isolated protein PKSI. Thus, the inhibitor PKSI is very likely encoded by this gene.     

Purification and characterization of a lipase from Staphylococcus aureus

An extracellular lipase from Staphylococcus aureus (strain FN 37) was purified to homogeneity. A cell-free culture broth was subjected to ammonium sulphate precipitation, and the lipase was isolated from the resuspended pellet by adsorption chromatography on octyl-Sepharose. The purification was 957-fold, and the recovery of the octyl-Sepharose chromatography was about 100%. The specific activity of the purified lipase was 546 mU of lipase activity per micrograms protein. The purity of the final product was documented by SDS-polyacrylamide gel electrophoresis in which a homogeneous protein band of 43 kDa was found. In gel chromatography on Sephadex G-200 the lipase eluted as a homogeneous peak with an apparent molecular mass of 110 kDa, suggesting that the lipase may exist as an oligomer in physiological media. Analysis of the amino-acid composition revealed a predominance of polar, non-charged amino acids, with serine accounting for 24 mol% of the amino-acid residues.     

The amino acid sequence of coagulogen isolated from southeast Asian horseshoe crab, Tachypleus gigas

The amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab (Tachypleus gigas) has been determined. The NH2-terminal sequence of the first 51 residues was obtained by automated Edman degradation. The intact protein was then treated with a Tachypleus clotting enzyme, to form a gel and to remove an internal peptide C (28 residues) located near the NH2-terminal portion. The gel protein, which consisted of A chain (18 residues) and B chain (129 residues), was S-alkylated and the resulting two chains were separated by acetone precipitation. Among these segments, A chain and peptide C were assigned to the NH2-terminal portion of whole coagulogen, as judged from their amino acid compositions. On the other hand, the covalent structure of B chain was determined by sequencing the peptides obtained from its tryptic digest. The alignments of the tryptic peptides were deduced from the sequence homology in comparison with the previously established B chain sequence of Japanese horseshoe crab (T. tridentatus) coagulogen. T. gigas coagulogen had a total of 175 amino acids and a calculated molecular weight of 19,770. When the sequence was compared with those of Japanese and American horseshoe crab (Limulus polyphemus) coagulogens, extensive structural homology was found: T. tridentatus/T. gigas, 87% and L. polyphemus/T. gigas, 67%. This comparison suggests that Japanese and Southeast Asian horseshoe crabs have a crab, based on amino acid sequence data.     

The amino acid sequence of Clostridium pasteurianum iron protein, a component of nitrogenase. II. Cyanogen bromide peptides

A total of 10 cyanogen bromide peptides were isolated from the S-beta-carboxymethyl iron protein of nitrogenase. Purification of these peptides was performed mainly by gel filtration on Sephadex G-50; by ascending paper chromatography using the solvent system of pyridine, isoamyl alcohol, 0.1 M ammonium hydroxide; and also, in some cases, with additional steps such as anion exchange column chromatography on Dowex 1-X2 or ascending paper chromatography in an acidic solvent system or by pyridine precipitation of the cyanogen bromide fragment. Sequenator analyses of three large cyanogen bromide peptides (53 to 72 residues) provided tryptic peptide overlap data for the inner portion of the protein. The cyanogen bromide peptides accounted for all of the 273 amino acid residues which were present in the tryptic peptides isolated from carboxymethyl-iron protein (Tanaka, M., Haniu, M., Yasunobu, K. T., and Mortenson, L. E. (1977) J. Biol. Chem. 252, 7081-7088).     

Protein-bound choline is released from the pneumococcal autolytic enzyme during adsorption of the enzyme to cell wall particles.

The inactive precursor form of the pneumococcal autolytic enzyme cloned in Escherichia coli was isolated by affinity chromatography on Sepharose-linked choline. The enzyme was recovered in an electrophoretically pure and activated form by elution from the affinity column with radioactive choline solution. When radioactive choline was used for elutions, the enzyme protein isolated contained protein-bound choline, at approximately 1 mol of choline per mol of enzyme protein, indicating the presence of a single choline recognition site. Radioactive choline remained bound to the enzyme protein during dialysis, precipitation by trichloroacetic acid or ammonium sulfate, and during gel filtration, but not during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incubation of the choline-labeled autolysin with pneumococcal cell walls at 0 degrees C resulted in the adsorption of the enzyme to the wall particles and a simultaneous release of free choline from the enzyme protein. It is suggested that the choline molecules that became bound to the enzyme protein during the activation of autolysin are expelled from the choline-binding site and replaced by choline residues from the wall teichoic acid as the autolysin molecules adsorb to their insoluble substrate before the onset of enzymatic wall hydrolysis.     

Isolation and characterization of a somatomedin-binding protein from mid-term human amniotic fluid

Human amniotic fluid is rich in a binding protein for somatomedins. This binding protein competes with human placenta membranes for labelled somatomedin A. Consequently, the placenta radioreceptorassay for somatomedin can be used for detection of the binding protein. The protein was isolated from human amniotic fluid by a three-step procedure: First, stepwise ammonium sulphate precipitation; second, hydrophobic chromatography (phenyl-Sepharose); and third, anion-exchange chromatography (fast protein liquid chromatography). The total recovery of binding protein calculated with the placenta radioreceptorassay was 50%. Polyacrylamide gel electrophoresis under native and denaturating conditions of the isolated protein disclosed a single band. The relative molecular mass was 35000, determined by exclusion chromatography, and 32000 under denaturating conditions in sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The isoelectric point was 4.3 according to chromatofocusing and the amino acid composition also disclosed a high content of acidic/amidated residues. The N-terminal amino acid sequence was Ala-Pro-Trp-Gln-Cys-Ala-Pro-Cys-Ser-Ala.     

Purification and characterization of a new antiviral protein from the leaves of Pandanus amaryllifolius (Pandanaceae)

A lectin, designated Pandanin, was isolated from the saline extract of the leaves of Pandanus amaryllifolius, using ammonium sulfate precipitation, affinity chromatography on mannose-agarose and molecular size exclusion by gel filtration. Pandanin is an unglycosylated protein with a molecular mass of 8.0 kDa both after gel filtration and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it is a single polypeptide chain. The first 10 residues of the N-terminal amino acid sequence are DNILFSDSTL. An analysis of the sequence of first 30 amino acids at the N-terminal region shows that Pandanin has about 50-60% homology to those of mannose-specific lectins reported from monocot plants. Pandanin exhibits hemagglutinating activity toward rabbit erythrocytes, and its activity could be reversed exclusively by mannose and mannan. Pandanin also possesses antiviral activities against human viruses, herpes simplex virus type-1 (HSV-1) and influenza virus (H1N1) with 3 day's EC50 of 2.94 and 15.63 microM, respectively.     

Odorant-Binding Protein from Culex tarsalis,the Most Competent Vector of West Nile Virus in California

We have identified and cloned an odorantbinding protein from the female mosquito, Culex tarsalis (CtarOBP). As expected for an olfactory protein, CtarOBP was detected by gel electrophoresis analysis in antennae but not in control tissues (legs). The isolated protein was identified by in-gel digestion and subsequent analysis of internal fragments by tandem mass spectrometry (MS-MS). Based on the amino acid sequences of two peptides generated by enzymatic digestion, degenerate primers were designed for cDNA cloning. The complete cDNA (cloned by RACE) encoded a protein with a signal peptide (24 residues) and a mature protein of 125 amino acid residues. The calculated molecular mass and isoelectric point of the mature protein were 14,515 Da and pl 5.5, respectively. CtarOBP showed the hallmark of odorant-binding proteins, 6 cysteine residues, and high sequence homology (61–96%) to previously characterized mosquito OBPs.     

Purification and partial characterization of a soluble hemagglutinin from Yersinia pseudotuberculosis

A soluble hemagglutinin (HA) produced by Yersinia pseudotuberculosis strain Inoue, serotype 5b, was purified by ammonium sulfate precipitation, gel filtration on Sepharose CL-6B and high performance liquid chromatography on a DEAE-5PW anion-exchange column. The purified HA was a 14.5 kDa protein with an isoelectric point of 4.5. Amino acid analysis indicated that the HA consisted of 133 residues, corresponding to the molecular weight of 14,100. The amino acid sequence of N-terminal 38 amino acid residues showed no homology with that of several fimbrial proteins from Escherichia coli.     

Membrane proteins of Rhodopseudomonas spheroides III. Isolation,purification, and characterization of cell envelope proteins

Cell envelopes (cell wall and cell membrane) from aerobically grown cells of Rhodopseudomonas spheroides were isolated and purified by a combination of differential centrifugation and centrifugation through 40% sucrose. Cell envelope protein from aerobically grown cells was resolved by dodecyl sulphate-polyacrylamide gel electrophoresis. Biochemical characterization of selected envelope membrane proteins demonstrated heterogeneity between different protein species. Amino acid analyses of individual proteins revealed between 50–60 mole% nonpolar residues.Envelope membranes derived from anaerobically grown cells were also isolated and purified by a combination of differential centrifugation, column chromatography on Sepharose 2B, and centrifugation in 40% sucrose. The dodecyl sulphate-polyacrylamide gel patterns of anaerobic and aerobic envelope membrane proteins were very similar and the results suggest a common protein structure.     

Purification and characterization of charantin, a napin-like ribosome-inactivating peptide from bitter gourd (Momordica charantia) seeds.

A peptide designated charantin, with a molecular mass of 9.7 kDa, was isolated from bitter gourd seeds. The procedure comprised affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on Mono S and gel filtration on Superdex 75. The N-terminal sequence of charantin exhibited marked similarity to that of the 7.8-kDa napin-like peptide previously isolated from bitter gourd seeds. Charantin inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC50 of 400 nm, a potency lower than that of the previously reported small ribosome-inactivating protein gamma-momorcharin (IC50 = 55 nm) which also exhibited an abundance of arginine and glutamate/glutamine residues. Charantin reacted positively in the N-glycosidase assay, yielding a band similar to that formed by the small ribosome-inactivating proteins gamma-momorcharin and luffin S.     

Purification and characterization of a low molecular weight zinc binding protein from human placenta

A low molecular weight, native zinc binding, cytosolic protein (LMZP) has been isolated, purified and characterized from human normal term placenta. Gel filtration of heat treated placental cytosol after sequential acetone precipitation (80% ppt) revealed a major zinc binding protein in the range of low molecular weight. This partially purified zinc binding fraction was further fractionated on DEAE-Sephadex A-25. The zinc was eluted in one of the three peak fractions. Further, the purity of zinc binding protein was confirmed on fast protein liquid chromatography (FPLC). The purified placental LMZP was homogenous on SDS-polyacrylamide gel electrophoresis with a single band. Ultraviolet (UV) spectrum of LMZP showed an absorption maximum at 257 nm which disappeared at pH 2. Molecular weight of LMZP as determined by gel chromatography, SDS-polyacrylamide gel electrophoresis and amino acid analysis was 6 kDa. It was calculated that 1 g atom of zinc was bound to 1 mole of the LMZP. Unlike in classical metallothionein, the amino acid composition of placental LMZP revealed the presence of aromatic amino acids, lower content of cysteine and higher content of histidine, glutamic acid and aspartic acid (10, 9 and 5 residues/mole, respectively).     

Isolation and characterization of a soluble, immunoactive peptide of glial fibrillary acidic protein

A soluble immunoactive peptide with a molecular weight of 16 000 was isolated and purified from the cyanogen bromide digest of the insoluble 50 000 dalton glial fibrillary acidic protein by Sephacryl S-200 gel filtration followed by DEAE-Bio-gel A chromatography. The homogeneity of the peptide was established by SDS-polyacrylamide gel electrohporesis and isoelectric focusing. The peptide from several species showed immunocrossreaction with rabbit antibody to intact glial fibrillary acidic protein. The peptide has a pI value of 5.32. The amino acid sequence of 28 residues from the amino terminus of the calf peptide has been determined.     

Rat adrenocortical carcinoma 494 autophosphorylating protein kinase, autophosphorylating protein kinase 500. Purification, biochemical and immunological characterization, and substrate specificity

A novel autophosphorylating protein kinase, autophosphorylating protein kinase 500, independent of cyclic AMP, cyclic GMP, calcium, and calmodulin was purified from rat adrenocortical carcinoma 494 by ammonium sulfate fractionation followed by the chromatographic steps of DEAE-cellulose, gel filtration, cyclic AMP-epoxy Sepharose, and phosphocellulose. Sometimes two additional chromatographic purification steps of chromatofocusing and gel filtration were necessary for complete purification. The enzyme was homogeneous as evidenced by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sucrose density sedimentation studies indicated that Mr of the enzyme was 490,000, while ultracentrifugal analysis demonstrated a value of 481,400 (+/-7%). The protein was composed of two identical subunits each with Mr = 250,000. The enzyme molecule was slightly asymmetric with frictional and sedimentation coefficients of 1.28 and 18.20, respectively, and a Stokes radius of 66 A. Isoelectric focusing electrophoresis revealed a single peak with pI 4.6, indicating acidity of the protein. The enzyme self phosphorylated one or more of its serine residues. The reaction utilized the terminal phosphate of ATP; GTP was inactive. Divalent cations (5 mM Mn2+ or 10 mM Mg2+) were essential for optimum activity. Autophosphorylating protein kinase 500 did not phosphorylate the commonly used exogenous substrates such as histones, casein, phosvitin, or protamine. Analysis of autophosphorylating protein kinase 500 with rabbit anti-autophosphorylating protein kinase 500 IgG by immunoelectrophoresis and crossed immune electrophoresis demonstrated single arcs of precipitation, confirming the biochemical demonstration of enzyme purification and homogeneity. Indirect immunofluorescence studies revealed an intracytoplasmic localization of the enzyme in cultured and freshly isolated adrenocortical carcinoma 494 cells. Both cell types revealed an intensity of perinuclear enzyme fluorescence, but an absence of the enzyme in the nuclei or nucleoli. The anti-autophosphorylating protein kinase 500 IgG blocked the self-catalyzed phosphorylation of autophosphorylating protein kinase 500, providing immunological support of the biochemical results that autophosphorylation is an intrinsic characteristic of the enzyme. When autophosphorylating protein kinase 500 was incubated with membrane-bound ribosomes, it phosphorylated a Mr = 31,000 protein. This phosphorylation was blocked by the anti-autophosphorylating protein kinase 500 IgG.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular characterization of limulin, a sialic acid binding lectin from the hemolymph of the horseshoe crab, Limulus polyphemus.

The sialic acid binding lectin, limulin, was isolated by gel filtration and ion-exchange chromatography from the hemolymph of Limulus polyphemus. When the purified protein was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of beta-mercaptoethanol, two major protein bands were observed. These two bands, subsequently found to contain carbohydrate as well, corresponded to molecular weights of 25 000 and 27 000. Amino acid sequence analyses were performed on both the intact protein and isolated cyanogen bromide fragments. The following primary structural features were noted in the amino-terminal region of limulin: (1) the absence of histidine and alanine from the NH2-terminal 50 residues; (2) the presence of five of the total eight prolines of the molecule between positions 13 and 30; and (3) a possible carbohydrate attachment site consisting of only the amino acids proline and serine between residues 13 and 19. The resultsof cyanogen bromide cleavage studies confirmed the presence of 2 methionine residues per subunit, at positions 25 and 58 respectively. No sequence heterogeneity was observed in this study. While it is quite possible that limulin plays some role in the defense mechanisms of the horseshoe crab, there is no obvious sequence homology between this invertebrate lectin and vertebrate immunoglobulins.     

Isolation, characterization, and biosynthesis of a phosphorylated glycoprotein from rat bone

A phosphorylated glycoprotein was purified from the mixture of proteins extracted by demineralization of rat bone with 0.5 M EDTA in 4 M guanidinium chloride. A high level of purity for the preparation was indicated by a single band on sodium dodecyl sulfate (SDS)-gradient gel electrophoresis, sedimentation equilibrium ultracentrifugal data, and by automated Edman degradation results. The molecular weight of the phosphoprotein was shown to be about 44,000 by sedimentation equilibrium analyses in 4 M guanidinium chloride, even though an Mr of 75,000 was obtained by 5-15% SDS-polyacrylamide gel electrophoresis. Subsequent analysis by 15% SDS-polyacrylamide gel electrophoresis gave an Mr of 45,000. Analytical data showed that the protein contained 16.6% carbohydrate, possibly including 1 N-linked oligosaccharide and 5-6 O-linked oligosaccharides. The aspartic acid- and glutamic acid-rich protein contained about 300 amino acid residues including 1 phosphothreonine and 12 phosphoserine residues. Alkaline beta-elimination/NaBH4 reduction data showed that the phosphate obtained by complete acid hydrolysis prior to amino acid analysis was equivalent to the phosphate subject to alkaline beta-elimination. In this experiment, the losses of serine plus threonine exceeded the amount of phosphate liberated by 5-6 residues/protein. These serine and threonine residues probably represent O-linked oligosaccharides, since the protein contained about this number of N-acetyl-galactosamine residues. That the phosphoprotein is synthesized and secreted by osteoblast-like cells was shown with cultures of clonal rat osteosarcoma cells. After pulsing with 32PO4 the proteins secreted into the medium were precipitated with trichloroacetic acid and the radiolabeled proteins were immunoadsorbed. A protein migrating in the same position, on 5-15% SDS-polyacrylamide gel electrophoresis (i.e. with an Mr = 75,000) and on 15% gels (Mr = 45,000), as the phosphoprotein obtained from bone could be specifically immunoprecipitated.     

The amino acid sequence of Clostridium pasteurianum iron protein, a component of nitrogenase. I. Tryptic peptides

A total of 25 tryptic peptides was isolated from the S-beta-carboxymethyl derivative of Clostridium pasteurianum iron protein (N2). In order to obtain the various peptides in pure state, a combination of gel permeation, cation and anion exchange column chromatographic methods, as well as various ascending paper chromatographic methods were adopted. Sequence studies of the tryptic peptides were carried out mainly by a modified manual Edman degradation procedure and also by automated analysis, carboxypeptidase digestion, and by hydrazinolysis. Thus, 242 residues (88.6%) out of a total of 273 amino acid residues were sequenced in the present study. The sum of the amino acid residues in the tryptic peptides isolated from iron protein (N2) accounted for the 273 amino acid residues present in the iron protein.     

PSI-O, a new 10-kDa subunit of eukaryotic photosystem I

A novel polypeptide with an apparent molecular mass of 9 kDa was detected after sodium dodecyl sulphate–polyacrylamide gel electrophoresis of Arabidopsis photosystem I (PSI) and was N-terminally sequenced. Corresponding cDNA clones encode a precursor protein of 140 amino acid residues which was imported into isolated intact chloroplasts and processed to the mature protein, designated PSI-O. The mature protein has two transmembrane helices and a calculated mass of 10 104 Da. The PSI-O protein was also shown to be present in PSI isolated from barley and spinach, and was essentially absent in chloroplast grana. Expressed sequences encoding similar proteins are available from many species of plants and green algae.     

Properties of a hydrophobin isolated from the mycoparasitic fungus Verticillium fungicola

Verticillium fungicola, isolated from Agaricus bisporus (commercial mushroom), produced significant extracellular hydrophobin when grown for 7 days in a static liquid culture of synthetic minimal medium. The hydrophobin was purified by precipitation with ammonium sulphate (80% saturation), Sephadex G-100 gel filtration, and hydroxyapatite column chromatography. The purified protein yielded a single band in polyacrylamide gel electrophoresis under native conditions, with an apparent molecular mass of 70 +/- 4 kDa, and also another single band in SDS-PAGE, with a molecular mass of 7 +/- 3 kDa. Molecular mass determined with matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) resulted in 7563.9 m/z. The same protein was extracted from the V. fungicola mycelium. Analysis of the amino acid composition revealed the presence of about 50% hydrophobic residues, detecting at least six cysteines, evaluated as cystines, and no free sulfhydryl groups. The protein did not show any glycosylation. On the basis of similarities in hydropathy patterns and solubility characteristics, V. fungicola hydrophobin can be included as a new member of Class II hydrophobins.