Physico-chemical and immunological properties of allophycocyanins

Allophycocyanins were purified from diverse cyanobacteria and one rhodophytan alga (Cyanidium caldarium). The native proteins are trimeric molecules with the structure ()₃. Representative native allophycocyanins and their and subunits were characterized with respect to molecular weight, amino acid composition, isoelectric point, absorption and fluorescence spectra and immunological properties. All of the allophycocyanins studied were strikingly similar with respect to each of these properties.Renatured and subunits of allophycocyanin were distinct immunologically from each other, and both cross-reacted with the antiserum to the native protein.Trimeric allophycocyanin was readily reconstituted from the purified and subunits. Formation of hybrid allophycocyanins was demonstrated by direct isolation and characterization of the hybrid proteins and by immunological techniques.The results support the view that allophycocyanins are a highly conserved group of proteins.Abbreviation Used SDS sodium dodecyl sulfate     

Purification and partial amino acid sequencing of bovine kidney alkaline phosphatase

Bovine kidney alkaline phosphatase (ALPase) was purified by the sequential application of monoclonal anti-bovine cartilage ALPase affinity, DEAE-cellulose, and Sepharose CL-6B chromatography. Sodium dodecyl sulfate-polyacrylamide-gel electrophoresis showed the presence of a single band corresponding to a molecular weight of 80,000. The N-terminal amino acid sequence of bovine kidney alkaline phosphatase was determined as follows: Leu-Val-Pro-Glu-Lys-Asp-Pro-?-Tyr-Trp-Arg-Asp-Gln-Ala-Gln.     

APDbase: Amino acid Physico-chemical properties Database

Physico-chemical properties of amino acids can be used to study protein sequence profiles, folding and function. We collated 242 properties for the 20 naturally occurring amino acids and created a dataset. The dataset is available as a database named APDbase( Amino acid Physico-chemical properties Data base). The database can be queried using either key words describing physico-chemical properties or pre-assigned database index number. The database contains corresponding references for each property value and facilitates deposition of new property values for processing and inclusion in the database.

Availability     

Purification and partial amino acid sequencing of alkaline phosphatase from rachitic rat epiphyseal cartilage

1. Alkaline phosphatase of rachitic epiphyseal cartilage was purified to apparent homogeneity by sequential application of monoclonal affinity, DEAE-cellulose, and Sepharose CL-6B chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the enzyme showed the presence of a dominant band corresponding to a molecular weight of 80,000. 2. The N-terminal amino acid sequence was determined as follows: Phe-Val-Pro-Glu-Lys5-Glu-Lys-Asp-Pro-Ser10-Tyr-Trp-Arg-Gln-+ ++Gln15-Ala-Gln-Glu- Thr-Leu20-Lys-Asn-Ala-Leu-Lys25-Leu-Gln-Lys-?-Asn-Val-Asn-?- Ala-Lys35-?-Ile-?- Met-Phe40-Leu-(Gly?)-Asp-(Ala/Gly?)-Met45-?-Val-?- (Val/Gly?).     

Physico-chemical properties and amino acid composition of a highly purified preparation of a specific estrogen-binding protein of the rat liver

The structure and properties of an unusual estrogen-binding protein (UEBP) from male rat liver that was purified by affinity adsorption chromatography was studied. A high degree of purity of UEBP (greater than 99%) associated with appreciable microheterogeneity was demonstrated. The latter seems to be due to partial proteolysis of the protein at the N-end in the course of the isolation procedure. The purified UEBP molecules have the following characteristics: Mr = 31 000 (data from SDS-PAAG electrophoresis), sedimentation coefficient 3.75 S, Stockes'radius 25.6 A, friction coefficient ratio 1.11. The protein absorbance maximum in the UV region lies at 276 nm; extinction coefficient--26, alpha-helix content is 25-30%. The value of equilibrium association constant for estradiol is 5 X 10(7) M-1. Estriol (greater than 100%) and, in a weaker degree, estrone and testosterone (approximately 10%) compete for the binding sites with [3H]estradiol; androsterone has no competitive effect. The amino acid composition of UEBP was determined. The protein was shown to possess a great number of residues carrying hydrophobic side groups (34.4%) and more acidic amino acids over basic ones as well as a low content of cystein, threonine and histidine.     

Purification of MEA, a mast cell growth-enhancing activity, to apparent homogeneity and its partial amino acid sequencing

A novel growth factor for bone marrow derived murine mucosal type mast cells has been isolated from the conditioned medium of the Mlsa-reactive mouse Th cell line MLS-4.2. In proliferation assays this growth factor synergizes, like IL-4, with IL-3 on established mast cell lines and was therefore termed MEA: mast cell growth enhancing activity. MEA was characterized as a glycoprotein with a Mr range between 37,000 and 43,000. Apparent homogeneity was obtained by using a four-step purification scheme including cation exchange chromatography, Procion red affinity chromatography, IEF, and gel filtration. Inasmuch as MEA was N-terminally blocked during automated Edman-degradation, peptide fragments after digestion with trypsin were used for partial amino acid sequence determination. All evaluable MEA peptide fragments showed complete sequence homology to a recently purified and cloned novel T cell growth factor (P40/TCGF III), the mouse homologue of human IL-9.     

Purification and partial amino acid sequencing of rat bone tumor (UMR106) alkaline phosphatase

Cultured rat osteosarcoma (UMR106) alkaline phosphatase was purified to apparent homogeneity by sequential application of polyclonal antibody affinity, DEAE-cellulose, and Sepharose CL-6B chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme preparation treated with sodium dodecyl sulfate and mercaptoethanol showed the presence of a dominant band (using silver staining) corresponding to a molecular weight of 80,000. The amino acid composition was similar to those of various alkaline phosphatases. The N-terminal amino acid sequence was determined as follows: Phe-Val-Pro-Glu-Lys-Glu-Lys- Asp-Pro-Ser-Tyr-Trp-Arg-Gln-Gln-Ala-Gln-Glu-Thr-Leu- Lys-Asn-Ala-Leu-Lys-?-Gln-Lys-?-Asn-Val-Asn-Ala-Lys.     

The primary structure of the lymphocyte pore-forming protein perforin: partial amino acid sequencing and determination of isoelectric point

The murine lymphocyte pore-forming protein (PFP) was purified to apparent homogeneity by successive steps of liquid chromatography. Monospecific antibodies were raised against purified PFP that detect only one protein band in murine CTL lines. 25% of the primary sequence of PFP (134 amino acids) was determined by amino terminal analysis of the purified protein and of some of its enzymatic cleavage products. These primary sequences were identical to sequences deduced by cDNA cloning. By isoelectric focusing, PFP was found to have a pI of 6.4. On the chromatofocusing column Mono P, however, PFP was found to elute at pH 4.7. This suggests a tertiary structure for monomeric PFP that is enriched in surface acidic amino acids.     

Subunit IV of yeast cytochrome c oxidase: cloning and nucleotide sequencing of the gene and partial amino acid sequencing of the mature protein.

The six small subunits (IV-VII, VIIa, VIII) of yeast cytochrome c oxidase are encoded by nuclear genes and imported into the mitochondria. We have isolated the gene for subunit IV from a yeast genomic clone bank and determined its complete nucleotide sequence. We have also isolated subunit IV from purified yeast cytochrome c oxidase and determined most of its amino acid sequence which confirms the positioning of approximately 90% of the amino acid residues. The sequence comparison shows that the coding sequence of the gene lacks introns and that subunit IV is made as a precursor with an amino-terminal extension of 25 residues, five of which are basic and none of them acidic. Precursor processing involves cleavage of a Leu-Gln bond.     

Physico-chemical properties of 7-aminodesacetoxycephalosporanic acid]

7-Aminodesacetoxycephalosporanic acid (7-ADCA) of 99 per cent purity was prepared by enzymatic hydrolysis of 7-phenylacetamidodesacetoxycephalosporanic acid. Its isoelecti point was determined by the electrophoretic method. Potentiometric titration of zwitterion of 7-ADCA was performed and the constants of its ionization were calculated. Minimum solubility of 7-ADCA zwitterion was determined and the curve of 7-ADCA solubility at wide pH ranges was estimated. Equilibrium of 3 forms of 7-ADCA was estimated.     

Purification and partial amino acid sequencing of a mycorrhiza-related chitinase isoform from Glomus mosseae-inoculated roots of Pisum sativum L.

Colonization of Pisum sativum L. cv. Frisson roots with the arbuscular mycorrhizal fungus Glomus mosseae leads to the induction of four acidic symbiosis-related chitinase (SR-chi) isoforms (EC 3.2.1.14). These isoforms were characterized as 30-kDa proteins with isoelectric points ranging between 5.2 and 5.85. One of these SR-chis was purified by affinity and anion-exchange chromatographies, and isoelectric focusing electrophoresis. The sequences of four internal peptides were obtained. They showed high homology to a class-I chitinase isoform from pea shoots. Parts of the conserved regions of class-I chitinases were found in this SR-chi. This result strongly supports the argument that this SR-chi isoform is of plant origin. The functional role of the SR-chis in arbuscular mycorrhizal symbiosis is discussed.     

棉铃虫卵内半胱氨酸蛋白酶的分离纯化、氨基酸序列分析及蛋白酶鉴定

采用阴离子交换层析法,从棉铃虫Helicoverpa armigera卵母细胞中分离纯化到一种半胱氨酸蛋白酶,SDS-PAGE电泳显示为一条带,分子量约为29 kD,原位水解电泳表明其具有蛋白水解活性。对其进行了部分氨基酸序列测定,初步确定这种蛋白酶属于半胱氨酸蛋白酶类中的组织蛋白酶B类。     

Purification and partial amino acid sequence of thuricin S, a new anti-Listeria bacteriocin from Bacillus thuringiensis

We report the isolation and characterization of a new bacteriocin, thuricin S, produced by the Bacillus thuringiensis subsp. entomocidus HD198 strain. This antibacterial activity is sensitive to proteinase K, is heat-stable, and is stable at a variety of pH values (3-10.5). The monoisotopic mass of thuricin S purified by high performance liquid chromatography, as determined with mass spectrometry ESI-TOF-MS, is 3137.61 Da. Edman sequencing and NanoESI-MS/MS experiments provided the sequence of the 18 N-terminal amino acids. Interestingly, thuricin S has the same N-terminal sequence (DWTXWSXL) as bacthuricin F4 and thuricin 17, produced by B. thuringiensis strains BUPM4 and NEB17, respectively, and could therefore be classified as a new subclass IId bacteriocin.     

The partial amino acid sequence of a non-histone chromosomal protein

The amino acid sequence of the first thirty nine residues of the nonhistone chromosomal protein HMG-17 has been determined. Results presented here give a molecular weight of 11,000 for the protein. Some interesting sequence homology with the trout specific histone, histone-T, is noted.     

The major androgen-dependent protease in dog prostate belongs to the kallikrein family: confirmation by partial amino acid sequencing

Free energy transduction in active transport resembles other protein-catalyzed processes, occurring by an ordered sequence of discrete bond-breaking and bond-making steps. The bonds that affect the transported ion directly are chelation bonds, which alter the chemical potential of the bound ion, but not its chemical identity. Available data for the sarcoplasmic reticulum Ca pump (admittedly incomplete) suggest that more than 50% of the free energy transfer may be localized to a single step of the reaction cycle.     

The partial amino acid sequence of a murine Ia molecule

Eleven of the amino terminal 14 amino acid residues have been assigned in the chain of the murine I-C^d subregion molecule. The murine chain shows no homology with P29 (the putative human chain equivalent), the chain of guinea pig Ia. 4, 5, or the murineI-A subregion chain.     

Amino acid/water interactions study: a new amino acid scale

Partition ratios of 8 free l-amino acids (Gln, Glu, His, Lys, Met, Ser, Thr, and Tyr) were measured in 10 different polymer/polymer aqueous two-phase systems containing 0.15?M NaCl in 0.01?M phosphate buffer, pH 7.4. The solute-specific coefficients representing the solute dipole/dipole, hydrogen-bonding and electrostatic interactions with the aqueous environment of the amino acids were determined by multiple linear regression analysis using a modified linear solvation energy relationship. The solute-specific coefficients determined in this study together with the solute-specific coefficients reported previously for amino acids with non-polar side-chains where used in a Quantitative Structure/Property Relationship analysis. It is shown that linear combinations of these solute-specific coefficients are correlated well with various physicochemical, structural, and biological properties of amino acids.