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1. The purification of a halidohydrolase from cell-free extracts of Pseudomonas dehalogenans NCIB 9061 grown on glucose and monochloroacetate as carbon source, is described. 2. This enzyme catalyses the hydrolysis of some 2-halogen substituents on aliphatic carboxylic acids, releasing a halide ion and a hydrogen ion and causing inversion of the configuration. 3. The molecular weight as determined by chromatography on Sephadex G-100 was about 15000. 4. The optimum activity was at pH 9.4. Activity was rapidly and irreversibly lost below PH6.0. 5. The amino acid composition of the purified enzyme in its native state and after photooxidation in the presence of methylene blue is presented. 6. The variation of the kinetic parameters, V and Km between pH 7.0 and pH 10.0 suggest that a group with a pKa of about 7.8 is involved in its base form in the catalytic reaction. 7. The enzyme is rapidly inactivated by reaction with iodine and upon photooxidation in the presence of methylene blue, but the effect of thiol reagents suggests that a thiol group is not directly responsible for catalysis. 8. It is suggested that a histidine residue is implicated in the catalytic reaction and a mechanism is proposed which is consistent with the experimental results. [ABSTRACT FROM AUTHOR]

1. Desmosine and isodesmosine were separated by ion-exchange and paper chromatography, after acid hydrolysis of purified elastin from beef ligamentum nuchae. The fractions obtained by ion- exchange chromatography were clearly mixtures of related compounds. The desmosine fraction could be resolved into seven compounds and the isodesmote into four by paper charmatography. 2. Desmosine was maximally degraded by irradiation at 274 nm and isodesmosine at 285 nm. These wavelengths did not correspond to the absorption maxima of the cross links, but to shoulders of the main absorption peaks. 3. When irradiated at their optimum wavelengths, but at various pH, both desmosine and isodesmosine seemed quite stable at pH greater than 8.5. Between pH 8 and 5, the photolytic rate was maximum and decreased slightly at more acidic pH. Below pH 4.0, one of the products of photolysis was free lysine. 4. In analogy to the mechanism of the photolytic degradation of N-methyl pyridinium chloride, it appears that the (iso)desmosines were degraded via the formation of an open amino aldehyde, which was hydrolysed at acid pH to give free lysine and a substituted glutaconic aldehyde. [ABSTRACT FROM AUTHOR]

The light chain fraction was separated from myosin extracted from chicken cardiac muscle. Two light chain components, L-1 and L-2 in the fraction were isolated by chromatography on a column of DEAE-cellulose (DE-52) in the presence of 4 M urea. After performic acid oxidation, the L-1 chain was digested with trypsin and the resulting peptides were isolated. The amino acid sequences of the peptides were established. The ordering of these tryptic peptides in the L-1 chain was deduced from the amino acid compositions and the partial sequences of peptic peptides from S-carboxymethylated L-1 chain. Comparing the whole sequence of the L-1 chain thus established with that of alkali light chain of rabbit skeletal muscle myosin, 67 amino acid substitutions and two insertions were recognized. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that Corynebacterium cyclohexanicum degrades cyclohex-anecarboxylic acid, a bacteriocide, through a pathway including the aromatization of 4-oxocyclohex-anecarboxylic acid to 4-hydroxybenzoic acid [Kaneda, T. (1974) Biochem. Biophys. Res. Commun. 58, 140–144]. Aromatization has now been shown to be catalysed by two desaturase enzymes. Under the action of desaturase I, 4-oxocyclohexanecarboxylic acid is converted to (+)-4-oxocyclohex-2-enecarboxylic acid which is then aromatized by desaturase II to 4-hydroxybenzoic acid. The latter reaction is presumed to occur via the unstable intermediate, 4-oxocyclohex-2,5-dienecarboxylic acid, which is spontaneously isomerized to 4-hydroxybenzoic acid. Desaturase I has been purified in an electrophoretically homogeneous form. It is monomeric with a molecular mass of 67 kDa and contains one tryptophan, one histidine and two cysteine residues per enzyme molecule. The enzyme produces an equivalent amount of 4-oxocyclohex-2-enecarboxylic acid and hydrogen peroxide from 4-oxocyclohexanecarboxylic acid. The properties of desaturase I have been studied in detail. Desaturase II is unstable and has been partially purified. Its characterization is therefore limited. However, the molecular mass of desaturase II was estimated to be 43 kDa by gel filtration chromatography. The characterization of both desaturase enzymes is described in this paper. The possible environmental importance of microbial aromatization in the biodegradation of compounds with the cyclohexane structure is discussed. [ABSTRACT FROM AUTHOR]

Two isoenzymes of phospholipase A (fraction DE-I and DE-JI) were isolated from the venom of Hemachatus haemachatus (Riughals) by gel filtration on Sephadex 0-50 and by ion-exchange chromatography on DEAE-cellulose. Fraction DE-I was homogeneous by various physicochemical criteria. The enzyme comprises 119 amino acid residues and is cross-linked by seven disulphide bridges. The values of the molecular weight of fraction DE-I from gel filtration in the presence of calcium ions, also by the dodecylsulphate gel method and calculated from the amino acid composition were close to 13500. The complete amino acid sequence of phospholipase A (fraction DE-I) was elucidated. The reduced and S-carboxymethylated enzyme was digested with trypsin, chymotrypsin, thermolysin and subtilisin and the peptides purified by DEAE-cellulose chromatography, gel filtration and chromatography or electrophoresis on paper. The amino acid sequences of the intact enzyme and the pure peptides were determined by the Edman procedure, either through the use of the automatic sequencer or by manual manipulation. The chymotryptic digest provided the necessary overlapping peptides for aligning the tryptic peptides. The amino acid sequence of Hemachatus haemachatus phos- pholipase A (fraction DE-I) shows a high degree of homology with the three isoenzymes of phospho- lipase A from Naja melanoleuca and is also homologous with the phospholipase A from Bitis gabonica and porcine pancreas. [ABSTRACT FROM AUTHOR]

Malonic acid as well as its model substrate methylmalonic acid are covalently bound to two chemically different sites of the fatty acid synthetase complex of yeast. When radioactively labeled malonyl CoA or methylmalonyl CoA are incubated with the multienzyme complex, radio- active malonyl and methyhnalonyl enzymes are formed and may be precipitated with trichloro- acetic acid. Treatment with performic acid splits only a minor proportion of the (methyl) malonyl enzyme bonds. Most of them are resistant to the oxidant and, hence, prove to be non-thioester linkages. Methyl- [3-14C]malonyl enzyme was digested with pepsin. By DEAE-Sephadex chromatography of the peptic hydrolysate the radioactive (methyl)malonyl peptides. were separated into three different fractions A, B and C. In peptides B and C (methyl)malonic acid is bound by performic acid stable linkages, whereas (methyl)malonyl peptide A apparently is a thioester and can be split by oxidation. Peptide C is always a minor sateffite of peptide B, the quantitative relation of both varying somewhat in different preparations. it is concluded that both are derived from a common original peptide sequence in the enzyme. Methyl- [3-14C]malonyl peptide B was further purified by the following procedures: Dowex-50X2 chromatography, subtilisin S digestion, DEAB-Sephadex chromatography, Dowex-50X2 chromatography, high voltage paper electrophoresis and paper chromatography. Two methylmalonyl peptides, B1and B2, resulted from subtilisin S treatment. They finally were obtained in analytically pure form and proved to be a pentapeptide and a heptapeptide, respectively, with the following amino acid composition: B1 = Leu,AIa,Gly,Ser,His and B2 = Leu, Ala,Gly(2 × ),Glu,Ser,His, Leucine was the C-terminal amino acid in peptide B1. No cysteine was found in either peptide. A preparation of [3-14C]malonyl peptide B was found to be analytically pure after DEAE- Sephadex, Dowex-50X2 and paper chromatography. Its amino acid composition was identical to that of methylmalonyl B1peptide B2. The acyl peptides B and C were highly sensitive against alkaline hydrolysis. It is concluded that the (methyl)malonic acid in peptides B and C is bound to serine by an O-ester linkage which is unusually activated by some surrounding amino acid, possibly histidine, [3-14C]Malonal peptide A was shown to contain stoichiometric amounts of cysteamine, β-alanine, and organic phosphate, corresponding to the quantity of malonate bound. These data suggest a thioester linkage between malonate and the SH-group of enzyme-bound 4'-phospho- pantetheine. 4'- Phospho-pantetheine, therefore, represents the so called "central" SR-group in yeast fatty acid synthetase, The other malonyl binding site is thought to be the active center of the malonyl transferase in the complex. After alkali treatment of the purified fatty acid synthetase under defined conditions 4' -phospho - pantetheine was isolated from the bydrolysate and characterized as S-benzoyl-4'-phospho- pantetheine. One molecule of the multienzyme complex contained between 3.5 and 6 molecules of 4'-phospho-pantetheine. According to the amount of malonyl enzyme formed, not all of the enzyme-bound phospho-pantetheine residues accept malonic acid. Some may be involved in other functions. [ABSTRACT FROM AUTHOR]

This paper is the first one of a series devoted to the primary structure of the major protein of cow's milk, αs1-casein, a single-chain phosphoprotein, formerly reposed to have arginine as an NH2-termimal, and Leu-TrpOH as COOH-terminal residues. ryptic digestion of maleyl αs1-casein (genetic variant B), in theory bruited to arginyl bonds, yields eight peptidic fragments, Tm1 to Tm8, which were fractionated by column chromatography and, in some cases, more thoroughly purified by preparative paper electrophoresis and/or chromatography. The ammo acid composition and the phosphate content of these fragments were determined. The fragments Tm3 and Tm8 arise from a partial non specific cleavage of Tm5. Thus, six fragments only—2, 4, 5, 6 and 7—arise from the specific cleavage of the αs1-casein chain at arginyl bonds, a result in accordance with the reported number of six arginyl residues in the chain, one being NH2-terminal. The fragment Tm7, with two arginyl residues, and Tm2 devoid of arginine appear to rcpresent respectively the NH2- and COOH-terminal parts of the αs1-casein chain. These peptidic fragments—except Tm4 which is devoid of lysine—were further digested by trypsin at lysyl bonds, after eliminating the blocking maleyl groups. The resulting peptides were purified by the same methods as mentioned above, and the composition of these tryptic peptides was established. As a control, the tryptic peptides of αs1-casein were simultaneously purified by the same methods from a hydrolysate of the protein which had not been treated with maleic anhydride. In summary, this first step of our work gives: (a) the composition of ail the bovine αs1-casein tryptic peptides; (b) partial information on the location of these peptides in the protein chain: (c) confirmation that the molecular weight of the bovine αs1-casein monomer is approximately 23600. [ABSTRACT FROM AUTHOR]

The partial ammo acid sequence of dihydrofolate reductase (DHFR. EC 1.5,1.3) from human KB/6b cells hi been determined by using 3.5 mg of protein. Peptides covering the entire polypeptide chain were recovered from preparative peptide maps generated by the combination of paper chromatography and electrophoresis at pH 4.4 Peptide maps from mouse L1210 DHFR were also generated for comparison. Amino acid sequence of 75% of the 186 amino acid residues in the polypeptide chain of human KB/6b DHFR was obtained from Edman degradation and the remaining sequence was deduced from the amino acid compositions, from electrophoretic mobilities of related peptides and from the sequence homologies with other known mammalian DHFR sequence. A comparison of the proposed human DHFR sequence with the previously known sequent of mouse enzyme [Stone, et al. (1979) J. Biol. Chem. 245, 480-488] indicates that 18 differences are located in the established sequence of 139 residues and that 5 additional differences are in the tentative sequence of the remaining 47 amino acids. Kinetic properties of human KB/6b and mouse L1210 DHFR, which were determined in parallel experiments, are also compared. The possible structural-functional relationships between human and mouse DHFR are discussed. [ABSTRACT FROM AUTHOR]

The amino acid sequence of the heme-binding domains of rat liver cytochromes b5 from outer mitochondrial membranes and from microsomes has been determined by a combination of automatic and manual degradation of fragments generated by trypsin digestion and by cleavage at tryptophan. Tryptic peptides were separated by high-pressure liquid chromatography. The sequence of microsomal cytochrome b5 is identical with the one published by Owls and Heinemann after completion of this study [Biochim. Biophys. Acta (1982) 704. 163- 173]. The sequence of outer membrane cytochrome b5 differs from the microsomal one at 38 positions out of 91. There are 40 positions invariant between this sequence and the eight micrasornal sequences published thus far. The non-conservative substitutions are located at the surface of the known three-dimensional structure of calf microsomal cytochrome b5 except for the substitution of histidine- 15 by arginine. This paper brings the final proof that two iso-cytochromes b5- exist in the same cell. Their high degree of similarity as well as their differential cellular localization raise some questions which are briefly discussed. [ABSTRACT FROM AUTHOR]

The results reported in the present paper and N-terminal sequence homologies established by other authors strongly support the assumption that the gastric protease previously called bovine pepsin I or bovine pepsin B belongs to the aspartate protease group and corresponds to gastricsin (or pepsin C) (EC 3.4.23.3) from other species. Bovine gastricsin was prepared from commercial extracts of adult bovine veils by a procedure involving DEAE-cellulose chromatography, gel filtration on Sephacryl S-200 and a further DEAE-cellulose chromatography. The preparation thus obtained was shown to be free of chymosin and bovine pepsin A by immunodiffusion, selective inactivation in urea and isoelectric focusing. Us molecular weight was estimated by gel filtration to be 32800. Bovine gastricsin displayed microheterogeneity on isoelectric focusing with pi values of the components ranging from 3.5 to 4.0. Chromatography of bovine gastricsin on hydroxyapatite separated three fractions, none of them being homogeneous by isoelectric focusing. Concanavalin-A—Sepharose 4B bound bovine gastricsin to some extent, but without any significant fractionation. Proteolytic activity could be detected directly on the isoelectric focusing gel for all the components of gastricsin and its fractions from hydroxyapatite and concanavalin-A—Sepharose 4B. Bovine gastricsin and its fractions from hydroxyapatite have similar amino acid compositions, different from those of bovine chymosin and pepsin A but obviously related to those of human, simian and porcine gastricsins. Bovine gastricsin which is inactivated by reaction with diazoacetyl-DL-norleucine methyl ester and with 1,2-epoxy-(p-nitrophenoxy)propane in a 1:1 and 1:2 stoichiometry, respectively, is able to hydrolyse a synthetic hexapeptide, Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe, used as reference substrate for aspartate proteases, and exhibits a low activity towards N-acetyl-L-phenylalanyl-L-diiodotyrosine. Its specific clotting activity with χ-casein as substrate is only half of that of chymosin and pepsin A. [ABSTRACT FROM AUTHOR]

A low-molecular-weight and a high-molecular-weight 1,4-β-glucan glucanohydrolase (Cχ enzyme) have been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus THchoderma vMde. The purification method for the isolation of the low-molecular-weight enzyme is a three-step procedure including chromatography on Bio-Gel P-10, chromatography on a dipolar adsorbent (arginine-fiepharose 6 B) and isoelectric focusing. The starting material for the isolation of the high-molecular-weight enzyme was pre-fractionated by chromatography on Bio-Gel P-10, by DEAE-Sephadex chromatography and by SE-Sephadex chromatography as described previously by us. Further fractionation of this material was achieved by affinity chromatography and repeated isoelectric focusing. Free zone electrophoresis of the low-molecular-weight enzyme indicated a homogeneous protein. The high-molecular-weight enzyme was homogeneous in sedimentation equilibrium analysis. The molecular weights of the enzymes were 12 500 and 50 000 ± 2000 respectively. The former value was determined by chromatography on a calibrated column of Bio-Gel P-100 and the latter value by sedimentation equilibrium analysis. The low-molecular-weight enzyme was isoelectric at pH 4.60 (10°C) and contained 21% carbohydrate. The corresponding values for the high-molecular-weight enzyme were pH 3.39 and 12%. Both enzymes were active in releasing free fibers from filter-paper. The low-molecular-weight enzyme was estimated to be about twice as effective as the high-molecular-weight enzyme in this regard. [ABSTRACT FROM AUTHOR]

Our previous studies [1,2] on maleyl bovine αS1-B casein were concerned with the isolation and amino acid composition of the tryptic peptides designated as Tm peptides. The sequence of the COOH-terminal Tm peptide containing 48 residues was also reported. In the present study, we report the overlaps of all the cyanogen bromide peptides as well as the Tm peptides from αs1-B casein. The αs1-B casein which contains five methionyl residues was cleaved with cyanogen bromide. The six expected peptides obtained by this treatment and designated as CN peptides, were separated initially on Dowex-50X2 and further purified by Sephadex column chromatography. The amino acid composition of these six purified CN peptides was determined. The results were in perfect agreement with the amino acid composition of αs1-B casein deduced from the analysis of Tm peptides. The three Tm peptides from αs1-B casek@ which contained methionyl residues were also cleaved by cyanogen bromide. The fragments obtained were purified by either paper chromatography or Sephadex column chromatography and the amino acid composition of these purified peptides was determined. Of the eight peptides thus obtained, three were found identical with the CN peptides obtained directly by BrCN treatment of the αs1-B ease@. The remaining three CN peptides from αs1-B casein for which identical fragments could not be located fun the Tm peptides, were digested with trypsin (in two eases after maleylation) and the resulting fragments were purified by Sephadex column chromatography. These trifle peptides provided the remaining gaps. This permitted in to locate the relative position of all the CN peptides in the αs1-B casein molecule. These results also indicate the location of all the Tm peptides except 2 in the αs1-B casein molecule. Further, the chymotryptic peptides from one of the CN fragments of αs1-B casein provided the missing overlaps. [ABSTRACT FROM AUTHOR]

In order to determine the primary structure of the bovine β-caseins, the A2 variant was first cleaved with trypsin and cyanogen bromide (CNBr). The tryptic hydrolyzate was separated by column chromatography on Dowex-50. After additional purifications, 13 peptides numbered Ti to T13 were isolated. The 14th tryptic peptide, T14, not eluted from the column, was directly obtained from the hydrolyzate by preparative paper electrophoresis. Amino-acid analyses were carried out on all these peptides. Peptide T1 contain 2 Arginyl residues, one of which is NH2-terminal. This peptide represents the NH2-terminal end of the β-casein. Its composition is identical to that reposed by Peterson et al. in 1958 [1] for a phosphopeptide isolated from the β-casein. Peptide T11 contains 2 Lysyl residues, one of which is NH2terminal. The COOH-terminal peptide was identified with peptide T4, devoid of basic aminoacids. The 14 tryptic peptides account for all the residues of the β-casein A2 when compared with the already published amino-acid analyses of the whole protein [2, 3]. Several other peptides, originating from incomplete or non-specific cleavages, were also obtained. Similarly, 7 peptides accounting for the 6 methionyl residues of the protein were obtained from the same variant of β-casein after cleavage with cyanogen bromide. Six of these peptides were separated on Sephadex G-50. The seventh peptide was obtained by chromatography on Dowex-50 of the whole CNBr-digest. After additional purifications, the ammo-acid compositions of the 7 pure peptides were determined. Peptide CN1 contains peptide T1. It represents the NH2terminal end of β-casein. The COOH-terminal end of β-casein was identified with peptide CN7. As in the case of the tryptic peptides, the 7 CNBr-peptides account for all of the amino-acid residues of β-casein A2. β-casein A2 has 208 amino-acid residues. Its molecular weight is very close to 24 000. [ABSTRACT FROM AUTHOR]

Poly(A)-specific ribonuclease was co-purified with poly(A) polymerase from Vigna unguiculata seedlings. Both activities were separated into two forms (enzymes 1 and II) by a final hydrophobic column chromatography. The enzyme I preparation, which was homogeneous as examined by SDS/PAGE, had both poly(A) polymerase and poly(A)-specific ribonuclcase activities. The antibody raised to the enzyme I preparation precipitated both enzyme activities. These indicate that a single polypeptide (Mr 63 000) is responsible for both poly(A)-polymerizing and poly(A)-hydrolyzing activities. The poly(A)-specific ribonuclease was a 3'-exonuclease specific to single-stranded poly(A), forming 5'AMP as the sole reaction product. The hydrolytic activity required either Mn2+ or Mg2+ with different optimum concentrations, whereas the polymenzing activity required Mn2+ but not Mg2+ ATP and PPi had little or no effect on the poly(A)-specific ribonuclease activity. [ABSTRACT FROM AUTHOR]

1. Different types of bacteriorhodopsin chromophores and their reactions are described. This includes intermediates of the reconstitution reaction of the purple complex, intermediates of the photochemical cycle and a photochemically active 500-nm chromophore. In contrast to the native chromophore (purple complex) some of these species are reducible by borohydride yielding stable retinyl proteins. 2. Hydrolysis and/or extraction of the retinyl proteins reveals that in the corresponding non reduced parent chromophores the retinal is bound either noncovalently or covalently to lysyl residues of the polypeptide chain. 3. Retinol, retinyl lysine and their retro isomers isolated from the various reduced chromophores are identified by thin-layer chromatography and mass spectrometry. 4. The absorption spectra of the retinyl proteins and binding studies with retinol and retinal indicate that the cyclohexene ring and the side chain of the retinyl moiety are forced into a coplanar conformation by interaction with the protein. The three peaked absorption band of the reduced chromophores is due to this planarized conformation and not to a retro configuration of the retinyl moiety. 5. Isomerization to retro compounds can be achieved by HCl as is known to be the case for retinyl compounds in solution. In addition photochemical isomerization is observed in the case of planarized retinyl moieties. Thus the native protein structure is responsible not only for a specific conformation of the retinyl moiety but also for its specific reactivity. [ABSTRACT FROM AUTHOR]

Glycogen synthase a was purified over 500-fold by a procedure which involved solubilisation of the enzyme from a protein-glycogen complex by the action of endogenous phosphorylase and debranching enzyme, followed by DEAE-cellulose chromatography, and either gel filtration on Sepharose 4B or fractionation with polyethylene glycol. 15 mg of protein could be obtained from 1000 g of muscle in five days, corresponding to a yield of 20%. The purity was over 90% as judged by gel electrophoresis and ultracentrifugal analysis. The amino acid composition was determined and the absorption coefficient, A1%280nm, measured refractometrically was 13.4. Glycogen synthase a sedimented as two major components, both of which were enzymatically active. The smaller species (13.3S) comprised 85% and the larger species (19.0S) 15% of the material. The molecular weight of the 13.3-S component was determined to be 377000 by high-speed sedimentation equilibrium centrifugation. The subunit molecular weight measured by gel electrophoresis in the presence of sodium dodecylsulphate was 88000 indicating that the 13.3-S species is a tetramer. The properties of the enzyme are compared to those obtained by other workers. [ABSTRACT FROM AUTHOR]

A low molecular weight product (Mr 7000) synthetized on rat liver mitoribosomes is selectively extracted by chloroform/methanol (&frac21;). This products characterized by an unusual hydrophobicity, as reflected by its chromatographic behaviour, its high content in hydrophobic amino acids and its strong capacity to bind lipids. Bound lipids contain a high percentage of linoleic acid. [ABSTRACT FROM AUTHOR]

For the elucidation of the amino-acid sequence of the carboxyl-terminal region of soybean trypsin inhibitor (Kunitz), fragments C and D were digested with trypsin, and the resulting peptides were separated by ion-exchange chromatography on Dowex 50X2 or by gel filtration on Bio-Gel P-4. Further fractionation and purification of the peptides were performed by ion-exchange chromatography on Dowex 1X2, by gel filtration on Bio-Gel P-2 or by high-voltage paper electrophoresis at pH 1.9 and 3.6. Three peptides were obtained in pure form from fragment C and ten peptides from fragment D, and their amino-acid sequences were determined by the direct Edman method and by carboxy- peptidase digestion technique. Overlapping peptides necessary for the alignment of the tryptic peptides from fragment D were obtained from a chymotryptic hydrolysate of fragment D. Nine main peptides and rune minor peptides were obtained. The amino acid composition and the partial amino-acid sequence of the 18 chymotryptic peptides of fragment D made it possible to establish the amino-acid sequence of the carboxyl-terminal region of the inhibitor. The complete amino-acid sequence of soybean trypsin inhibitor (Kunitz) deduced here was compared with that of Bowman-Birk inhibitor, another well-known soybean proteinase inhibitor. [ABSTRACT FROM AUTHOR]

For the elucidation of amino acid sequence around the reactive site of soybean trypsin inhibitor (Kunitz), fragments A and B were digested with trypsin, and the resulting peptides were separated by ion-exchange chromatography on Dowex 50X2 or by gel filtration on Bio-Gel P-4. Further purification of the peptides was carried out by gel filtration on Sephadex G-25 or by high-voltage paper electrophoresis at pH 3.6 and 6.5. Nine peptides were obtained in pure form from fragment A and three peptides from fragment B, and their amino acid sequences were determined by the direct Edman method and by carboxy-peptidase digestion technique. Overlapping peptides necessary for the alignment of the tryptie peptides from fragments A and B were obtained from a chymotryptic hydrolysate of fragment AB by gel filtration on Bio-Gel P-4, and their ammo acid compositions and sequence analyses of some peptides made it possible to establish the amino acid sequence of an amino-terminal region of the inhibitor consisting of AB amino acid residues. The reactive site (Arg63-Ile64) of the inhibitor to trypsin was involved in this region. [ABSTRACT FROM AUTHOR]

The purification of eleven neurotoxins from the venom of three scorpions (Androctonus australis Hector, Buthus occitanus tunetanus and Leiurus quinquestriatus quinquestreatus) has been performed by Sephadex G-50 filtration (with and without recycling) and equilibrium chromatography on Amberlite CG-50, CM-Sephadex C-50 and DEAE-Sephadex A-50. All the chromatographic steps were carried out in ammonium acetate buffers. Purity of the neurotoxins was assessed by amino acid analysis and zone electrophoresis. All have a molecular weight of about 7000 daltons and do not contain methionine. As shown by amino acid composition and chain length scorpion neurotoxins show a high degree of homology. [ABSTRACT FROM AUTHOR]

A new sulfur-rich basic polypeptide. so called ω-hordothionin, has been isolated from barley endosperm by extractions with NaCl and ammonium bicarbonate followed by reverse-phase high performance liquid chromatography. Purified ω-hordothionin was found to be homogeneous by SDS/polyacrylamide gel electrophoresis, N-terminal amino-acid sequencing and electrospray-ionization mass spectrometric analysis. The complete primary structure of ω-hordothionin was determined by automatic degradation of the intact molecule and peptides obtained by proteotytic cleavage. ω-hordothionin consists of a single polypeptide chain of 48 amino acids with a molecular mass of 5508 Da deduced from its amino acid sequence, which fully coincides with the 5508.2 Da determined by elcctrospray-ionization mass spectrometry. The isolated polypeptide showed a characteristic composition with a high content of basic amino acids (five arginine residues, two lysine residues and six histidine residues) and eight cysteine residues. and has strong sequence identity (66%) with the sorghum SIα1 α-amylase inhibitor. ω-hordothionin. like γ-hordothionin. exhibited translation inhibitory activity on both cukaryotic cell-free systems from mammalian (rat liver and rabbit reticulocyte lysates) and prokaryotic cell-free systems (Escherichia coli). However, in contrast to γ-hordothiomn, ω-hordothionin did not inhibit plant systems such as Triticum aestivum, Cucumis sativus, Vicia sativa and Hordeum vulgare. ;γ-hordothionin also inhibited the α-amylase activity from human saliva, while ω-hordothionin and the other different genetic variants of thionins, α- hordothionin and β-hordothionin. failed to show any inhibitory effect. [ABSTRACT FROM AUTHOR]

The cDNA clones corresponding to soybean β-amylase mRNA were isolated and sequenced. The cDNA contained an open-reading frame composed of 496 amino acids. The comparison of the amino acid sequence deduced from the cDNA with the N-terminal peptide sequence from mature enzyme proved that β-amylase had no leader sequence. Employing the cDNA, the β-amylase was directly synthesized in Escherichia coli by the expression coli by the expression vector pKK233-2 controlled by the tac promoter. The enzyme activity detected in E. coli lysate drastically increased with a lower cultivation temperature, and the total activity and specific activity of the enzyme in E. coli lysate cultured at 13°C was 130-fold and 280-1bid. respectively, the value at 37°C. The enzyme produced in E. coli was purified by the affinity column chromatography of cyclomaltohexaose-immobilized Sepharose 6B. Employing the established expression and purification system of the enzyme, the functional ionizable groups in the active site were searched. His93. involving an imidazole, and Asp348. involving a carboxylate, in the highly conserved regions within the β-amylases were replaced by Arg (H93R) and Asn (D348N) by site-directed mutagenesis, respectively. All β-amylases. Including the non-mutant and mutant β-amylases, produced in E. coli exhibited lower Vmax values than that of β-amylase isolated conventionally from soybean seeds. Especially the Vmax value of IH93R]β-amylase was reduced drastically compared to that of the non-mutant: however, none of them lost their enzyme activities completely. Therefore. neither His93 nor Asp348 may participate in the catalytic reaction directly. [ABSTRACT FROM AUTHOR]

A purification procedure consisting of DEAE-Sephacel chromatography, heparin-Sepharose CL-6B chromatography and Mono-S chromatography led to the isolation of three proteins endowed with RNase activity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. They were referred to as p1, p2 and p3, according to their elution order from the Mono-S column. Complete amino acid sequence of p2 and partial sequence of p3 displayed high sequence similarity to the 7-kDa DNA-binding proteins previously isolated in Sulfolobus strains [Choli, T., Wittman-Liebold, B. & Reinhardt, R. (1988) J. Biol. Chem. 263, 7087-7093]. The molecular mass of p2, calculated from sequence data, was 7.02 kDa, which compares fairly well with the value of 7.4 kDa determined by SDS/PAGE. Gel filtration of the molecule under native conditions displayed, however, a largely prevailing form with an assessed molecular mass of 13.0 kDa, which points to a dimeric structure. Kinetic characterization of protein p2 showed a broad pH optimum in the range 6.7 - 7.6 using yeast RNA as substrate; also, it was shown that activity was unaffected by EDTA, Mg2+ and phosphate. The enzyme did not accept as substrate any homopolyribonucleotide, which points to a rather narrow substrate specificity. This was also confirmed by incubating p2 with tRNAfMet Met (fMet, N-formylmethionine) from Escherichia coli: the hydrolysis products were thus identified as 3'-phosphooligonucleotides. [ABSTRACT FROM AUTHOR]

Genes encoding pre-protein and prepro-protein of wheat germ agglutinin isolectin 2 (WGA2) were chemically synthesized and expressed in the yeast $accharomyces cerevisiae under the control of the ENO1 promoter. Yeast harboring either a pre-WGA2 or a prepro-WGA2 gene expression plasmid secreted a mature form of WGA2 into the culture medium. The amount of WGA2 secreted by the strain KS58-2Ddel, which has a ssll mutation causing a supersecretion of human lysozyme [Suzuki, K., Ichikawa, K. & Jigami, Y. (1989) Mol. Gen. Genet. 219, 58–64], was 20-fold greater than that secreted by the wild-type strain KK4. The recombinant WGA2 from the cells containing the prepro-WGA2 gene expression plasmid was purified to homogeneity by a three-step ion-exchange chromatography scheme. As in wheat, the N-terminal signal peptide of recombinant WGA2 purified from yeast culture was processed to form an N-terminal 5-oxoprolyl (pyroglutamyl) residue. Likewise, we found that the C-terminal pro-region of recombinant WGA2 had also been processed in yeast. Using electrospray ionization mass spectrometry, we found the processed C-terminus to be heterogeneous in both recombinant WGA2 purified from yeast and in authentic WGA2. The major component of the recombinant WGA2 contained two additional amino acids at its C-terminus compared to that of authentic WGA2. In spite of this difference in the C-terminus, the recombinant WGA2 exhibited a sugar binding activity that was indistinguishable from that of authentic WGA2. [ABSTRACT FROM AUTHOR]

Thermus sp. strain Rt41A produces an extracellular thermostable alkaline proteinase. The enzyme has a high isoelectric point (10.25–10.5) which can be exploited in purification by using cation- exchange chromatography. The proteinase was purified to homogeneity and has a molecular mass of 32.5 k Da by SDS/PAG E. It is a glycoprotein, containing 0.7% carbohydrate as glucose equivalents, and has four half-cystine residues present as two disulphide bonds. Maximum proteolytic activity was observed at pH 8.0 against azocasein and greater than 750 of this activity was retained in the pH range 7.0–10.0. Substrate inhibition was observed with casein and azocasein. The enzyme was stable in the pH range 5.0–10.0 and maximum activity, in a 10-min assay, was observed at 90°C with 5 mM CaCl2 present. No loss of activity was observed after 24 h at 70°C and the half-lives at 80°C and 90°C were 13.5 h and 20 min, respectively. Removal of Ca2+ reduced the temperature for maximum proteolytic activity against azocasein to 60°C and the half-life at 70°C was 2.85 rain. The enzyme was stable at low and high ionic strength and in the presence of denaturing reagents and organic solvents. Rt41A proteinase cleaved a number of synthetic amino acid p-nitrophenol esters, the kinetic data indicating that small aliphatic or aromatic amino acids were the preferred residue at the PI position. The kinetic data for the hydrolysis of a number of peptide p-nitroanilide substrates are also reported. Primary cleavage of the oxidized insulin B chain occurred at sites where the P1′ amino acid was aromatic. Minor cleavage sites (24 h incubation) were for amino acids with aliphatic side chains at the P1′ position. The esterase and insulin cleavage data indicate the specificity is similar for both the P1 and P1′ sites. [ABSTRACT FROM AUTHOR]

The squalene-hopene cyclase from Bacillus acidocaldarius cytoplasmic membrane, was purified to homogeneity by solubilization with Triton X-100, chromatography on DEAE-cellulose, phenyl Sepharose and two gel-filtration columns. The enzyme monomer had a molecular mass of 75 kDa. The sequence of the first 23 amino acids was determined by Edman degradation. The enzyme activity was efficiently inhibited by n-alkyldimethylammonium halides with alkyl chain lengths between 12 and 18 C atoms. Inhibition was also observed with (5-hydroxycarvacryl)trimethylammonium chloride 1-piperidine carboxylate, dodecyldimethylamine N-oxide, azasqualene and farnesol. Competitive inhibition with dodecyltrimethylammonium bromide, (5-hydroxycarvacryl)trimethylammonium chloride 1-piperidine carboxylate and dodecyldimethylamine N-oxide was demonstrated by Lineweaver-Burk plots. [ABSTRACT FROM AUTHOR]

High-mobility-group protein 17 (HMG-17) was identified by reversed-phase high-performance liquid chromatography analysis as a major component in acidic extracts of transplantable rat glucagonoma tissue but not in insulinoma tissue of similar origin. The peptide was purified in a single step and the entire sequence of 89 amino acids was determined. Rat HMG-17 has a molecular mass of 9238 Da and shows strong similarity to human, bovine (94.4%) and chicken (88.8%) HMG-17. Six of the seven residues which vary among the mammalian sequences are located within a short segment (positions 64–83) present in the acidic, non-DNA-binding C-terminal part of HMG-17. This region shows least similarity to the otherwise related proteins HMG-14 and H6 (a trout HMG protein). Interestingly, four of the six variable positions are Asp in rat HMG-I 7 which results in an overall net increase in the negative charge of the C-terminal region. The nature of selective hyper-expression of HMG-17 in glucagon but not in insulin-producing tumor tissue remains to be clarified. [ABSTRACT FROM AUTHOR]

A thermophilic and thermostable/β-galactosidase activity was purified to homogeneity from crude extracts of the archaebacterium Sulfolobus solfataricus, by a procedure including ion-exchange and affinity chromatography. The homogeneous enzyme had a specific activity of I 16.4 units/mg at 75°C with o-nitrophenyl β-galactopyranoside as substrate. Molecular mass studies demonstrated that the S. solfataricus β-galactosidase was a tetramer of 240 ± 8 kDa composed of similar or identical subunits. Comparison of the amino acid composition of β-galactosidase from S. solfataricus with that from Escherichia coli revealed a lower cysteine content and a lower Arg/Lys ratio in the thermophilic enzyme. A rabbit serum, raised against the homogeneous enzyme did not crossreact with β-galactosidase from E. coli. The enzyme, characterized for its reaction requirements and kinetic properties, showed a thermostability and thermophilicity notably greater than those reported for β-galactosidases from other mesophilic and thermophilic sources. [ABSTRACT FROM AUTHOR]

Pravastatin sodium (CS-514) is a tissue-selective inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme in cholesterol biosynthesis. This compound is obtained by microbial hydroxylation of sodium ML-236B (compactin) carboxylate. The soluble cytochrome P-450 was induced by sodium ML-236B carboxylate in Streptomyces carhophilus of Actinomycetes as detected in its cell-free extract. This cytochrome P-450 was designated as cytochrome P-450sca after its origin. Cytochrome P-450sca was purified by successive chromatography on anion-exchange, gel filtration and hydroxyapatite columns. On hydroxyapatite cytochrome P-450sca was further separated into minor and major peaks, designated cytochrome P-450sca-1 and cytochrome P-450sca-2, respectively. Each peak yielded a single band on sodium dodecyl sulfate/polyacrylamide gels with molecular masses of 46 ± 1 kDa. The activity hydroxylating sodium ML-236B carboxylate to pravastatin sodium was reconstituted in the presence of an electron transport system, an NADPH-generating system and oxygen. The Ks values of the cytochromes P-450sca-1 and P-450sca-2 for sodium ML-236B carboxylate were 179 μM and 229 μM, respectively. The CO versus reduced difference spectra of both cytochromes P-450 showed an absorption maximum at 448.5 nm. Their substrate difference spectra with sodium ML-236B carboxylate showed an absorption maximum at 386 nm. Amino acid analysis indicated that cytochrome P-450sca-1 and P-450sca-2 contained 46% and 47% hydrophobic residues, respectively. On Western blotting, cytochromes P-450sca-1 and P-450sca-2 were immunologically identical. [ABSTRACT FROM AUTHOR]

Tyrosine-protein kinase, phosphorylating tyrosine residues of transmembrane band 3 protein, has been partially purified from human erythrocyte cytosol by DEAE-Sepharose chromatography followed by heparinSepharose chromatography. Such a Tyr-protein kinase (36 kDa), as distinct from the Ser/Thre-protein kinases (casein kinase S and TS), appears to display a broader site specificity than does the previously described human erythrocyte P-Tyr-protein phosphatase, dephosphorylating band 3 protein. That is, it is able to phosphorylate not only the highly acidic copolymer poly(Glu-Tyr)4:1 but also angiotensin II, lacking an acidic amino acid sequence around the target Tyr residue. Moreover, the phosphorylation of these two substrates exhibits a different pH dependence and a different response to NaC1 and 2,3-bisphosphoglycerate. These results suggest that in intact erythrocytes the cytosolic Tyr-protein kinase might phosphorylate band 3 not only on Tyr-8, surrounded by several acidic side-chains (as demonstrated preferentially to occur in isolated ghosts), but also on other Tyr residues surrounded by other amino acid sequences. [ABSTRACT FROM AUTHOR]

The toxin preparations ATX I, ATX Ia and ATX Ib from Anemonia sulcata were investigated by proton nuclear magnetic resonance (NMR). High-resolution phase-sensitive two-dimensional NMR experiments were used to monitor the separation by high-performance liquid chromatography of the two isoproteins AIX la and ATX Ib. For ATX Ia complete sequence-specific resonance assignments were obtained and the secondary structure was determined. To obtain the NMR assignments we used a variant of the sequential assignment technique which relied extensively on cross-peak fine-structure analysis in phase-sensitive spectra, using spectrum simulations based on density matrix calculations with the program SPHINX. These procedures, which resulted in extensive amino acid spin system identifications prior to the sequential assignments, should be generally applicable for small proteins with relatively narrow ¹H-NMR lines. The secondary structure of ATX I includes a β sheet consisting of four strands. No evidence was found for the presence of regular helical segments. The four β strands are connected by two extended loops and a tight turn, for which further characterization has to await the complete determination of the three-dimensional structure. [ABSTRACT FROM AUTHOR]

A juvenile-hormone-binding protein (JHBP) has been isolated from Galleria mellonella hemolymph by gel filtration, phosphocellulose chromatography, and by chromatofocusing. The isolated protein is homogenous as judged by column chromatography and gel electrophoresis in the presence and absence of denaturing agent. It has a relative molecular mass of 32 000, Stokes radius 2.4 nm, sedimentation coefficient of 2.3 S, molar absorption coefficient at 280 nm ε = 2.34 x 104 M-1 cm-1, and is composed of a single polypeptide chain. Chromatofocusing analysis (pI 8.6) and isoelectric focusing (pI 8.1) indicate that the JHBP is an alkaline protein. Its amino acid composition and fluorescence absorption spectra indicate that the protein does not contain tryptophan residues. The protein exhibits one class of binding sites for juvenile hormone (JH), 0.8 per molecule, with the following dissociation constants: JH I, 8.5 × 10-8 M; JH II, 7.2 x 10-8 M; JH III, 47 x 10-8 M. The JHBP binds (10R, 11S)-JH II enantiomer with 2.3-times higher affinity then (10S, l lR)-JH II enantiomer. The pH optimum of binding is 7.0. [ABSTRACT FROM AUTHOR]

Trypanosoma brucei has two phosphoglycerate kinase (PGK) isoenzymes, one is particle-bound and localized in glycosomes while the other is present in the cytosol. The cytosolic isoenzyme (cPGK) was 900-fold purified from cultured procyclic trypanosomes by hydrophobic interaction chromatography on phenyl-Sepharose followed by affinity chromatography on 2',3'-ATP-Sepharose and had a specific activity of 275 units/rog protein, cPGK was compared with the purified glycosomal isoenzyme (gPGK) from bloodstream-form trypanosomes as well as with the commercially available PGKs from yeast, rabbit muscle and Spirulina platensis, a blue-green alga. Like all other PGKs, cPGK was a monomeric protein with a molecular mass of approximately 45 kDa similar to that of the PGKs from other organisms but 2 kDa smaller than that of gPGK. Despite this difference in length and a great difference in isoelectric point, the two trypanosome isoenzymes strongly resembled each other in several respects. (a) The kinetic parameters did not differ significantly from each other or from the PGKs of other organisms. (b) Both trypanosome enzymes resembled the enzyme from S. platensis in that they had an almost absolute requirement for ATP, contrary to the enzymes from yeast and rabbit muscle, which were capable of utilizing GTP and ITP also. This difference in substrate specificity may be related to the amino acid substitutions, Trp 308→His and Ala 306→Glu in the adenine-binding site, which are only found in the two Trypanosorna isoenzymes. Kinetic analysis showed that these substitutions do not prevent binding of the ATP analogoues, but probably prevent phosphoryl-group transfer. (c) Both isoenzymes displayed an activity optimum at pH 6.0-9.0 similar to that for the enzyme of yeast. Both gPGK and cPGK were inhibited by the trypanocidal drug Suramin. This inhibition could be described as competitive both with ATP and 3-phosphoglycerate with two inhibitor molecules binding to one molecule of enzyme. The gPGK, however, was much more sensitive (Kiapp. = 8.0 µM) to Suramin than either the cPGK (Kiapp. = 20 µM) or the enzymes from rabbit muscle (Kiapp. = 55 µM), yeast (Kiapp. = 167 µM) or S. platensis (Kiapp. = 250 µM). It is suggested that positive charges on the enzyme's surface may play an important role in the potentiation of the binding of the negatively charged Suramin molecule. [ABSTRACT FROM AUTHOR]

Human protamine P2 was purified to homogeneity by solubilizing whole spermatozoa in guanidinium HCL containing 2-mercaptoethanol, alkylating the resulting protamine thiols with vinylpyridine, removing acid-insoluble material by acid dialysis and using CM-cellulose chromatography to remove non-protamine basic proteins and separate protamines P1 and P2. The P2 preparation contained two components, P2a and P2b, which were sequenced completely without being separated. The peptides obtained from thermolysin and endoproteinase Lys- C digestions were purified by reverse-phase high-pressure liquid chromatography and sequenced using a gas- phase sequencer. P2a contains 57 amino acids and has a relative molecular mass of 7636 while P2b contains 54 amino acids, which are identical to residues 4-57 of P2a, and has a relative molecular mass of 7242. Protamine P2a is approximately 50% homologous with human protarnine P1. The amino acid sequcnce of P2a is: [ABSTRACT FROM AUTHOR]

The adenylate kinase isoenzyme located in the intermembrane space of mitochondria, AK2, is a monomeric protein of Mt 30000 which catalyzes the reaction ATP + AMP ⇌ 2ADP. 1. The protein was reduced and S-carboxymethylated with iodo[14C2]acetate. Using a Laursen sequenator, the N-terminal sequence of S-carboxymethylated AK2 was determined as Ala-Pro-Asn-; in some batches of the isolated protein the N-terminal dipeptide portion was missing. The C-terminus of AK2 was found to be Met. 2. Cleavage with CNBr yielded eight fragments which could be isolated in one step using high-performance size-exclusion chromatography. They ranged in size over 4-88 amino acid residues, the total being approximately 270 residues. All CNBr fragments were overlapped with Met-containing tryptic peptides of AK2. 3. The N-terminal 111 residues of AK2 were sequenced. Except for an N-terminal extension of nine residues, this segment of AK2 could be aligned with the sequence 1-104 of cytosolic AK1. Allowing for two deletions in AK2, 43 of the 102 aligned residues are identical. Since this section contains the catalytic residues such as His-36 and Asp-93, we conclude that AK1 can serve as a three-dimensional model of AK2 in mechanistic and drug-designing studies. 4. Preliminary sequence results on AK2 beyond position 104 show that AK2 here contains a wing of approximately 50 residues which has no counterpart in AK1. The chain folds of the adenylate kinase isoenzymes are similar again from a position corresponding to residue 115 of AK1 onwards. The additional structural motifs of AK2 are probably related to the location of this isoenzyme in the mitochondrion. [ABSTRACT FROM AUTHOR]

Three fractions (DE-I, DE-II and DE-III) of Z-protein (fatty acid binding protein) have been isolated from rat liver cytosol by DEAE-cellulose chromatography and characterized. They had the same molecular weight of 14000 and essentially identical amino acid composition. However, composition of endogenous fatty acids were found to differ strikingly from one another. Long-chain fatty acids detected in DE-II were palmatic, stearic, oleic, linoleic and arachidonic acids. In contrast to DE-II, DE-III contained mainly arachidonic acid. Molar ratios of endogenous long-chain fatty acids to both DE-II and DE-III were estimated to be around 1.0. Unlike the latter two fractions, DE-I was virtually lipid-free. Analyses of the three fractions by polyacrylamide gel electrophoresis, electrofocusing and DEAE-cellulose chromatography before and after delipidation suggested that the difference between DE-I and DE-II was in part due to fatty acids bound to DE-II. In contrast, DE-III appeared to be somewhat different from these forms in its protein structure, though tryptic peptide mappings of the three fractions did not reveal clear differences among them. Analysis of the primary structure was made on the most abundant fraction, DE-II, to investigate the relationship among the three fractions and to other proteins. The protein was single chain consisting of 127 amino acid residues and had a mostly acetylated NH2 terminus and a free sulfhydryl group. The complete sequence of Z-protein showed striking homology to cellular retinoid binding proteins and peripheral nerve myelin P2 protein, which indicated the presence of a new family of cellular lipid-binding proteins diverged from a common ancestor. A possible intragenic duplication of Z-protein was also suggested. [ABSTRACT FROM AUTHOR]

tRNAArg and argiuyl-tRNA synthetase have been purified to homogeneity from brewer's yeast by chromatographic methods. Arginyl-tRNA synthetase is a monomeric enzyme with a molecular weight of 72000. Two active forms of the enzyme can be found, they are interconvertible. The more stable conformation is probably the natural one. Arginyl-tRNA synthetase seems to recognize arginine very specifically. No evidence for any proof-reading mechanism could be found. The steady-state mechanism is somewhat different from the types found with arginyl tRNA synthetase from other sources. However, all these results are compatible with a concerted reaction. Simultaneously with the release of AMP or pyrophosphate an allosteric rearrangement occurs. This conversion seems to be determining for the reaction mechanism. [ABSTRACT FROM AUTHOR]

Two forms of adrenodoxin reductase, differing in isoeleclric properties and thermal stability, have been highly purified from bovine adrenal cortex mitochondria. Both preparations appear to be homogeneous based upon polyacrylamide disc-gel electrophoresis, isoelectric focusing, and ion-exchange chromatography. Both are monomeric proteins containing one FAD per molecule (Mr 50000) and exhibit spectra typical of flavoproteins. The two preparations are indistinguishable by their amino acid compositions and N-terminal (serine) and C-terminal (leucine) amino acids, but reveal distinct sugar contents. Electrophoretic mapping of the protease digests of the two preparations indicates that the two have identical, or closely similar, primary structures. Each form functions equally as an adrenodoxin-dependent electron transport component from NADPH to the P-450 cytochromes in the two different reconstituted steroid monooxygenase systems which catalyze cholesterol side-chain cleavage reaction and steroid 11β-(18-)hydroxylase reaction, respectively. In all adrenodoxin reductasecontaining fractions from adrenal cortex so far examined, i.e. the soluble fraction, and the sonic-solubilized and detergent-solubilized mitochondrial fractions, a constant ratio of about 2:5 between the two forms is found as judged by the DEAE-cellulose chromatogram. No evidence has been obtained for mutual conversion of the two forms of the reductase, or for dissociation of the sugar moiety during the purification process and under various conditions examined. [ABSTRACT FROM AUTHOR]

Two trypsin inhibitors from the seed or Acacia elata, a legume of the subfamily Mimosoideae, were isolated by affinity chromatography on trypsin-Sepharose 48 and separated by chromatography on SP-Sephadex C-25 and Sephadex G-100. The two inhibitors, with molecular weights of about 20000, were composed of two polypeptide chains linked by a disulfide bond. The two inhibitors had essentially the same amino acid compositions and both contained four half-cystine residues, no methionine and were rich in aspartic acid, glutamic acid, glycine and leucine. The inhibitors had isoelectric points of 6.4 and 5.9. The inhibitors stoichiometrically inhibited trypsin in the molar ratio of 1: 1, α-chymotrypsin was inhibited also in a 1: 1 molar ratio but the binding of the enzyme by the inhibitor was weaker and the inhibitor-chymotrypsin complex dissociated during the assay. Both enzymes are probably inhibited at an identical site. Amino-terminal sequence analysis of the two polypeptide chains of the inhibitors revealed extensive homology with the trypsin inhibitor from the silk tree (another Mimosoideae legume); both these inhibitors are homologous with the soybean trypsin inhibitor (Kunitz). [ABSTRACT FROM AUTHOR]

High-molecular-weight (high-MT) kininogen was purified from horse plasma by chromatography on columns of DEAE-Sephadex A-50, CM-Sephadex C-50, p-chlorobenzylamine-Sepharose and Sephadex G-150. The yield was about 151 mg from 8.1 of fresh plasma. The purified material gave a single band on sodium dodecylsulfate/ polyacrylamide gel electrophoresis and a single precipitin line on immunodiffusion and immunoelctrophoresis. The molecular weight of horse high-MT kininogcn was estimated to be 78000 by dodecylsullate gel electrophoresis using the Ferguson plot. Its polypeptide content was determined to be 86% by amino acid analysis and there was a total of 581 amino acid residues/molecule of protein The kininogen contained a total of 13.9% carbohydrates, consisting of hexoses (7.8%), glucosamine (1.9%), galactosamine (0.6%) and sialic acid (3.6%). On incubation of horse high-MT, kininogen with bovine and horse plasma kallikreins, several fragments winch contained extremely high levels of histidine, were liberated, in addition to kinin. After the liberation of kinin and histidine-rich fragments, a protein free of kinin and its fragments was isolated. This protein consisted of two polypeptide chains, heavy chain and light chain, which are bridged by disullide bonds. The molecular weight and amino acid composition of the heavy chain anti the light chain from horse high-MT kininogen were very similar to those of the heavy and light chains from bovine high-MT kininogen. respectively. From these results, it was revealed that horse high-MT kininogen is quite similar to bovine high-MT kininogen in terms of their physicochemical and chemical properties, although they are immunologically distinguishable. [ABSTRACT FROM AUTHOR]

Two groups of crosslinked polyacrylic gels with immobilized lysine and lysine peptides (Lys)5 and (Lys)5-Pro have been used as models for the chromatographic investigation of lysine-peptide-oligonucleotide interactions. One group carries carboxylic groups in addition to the peptide residues in the gel matrix; the other gel type contains no such carboxylic groups in the gel matrix. Nucleotides of the series (dT)2–5, p(dT)1–4, p(dT)1–4p, (dA)2–5, p(dA)1–5 and p(dA)1–4p were chromatographed on these gels under various conditions in an aqueous buffer. On the gels of the first group the nucleotides were retarded only slighly, the positive charges of the e-amino groups being compensated partially or totally by the negatively charged carboxyl groups of the polymer matrix. On the gels of the second group, however, the oligonucleotides underwent specific interactions. These interactions were based primarily upon electrostatic forces between the positively charged ϵ-amino groups of the immobilized peptides and the negatively charged phosphate groups of the oligonucleotides. Our results indicate that, in addition to the electrostatic interaction, the conformation plays a crucial role. We explain the selectivity of the interaction with a conformation-fit mechanism. The origin of this mechanism, which creates specific interactions from unspecific forces, is discussed. [ABSTRACT FROM AUTHOR]

Enzymes can be reconstituted with high yield after dissociation and deactivation in strong denaturants. This finding can be utilized to separate proteases and other impurities (with molecular weights differing under denaturing conditions from the subunit molecular weight of the enzyme to be purified) from the desired enzyme. Applying this approach to tryptophan synthase from yeast, a homogeneous preparation with a molecular weight of 154000 ± 5000 and a specific activity of 1200 ± 100 Yanofsky units/mg has been obtained. The latter value exceeds the previously reported maximum activity by a factor of 10, and characterizes the enzyme in its native state. The Km values for D-glyceraldehyde 3-phosphate and indole of the indole-glycerolphosphate synthesis (reaction 2) and the pH optimum, as well as the Km values for L-serine, indole, and pyridoxal 5′-phosphate of the L-tryptophan condensation (reaction 3) have been determined. At least two tryptophan-synthase-inactivating proteinases from yeast have been well characterized in the past. Therefore, the low activity of previous preparations may be assumed to be caused by proteolytic cleavage. To prove this hypothesis, tryptophan synthase was prepared following a conventional procedure. Using ultracentrifugation and gel chromatography under quasi-physiological conditions, this enzyme has been found to be a homogeneous species of Mr = 75000 ± 3000 and s20, w = 3.3 ± 0.2 S, which is still capable of catalyzing both reactions 2 and 3 with a specific activity for the latter reaction of 120 Yanofsky units/mg. Comparing the Km values for all relevant ligands, only the Km value for pyridoxal 5′-phosphate shows a marked difference with respect to the value observed for the native enzyme. At -18°C the nicked enzyme is stable over a period of several weeks. As measured by sedimentation equilibrium in 6M guanidine · HCl at pH 2.3, the molecule consists of two large polypeptide fragments, Mr,1 = 22000 ± 3000 and Mr,1 = 50000 ± 5000. These results suggest tryptophan synthase from yeast to be a dimer of two bifunctional subunits; obviously the specific proteinases preferentially attack a region forming a link between two domains of the enzyme corresponding to the α and β subunits of the Escherichia coli enzyme. [ABSTRACT FROM AUTHOR]

A protein with properties similar to apoprotein A-IV of rat high-density lipoproteins has been isolated from the lipoprotein fraction (e < 1.006 g/ml) of individual non-fasting hypertriglyceridaemic subjects. The Mr of human apolipoprotein A-IV determined by polyacrylamide-gel electrophoresis in sodium dodecylsulfate and by column chromatography in 6 M guanidine hydrochloride is 46000. The amino acid composition is significantly different from any previously characterised human apolipoprotein but resembles that of rat apolipoprotein A-IV. The N-terminal amino acid of human and rat apolipoprotein A-IV is glutamate or glutamine. The pI of the major human A-IV band in 6 M urea is &ap; 5.15. Immunochemically apolipoprotein A-IV is not identical with any of the well known human apolipoproteins. Human apolipoprotein A-IV is present in all fractions of e < 1.006 g/ml isolated by a single ultracentrifugal spin from non-fasting subjects, but is lost from lipoproteins of e < 1.006 g/ml upon recentrifugation. Irnmunoelectrophoretic studies show that most of human apolipoprotein A-IV is present in the fraction of e < > 1.21 g/ml and is unassociated with the major lipoprotein fractions in serum. Charge- shift electrophoresis however clearly demonstrates the hydrophobic character of human apolipoprotein A-IV. There is evidence that apolipoprotein A-IV is a constituent of chylomicrons that is removed from the particle surface when chylomicrons enter the plasma compartment. [ABSTRACT FROM AUTHOR]

A procedure utilising affinity chromatography is described for the large-scale purification of pigliver catechol-O-methyl transferase. The enzyme prepared by this method appears to be homogeneous by polyacrylamide gel electrophoretic criteria and gel chromatography. It is stable for prolonged periods when stored at -5 °C in 20% (v/v) glycerol. The enzyme has a molecular weight of about 23 000 and does not appear to be a compound of subunits, or to associate to any appreciable degree. The pH optimum of the enzyme's activity is approximately pH 7.1-7.4, it does not catalyse the methylation of benzimidazole and has a Km of 0.64 mM and 0.056 mM towards 3,4-dihydroxyphenylacetic acid and S-adenosyl-L-methionine, respectively. Amino acid analysis showed the presence of five cysteine residues. [ABSTRACT FROM AUTHOR]

The properties of porcine follitropin and its subunits which have not yet been characterized are presented. The porcine follitropin obtained has a biological potency of 81 times the National Institutes of Health Porcine Follitropin P-1 preparation. Its contamination by lutropin and thyrotropin amounted to l and 0.5 percent by weight respectively, as measured by radioimmunoassay. The α and β subunits of porcine follitropin were obtained by incubation in an acidic urea solution followed by anion exchange chromatography. The amino acid composition of porcine follitropin α subunit was found to be identical to that of a chain of porcine lutropin and thyrotropin. These porcine α chains differ, nevertheless, markedly in their carbohydrate composition partic- ularly with respect to their mannose and galactose contents. The amino-terminal residue of the follitropin α subunit is threonyl. The carhoxy-terminal end of the α chain is of variable length. Cysteyl residue was detected at the aminoterminal end of the follitropin β chain with glutamic acid at its carboxy-terminal end. Cross-contamination of the α and β subunit preparations was measured by specific radioimmunoassay and amounted to 0.5 and 0.1 percent by weight respectively. [ABSTRACT FROM AUTHOR]

The molecular weight, the amino acid composition and the N-terminal and C-terminal amino acids of two allosteric (Lactobacillus casei, L. curvatus) and two non-allosteric (L. plantarum, L. aeidophilus) L-lactate dehydrogenases, purified to homogeneity by affinity chromatography, were determined. The amino acid composition of the only tryptic peptide unequivocally common to the fingerprints of the 4 enzymes is virtually identical with that of the arginine peptide, called Arg6 of the substrate-binding site of the L-lactate dehydrogenase of several animals. However, the 'essential' cysteine residue 165 is replaced by threonine, as it is in the L-lactate dehydrogenase of lobster. In addition, the 4 bacterial peptides differ by one or two changes in single amino acid residues from each other as well as from those of animals. The data indicate that not only the animal L-lactate dehydrogenases, but also the allosteric and the non-allosteric lactate dehydrogenases from bacterial sources may have evolved from a common gene. [ABSTRACT FROM AUTHOR]

The phospholipid exchange proteins of rat liver that catalyze the transfer of phosphatidylcholine and phosphatidylinositol from rat liver microsomes to liposomes, have been purified and characterized. Two proteins were detected with dual specificities catalyzing the transfer of both phosphatidylinositol and phosphatidylcholine. Both proteins showed a strong preference for phosphatidylinositol transferring 8 to 9 times as much of the microsomal phosphatidylinositol pool as the microsomal phosphatidylcholine pool. The two proteins had iso-electric points of 5.1 and 5.3 and were purified 300-fold and 500-fold. respectively. A protein that catalyzed specifically the transfer of phosphatidylcholine, was purified 7000-fold. This protein had an iso-electric point of 8.4 and a molecular weight of approximately 16000 calculated from Sephadex G-50 chromatography and sodium dodecylsulfate-polyacrylamide gel electrophoresis; the amino acid composition was determined. An antiserum against this protein was raised in rabbits. Treatment of a rat liver supernatant fraction with the antiserum immunoglobulin fraction demonstrated that 60%, of the phosphatidylcholine transfer activity is due to this protein. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus contains cell wall protein A as well as extracellular protein A. The two types of protein A have very similar amino acid compositions, electrophoretic mobilities and sizes. The release of extracellular protein. A from exponentially growing bacteria is dependent on protein synthesis de novo and protein A is released directly after being synthesized on the ribosomes. Bacteria in the stationary phase, however, release protein A as a result of cell lysis. Protoplasts have been isolated which produce protein A as extensively as the intact bacteria but because of the absence of cell wall all the protein A is of the extracellular type. In the presence of puromyein, an enhancement of the formation of extracellular protein A is observed from cells also producing cell wall protein A. [ABSTRACT FROM AUTHOR]

1. A simple method is described for the purification of phenylalanine hydroxylase using affinity chromatography. 2. Two forms of enzyme are separated by this method. One of these is only partially purified and the other is highly purified. Attempts to interconvert these forms were unsuccessful. [ABSTRACT FROM AUTHOR]

Lysozyme (mucopeptide N-acetylmuramylhydrolase) from Asterias rubens was obtained in a chromatographically and electrophoretically pure state by gel filtration and affinity chromatography. The quantitative amino acid composition, the molecular weight and the N-terminal sequence determined by a sequencer are reported. This new invertebrate enzyme presents important differences when compared to previously studied lysozymes. [ABSTRACT FROM AUTHOR]