Subunits of the alkaline phosphatase of Bacillus licheniformis: chemical, physicochemical, and dissociation studies. (1/91)

The alkaline phosphatase (orthophosphoric monoester phosphydrolase, EC 3.1.3.1) of Bacillus licheniformis MC14 was studied in an attempt to determine the number of subunits contained in the 120,000-molecular-weight native enzyme. Two moles of arginine was liberated per mole of native enzyme by carboxypeptidases A and B in the presence of sodium dodecyl sulfate. The effect on the native enzyme of progressively lowering the solvent buffer pH was monitored by determining the molecular weight by sedimentation equilibrium analysis, the sedimentation coefficient, the frictional coefficient, and the percent alpha-helix content of the enzyme. The alkaline phosphatase dissociates into two subunits around pH 4. At pH 2.8 a further decrease in S value, but no change in molecular weight, is observed, indicating a change in conformation. The frictional coefficients and percent alpha-helix content agree with this interpretation. A subunit molecular weight of 59,000 was calculated from sodium dodecyl sulfate gels.  (+info)

Chemical modification of yeast 3-phosphoglycerate kinase. (2/91)

Sulfhydryl reagents, as well as mild hydrogen peroxide oxidation, do not inhibit the activity of yeast phosphoglycerate kinase, indicating that the single thiol group and 3 methionine residues present in the enzyme are not essential for activity. Nitration of phosphoglycerate kinase by tetranitromethane inhibits the enzyme by reaction with a single tyrosine residue. Substrates provide partial protection against inactivation by nitration. Circular dichroism spectra indicate that no conformational changes occur upon nitration. However, perturbation of the microenvironment surrounding the aromatic amino acid residues, particularly tyrosine, was observed. The same perturbation was observed on addition of the substrate 3-phosphoglycerate kinase to native phosphoglycerate kinase. The role of lysine in the action of yeast phosphoglycerate kinase has been studied by modification with O-methylisourea, 2-methoxy-5-nitrotropone, and pyridoxal phosphate. Guanidination shows that there are lysines essential for phosphoglycerate kinase; extrapolation to zero activity indicates that there are three essential lysines as judged by nitrotroponylation and three essential lysines when the enzyme is reacted with pyridoxal phosphate. Substrates afford partial protection and extrapolation to total protection indicates that up to three lysines are protected by MgITP and one lysine by 3-phosphoglycerate. Spectrofluorescence and optical rotatory dispersion measurements show that there is no detectable conformational change for the guanidinated phosphoglycerate kinase and that there are slight changes in the spectra suggesting that there may be slight conformational changes for the nitrotroponylated and the pyridoxal phosphate-modified enzymes.  (+info)

Synthesis of pppGpN type dinucleotide derivatives: the 5' end sequence of some RNAs. (3/91)

A rapid and simple synthesis of pppGpN type /N equals C, U or A/ diribonucleotide derivatives is described by coupling guanosine 2', 3'-cyclic phosphate 5'-triphosphate with the appropriate ribonucleoside in the presence of ribonuclease T-1.  (+info)

The use of s-2-cyanoethyl phosphorothioate in the preparation of oligo 5'-deoxy-5'-thiothymidylates. (4/91)

An improvement of our strategy for the stepwise synthesis of oligo 5'-deoxy-5'-thiodeoxyribonucleotides [Chladek and Nagyvary (1972) J. Amer. Chem. Soc. 94, 2079] involves the use of 5'-O-tosylthymidine 3'-S-2-cyanoethyl phosphorothioate. The displacement of the tosylate by thymidine 3'-phosphorothioate and subsequent alkaline deblocking afforded the dinucleotide (Tps)2. The process of displacement and deblocking was repeated three more times at an average yield of 30 percent per step. The corresponding bifunctional derivative of deoxyadenosine was found much less reactive and practically unsuitable for repeated chain elongation. The ORD and CD spectra of the analogs are similar to those of the natural oligonucleotides.  (+info)

Broad-specificity proteinase inhibitors in Scopolia japonica (Solanaceae) cultured cells. Isolation, physicochemical properties, and inhibition kinetics. (5/91)

Proteinase inhibitors were isolated from Scopolia japonica cultured cells. Isolation procedures involve concentration by a hydrophobic resin of Diaion HP-20, decolorization by Duolite A-7, affinity chromatography on trypsin-Sepharose, and Bio-Gel P-4 chromatography. It was found that the proteinase inhibitors from S. japonica cells are a mixture of at least five components. For the inhibitory components except one, amino acid analyses, measurements of sedimentation equilibrium and optical rotatory dispersion (ORD) were carried out. The inhibitors were shown to be the polypeptides with molecular weights in the range of approximately 4000 to 6000. In addition, one of them was found to have approximately 15% alpha-helical conformation by the Moffitt-Yang analysis of ORD data. The inhibitors were found to have potent inhibitory activity for trypsin, chymotrypsin, plasmin, kallikrein and pepsin but not for papain with synthetic and natural substrates. These inhibitors formed stable complexes with trypsin and chymotrypsin in an equimolar ratio, and their inhibitory mechanisms for both enzymes were of non-competitive type.  (+info)

The extraction and characterization of bovine epidermal alpha-keratin. (6/91)

1. The alpha-fibrous protein (alpha-keratin) component of bovine epidermis has been extracted and characterized. 2. Prekeratin, a multichain unit of the epidermal tonofilaments, was shown to consist of six different polypeptide chains on polyacrylamide-gel systems containing sodium dodecyl sulphate or sodium decyl sulphate with discontinuous gel buffers, but only three chains were seen when a gel system containing sodium dodecyl sulphate with a continuous gel buffer was used. 3. Extraction of the 'keratinized' stratum corneum and the living part of the epidermis with urea buffers at pH 7.6 or 9.0 released 60% of the total dry weight of the tissues in the form of alpha-helical polypeptides. 4. The numbers, relative amounts and properties of the extracted polypeptides were the same as the subunits of prekeratin and thus are derived from the tonofilaments in situ. 5. The subunits of prekeratin and the polypeptides extracted from the living cell layers contained an average of six cysteine residues, but those from the stratum corneum contained an average of three intrachain disulphide bonds. 6. The polypeptide chains aggregated through non-covalent interactions in vitro into filaments that were similar to the tonofilaments. 7. Since the polypeptides could be released from the stratum corneum without breaking covalent bonds, it is concluded that such bonds do not cross-link the tonofilaments and non-fibrous keratohyalin. It is suggested that the tonofilaments and keratohyalin of bovine epidermis are associated by secondary bonding forces.  (+info)

Complexes of poly(adenylic acid) with complementary monomers. (7/91)

The interaction of a number of potentially complementary monomers with poly(A) has been investigated by equilibrium dialysis, optical rotatory dispersion and ultraviolet absorption measurements. Experiments were conducted at pH 7.0, 0.15 M Na+, where poly(A) exists as a random coil with some degree of base-stacking, and at pH 6.0, 0.15 M Na+, where poly(A) adopts the protonated double-helical acid form structure below 15 degrees C. Binding isotherms show that, at 3.5 degrees C, poly(A) forms a 1 : 1 complex with xanthine at pH 6, and a 2:1 complex at pH 7, while oxoformycin forms a 1:1 complex with poly(A) at both pH 6 and pH 7. Poly(A) forms a complex, tentatively assigned 1:1 stoichiometry, with 8-azaxanthine at pH 6, but no complexing occurs at pH 7. The complexes have been characterized by their optical rotatory dispersion and ultraviolet spectra, their thermal stabilities, and their rates of formation at low temperature. All the complexes are laevorotatory at long wavelengths (greater than 300 nm) and unplex formation at low temperature is a slow process requiring many hours for completion. The complexes of poly(A) with 3-methylxanthine have been reinvestigated and shown to undergo normal helix-coil transitions; the anomalous melting behaviour noted previously [Biopolymers, 10, 21 -- 33 (1971)] has been explained. From a comparison of optical rotatory dispersion spectra, it is concluded that the poly(A) with 3-methylxanthine have similar structures, which are quite different from the structures of the corresponding complexes with 7-methylxanthine. The structures and properties of the poly(A) - monomer complexes are discussed, and compared with those of other polynucleotide - monomer complexes. No significant interaction was observed between poly(A) and hypoxanthine, allopurinol, 6,8-dihydroxypurine, 1-methylxanthine, 9-methylxanthine, theophylline, theobromine or 3,9-dimethylxanthine.  (+info)

Conformation, enzymic activity, and immunochemistry of a lysozyme derivative modified at tryptophan 123 by reaction with 2,3-dioxo-5-indolinesulfonic acid. (8/91)

Reaction of hen egg-white lysozyme with 2,3-dioxo-5-indolinesulfonic acid (DISA) yielded a homogeneous derivative which was modified at a single tryptophan residue. The modification was located at Trp-123. The absorption spectrum of the derivative showed a new peak in the visible range with lambdamax at 365 nm. In addition, the absorption maximum in the ultraviolet which appears in lysozyme at 280 nm was shifted to 270 nm in the derivative and appreciably enhanced. In ORD measurements, the rotatory behaviors of lysozyme and its derivative were identical at the 233 nm negative minimum and the 199 nm positive extremum. CD measurements gave equal [theta] values for lysozyme and derivative at the two negative ellipticity bands at 208 and 220 nm. Although no conformational differences between lysozyme and derivative were observed by ORD and CD measurements, some changes were detectable by chemical methods. Accessibility to tryptic hydrolysis and susceptibility of the disulfide bonds to reduction were increased in the derivative relative to lysozyme. The lytic activity of the derivative, which retained the same pH optimum as native lysozyme, was greatly (50%) decreased, probably as a result of the slight conformational change. With several antisera to lysozyme, the native protein and its derivative had equal antigenic reactivities. The findings were instrumental in further delineation of an antigenic reactive site in lysozyme.  (+info)

• 1
• 2
• 3
• 4
• 5
• 6
• 7
• 8
• 9
• 10