Purification and properties of several transfer RNA methyltransferases fromS. typhimurium

Summary A fast method for a single-step fractionation of a number of tRNA methyltransferases fromSalmonella typhimurium is described. The method basically consists of ion-exchange chromatography on a phosphocellulose column and permits the separation of the enzymes forming mt⁶A, m¹G, m⁵U, m⁷G. The enzyme fractions appear sufficiently purified to allow the estimation of some molecular and kinetic properties. The apparent K_M for adenosylmethionine range between 1.5 to 3.2×10⁻⁵ M, whereas K_M for undermethylated tRNA range between 3.1×10⁻⁵ M to 3.1×10⁻⁴ M. Glycerol gradient determination indicates the following M_r for the native proteins: 25×10³, 40×10³, 50×10³ and 65×10³ for m⁷G-, mt⁶A-, m¹G- and m⁵U-forming enzymes, respectively. A complete analysis of methylated nucleosides formedin vivo inS. typhimurium has been obtained: it also allowed us to infer the pattern of the various tRNA methyltransferases for this prokaryote. The tRNA methyltransferase forming mt⁶A has been isolated for the first time from any type of cell.     

Structural Aspects of Photosystem I from Dunaliella salina

A native PSI complex and a PSI core complex have been isolated from the halophilic green alga, Dunaliella salina. The composition and properties of these complexes are similar to previously described PSI complexes from spinach membranes. By growth on ¹⁴C-NaHCO₃, it has been possible to isolate uniformly labeled ¹⁴C-PSI complexes in order to determine PSI subunit stoichiometry. This analysis has shown a ratio of one copy of three low molecular weight subunits (22,000; 15,000; 8,000) per two copies of high molecular weight subunits (84,000). Using a ¹⁴C-labeled cytochrome b₆-f complex as an internal protein standard, it has been possible to estimate the molecular weight of a PSI core complex as about 330,000. This complex contains one P700, two 84,000 subunits, and one subunit of 22,000, 15,000, and 8,000.     

Islanditoxin, α Toxic Metabolite Produced by Penicillium islandicum Sopp

Islanditoxin¹,²¹ has been isolated from the cultured broth of Penicillium islandicum, and C₂₅H₃₃O₈N₅Cl₂ was proposed as its molecular formula. The presence of the peptide linkage in islanditoxin was ascertained and the general properties were described.     

Potential anti-diabetic applications of a new molecule with affinity for β3-Adrenoceptors

β₃-adrenoceptors have been described through both molecular and pharmacological studies. In this context, it has been characterized the β₃-adrenoceptors agonist activity of a new compound (Trecadrine^®), and evaluated whether it exhibits additional activity at β₁ and β₂- adrenoceptor subtypes. In addition, it has been tested its potential anti-diabetic properties in a model of alloxan-induced diabetes in rats. Our data show that Trecadrine^® induces oesophageal muscularis mucosae relaxation, indicating a putative action on β₃-adrenoceptors. In the absence of β₃-adrenoceptors antagonists, assays with β₁ and β₂-adrenergic antagonists reveal a quite remarkable selectivity for β₃-adrenoceptors. Furthermore, it has been suggested that this molecule has hypoglycaemic and lipomobilizing effects, although hypoglycaemia was not related to an increase in insulin secretion in diabetic rats. It is concluded that Trecadrine^® mainly acts through β₃-adrenoceptors, and it may constitute a potential drug in the treatment of diabetes.     

Comparison of the effects of calcium and magnesium on the conformation of tetra cycline in Me2SO solution

The effects of anhydrous Ca(NO₃)₂ on the ¹³C nuclear magnetic resonance (nmr) and circular dichroism spectra of tetracycline in Me₂SO-d₆ solution have been investigated in order to make a comparison with the results of a previous study in which the effects of Mg(NO₃)₂ were determined under the same conditions. The results of experiments described in this article provide strong evidence that Ca²⁺ and Mg²⁺ bind tetracycline at the same sites in Me₂SO and that both ions induce the same change in molecular conformation of tetracycline upon binding. The Ca²⁺ complex, in contrast to the Mg²⁺ complex, has a lifetime that is short on the nmr time scale.     

Diphosphopyridine nucleotide-linked D-lactate dehydrogenases from the horseshoe crab, Limulus polyphemus and the seaworm, Nereis virens. I. Physical and chemical properties

d-lactate dehydrogenase has been purified from horseshoe crab (Limulus polyphemus) skeletal muscle and the seaworm (Nereis virens). The purified Limulus dehydrogenase was shown to be a dimer, with a molecular weight of approximately 70 000. Sephadex gel filtration and equilibrium sedimentation yield molecular weights of about 80 000 and 70 000 respectively. Acid dissociation yields a molecular weight species of about 35 000. The native enzyme has an s^o₂₀^w of 3.95. Extrapolation of para-hydroxymercuribenzoate inhibition curves to 100% inhibition corresponds to two molecules of para-hydroxymercuribenzoate bound per molecule of enzyme. Studies on the stoichiometric binding of reduced coenzyme show two molecules bound per molecule of enzyme. The number of tryptic peptides has been found to be one-half that expected from the amino acid composition. The electrophoretic pattern of isoenzymic forms can be best interpreted as suggesting that the enzyme is dimeric. In vitro high salt, freeze-thaw hybridizations of the isolated Limulus muscle isoenzymes yield the electrophoretic pattern predicted by a dimeric structure.The physical properties ot Nereis lactate dehydrogenase have been found to be similar to those for the Limulus muscle lactate dehydrogenase.     

Two enzymatically active forms of valyl-tRNA-synthetase from E. coli.

This paper reports the purification, kinetic properties and molecular weights of two active forms of valyl-tRNA synthetase from E. coli. Whereas form I catalyses the valine dependent ATP-[³²P] pyrophosphate exchange reaction as well as the esterification of tRNA_I^Val, form II has a different K_m-value for the aminoacylation but still catalyses the pyrophosphate exchange reaction with the same K_m-value as form I. While form II, with a molecular weight of 125,000, can be dissociated into two unequal parts with molecular weights of M_I = 69,000 and M_II = 46,500, respectively, form I consists of one polypeptide chain with a molecular weight of 115,000. The data obtained from amino acid analysis indicate that the two fragments also differ in their electrostatic charge capacity.     

Continuous measurement of 14C-labeled carbon dioxide and lactic acid by cultured cells grown in Leighton tubes.

A procedure for continuous measurement of ¹⁴CO₂ production by cultured cells grown in Leighton tubes has been described. The apparatus developed also permits aliquots of the incubation medium to be taken during the experiments for analysis of labeled metabolites released into the solution. A simple method for determination of [¹⁴C]lactic acid in such aliquots has been described. The reproducibility and usefulness of the apparatus has been demonstrated by incubating fibroblasts with glucose labeled in the C-1 or C-6 position, and examining the effects of selected drugs on CO₂ and lactic acid production.     

Myonemal contraction of spirostomum. II. Some mechanical properties of the contractile apparatus

In several respects, notably the high velocity of shortening, Ca²⁺ dependence, and ATP independence, contraction of Spirostomum resembles the spasmonemal mechanism of the peritrich ciliates. In this report further mechanical properties of the contractile apparatus are described that extend this comparison. The velocity-load characteristic is more appropriate to an elastomer than to a muscle where contraction force is load-dependent. Active tension is found to relate linearly to cell length for extensions up to and beyond resting length (l_r), an elastic limit is reached around 1.5 l_r. At resting length this tension, measured by the deformation of a glass microbalance, is similar to that predicted from consideration of the hydrodynamic forces normally resisting shortening. The tension-length relation for the unstimulated (passive) cell is also linear between l_r and the elastic limit, but is displaced from the active tension-length curve and is of reduced stiffness. Kinetic studies suggest that maximum tension and maximum velocity coincide. Calculations are presented that support a model of contraction in Spirostomum in which the myonemes behave as a mechanochemical engine powered directly by the chemical potential of Ca²⁺.     

Pattern recognition based on color-coded quantum mechanical surfaces for molecular alignment

A pattern recognition algorithm for the alignment of drug-like molecules has been implemented. The method is based on the calculation of quantum mechanical derived local properties defined on a molecular surface. This approach has been shown to be very useful in attempting to derive generalized, non-atom based representations of molecular structure. The visualization of these surfaces is described together with details of the methodology developed for their use in molecular overlay and similarity calculations. In addition, this paper also introduces an additional local property, the local curvature (C _L), which can be used together with the quantum mechanical properties to describe the local shape. The method is exemplified using some problems representing common tasks encountered in molecular similarity. Figure Molecular surfaces for Lorazepam (left) and Diazepam (right)     

A monolayer and freeze-etching study of charged phospholipids. I. Effects of ions and pH on the ionic properties of phosphatidylglycerol and lysylphosphatidylglycerol

A comparative study of the ionic properties of phosphatidylglycerol (PG) and lysylphosphatidylglycerol (LPG) has been carried out using monolayer and freeze-etch techniques. It is shown that the ionization state of the PG monolayer is strongly dependent on the subphase ionic strength. Mg⁺⁺ and Ca⁺⁺ induced a marked condensing effect. With Ca⁺⁺ a typical cylindrical structure could be observed by freeze etching, this structure being assumed to be generated by Ca⁺⁺ binding to PG. These phenomena were observed with both didodecanoyl-PG and PG from S. aureus. With respect to LPG it has been shown that the anion MoO₄⁼ gave a strong film condensation of didodecanoyl-LPG monolayers at pH 6 corresponding to a change in the bulk morphology from a transparent gel to a lamellar liposomal structure. A similar decrease in the molecular packing of S. aureus LPG was induced by MoO₄⁼, without a change in the freeze etch morphology of the dispersion. Evidence is presented on the important role that the polar head groups of these charged phospholipids and their ionic environment have on the overall molecular packing. Differential scanning calorimetric measurements demonstrated that the liquid-crystalline to gel transition of didodecanoyl-PG is strongly dependent on the cations in the suspension. These phenomena may be relevant to the physical state of lipids in biological membranes.     

A novel aminopeptidase from Clostridium histolyticum

An aminopeptidase was found in the culture filtrate of Cl. histolyticum and purified to homogeneity (130 times) in a two-step procedure. All types of N-terminal amino acids, including proline and hydroxyproline are cleaved by the enzyme from small peptides and from polypeptides. A low rate of hydrolysis was observed for β-naphthylamides and for alanine amide; p-nitroanilides were not hydrolyzed. Kinetic parameters (K_m and V_max) for several tripeptides and the tetrapeptide Pro-Gly-Pro-Pro were determined. The enzyme has a pH optimum at 8.6. The presence of either Mn⁺⁺ or Co⁺⁺ is essential for its activity. Only slight activation was observed with Ni⁺⁺ and Cd⁺⁺, while Zn⁺⁺ and Cu⁺⁺ were inhibitory. The molecular weight of the native enzyme is about 340,000, and a molecular weight of about 60,000 was determined for the reduced and denatured enzyme by gel electrophoresis in sodium dodecyl sulfate (SDS).The culture filtrate of Cl. histolyticum has been shown to contain various proteolytic enzymes, in addition to collagenase^1–5. In a search for enzymes acting on proline-rich peptides, we tested the crude filtrate with (Pro-Gly-Pro)_n, (Pro-Gly-Pro)_n-OMe, α,DNP-(Pro-Gly-Pro)_n and poly-L-proline as substrates. Proline was formed only from (Pro-Gly-Pro)_n and its methyl ester. This showed the presence in Cl. histolyticum filtrate of an aminopeptidase which cleaves N-terminal proline from polypeptides but not from polyproline. The purification and some of the properties of this clostridial aminopeptidase (CAP) are described in this communication.     

Rigidity of α-helical polypeptides. A new method of obtaining the persistence length from viscosimetric data

The hydrodynamic properties of α-helical poly(L -glutamic acid), (Glu)_n in aqueous solutions and in mixtures of water with organic solvents have been interpreted in terms of the persistence length of the macromolecule. A modification of the method of Vitovskaya and Tsvetkov has been proposed in order to allow a more accurate determination of this parameter. The addition of an organic solvent increases strongly the rigidity of the helical conformation of (Glu)_n. A comparison is made with some data of the literature of poly[N⁵-(3-hydroxy propyl)L -glutamine], [Gln(CH₂)₃OH]_n, and poly(γ-benzyl-L -glutamate), [Glu(OBzl)]_n.     

Radiation inactivation of enzymes at low dose rates: Identical molecular weights of rat liver cytosolic and lysosomal neuraminidases

A low-dose-rate gamma source (⁶⁰Co) was calibrated with enzymes of known radiation inactivation behaviors and used for molecular weight determination of rat liver neuraminidase (EC 3.2.1.18). The method allows direct comparison of radiation inactivation of standard and unknown enzymes under identical experimental conditions. The membrane-bound lysosomal neuraminidase had the same molecular weight (M_r = 56,000 ± 8500) as the soluble cytosolic neuraminidase (M_r = 56,000 ± 7000) although they differ in their kinetic properties and substrate specificity.     

Preparation,purification, and properties of E. coli virus T2

1. A method for the preparation of 8 to 10 liter quantities of T₂ virus lysates, titering 2 to 5 x 10¹¹ infectious units per ml. has been described. 2. Procedures have been developed for the concentration and purification of virus to a high specific infectivity. No fractionation procedure of the several used succeeded in further raising the specific infectivity of these purified preparations. 3. Some of the general properties of the better preparations have been determined. They exhibited titers of 2 x 10¹⁵ infective units per gm. of material or 1.2 x 10¹⁶ per gm. of nitrogen. 4. A study of the distribution of nitrogen among the various fractions of the virus showed that about 6 per cent of the total nitrogen is soluble in 4 per cent trichloracetic acid; that the protein nitrogen is about 40 per cent of the total and the nucleic acid nitrogen is 53 per cent. At least 96 per cent of the total phosphorus is in the nucleic acid fraction. Less than 0.5 per cent quantities of lipid and PNA were found.     

Studies on γ Globulin of Rice Embryo

An improved method has been described for the isolation and purification of γ globulin from rice embryo. The method involves the extraction with phosphate buffer, pH 7.0 and ionic strength 0.1, the fractionation in saline solution of ionic strength 0.31, the removal of nucleic acids by precipitation with ammonium sulfate and the gel filtration chromatography on a Sephadex G-200 column. Although the preparations exhibited homogeneous patterns in sedimentation analysis, the electrophoretic patterns on polyacrylamide gels at pH 8.35 and ionic strength 0.11 exhibited at least two components. Three major components, γ₁, γ₂ and γ₃ globulins, were isolated by ion exchange chromatography on a DEAE Sephadex A-50 column. These components were revealed to be homogeneous in electrophoresis as well as sedimentation. N-Terminal amino acid compositions have also been described.

The molecular weight of γ₁ globulin was determined as 2.0 × 10⁵ by the Archibald method, and the intrinsic viscosity, [η], and the sedimentation coefficient, s_{20, w}^°, were found to be 0.0424 dl/g and 7.26S respectively. These values indicated the large asymmetry of the protein. The protein was composed of 18 residues of hexose, 3 residues of pentose, 6 residues of hexosamine and 1751 residues of amino acids: Lys₅₈, His₄₇, Arg₁₄₈, Asp₁₂₆, Glu₂₇₃, Gly₁₆₁, Ala₁₄₄, Val₁₂₁, Leu₁₀₆, Ile₇₂, Pro₈₃, Ser₁₃₆, Thr₄₈, Hyp₆₈, Cys₁₇, Met₁₆, Tyr₄₄, Trp₈, Phe₇₅ and amide ammonia₁₆₃. The N-terminal amino acid analysis suggested that the protein was composed of ten subunits. The properties and the composition were discussed in comparison with those of the 7S globulin of soybean cotyledon.     

Zinc transport by transferrin in rat portal blood plasma.

Zinc transport in mesenteric lymph and zinc distribution in portal plasma and venous plasma were examined in rats that had been given an oral dose of ⁶⁵Zn. Less than 1% of an oral dose of ⁶⁵Zn appeared in the mesenteric lymph over a period of 8 hr. In portal plasma, approximately 70% of the isotope recovered after gel-filtration chromatography was bound to a protein that was identified as transferrin on the basis of molecular weight and electrophoretic properties. In venous plasma, the major fraction of ⁶⁵Zn was bound to albumin while the remainder of the isotope was associated with higher molecular weight proteins including transferrin and α₂-macroglobulins. These results demonstrate that zinc is transported from the intestine to the liver via the portal blood, and the results demonstrate that zinc is transported in portal plasma bound to transferrin.     

THE KINETICS OF PENETRATION : XII. HYDROGEN SULFIDE

The rate of entrance of H₂S into cells of Valonia macrophysa has been studied and it has been shown that at any given time up to 5 minutes the rate of entrance of total sulfide (H₂S + S^-) into the sap is proportional to the concentration of molecular H₂S in the external solution. This is in marked contrast with the entrance of ammonia, where Osterhout has shown that the rate of entrance of total ammonia (NH₃ + NR₄ ⁺) does not increase in a linear way with the increase in the external concentration of NH₃, but falls off. The strong base guanidine also acts thus. It has been shown that the rate of entrance of H₂S is best explained by assuming that it enters by diffusion of molecular H₂S through the non-aqueous protoplasmic surface. It has been pointed out that the simple diffusion requires that the rate of entrance might be expected to be monomolecular. Possible causes of the failure of H₂S to follow this relationship have been discussed.     

Defining the "fast-reacting" thiols of myosin by reaction with 1, 5 IAEDANS.

The specificity of the fluorescent reagent N-iodoacetyl-N-(5-sulfo-1-naphthyl)ethylenediamine (1,5 IAEDANS) for a specific thiol group of myosin has been characterized by a comparison with iodoacetamide (IAA) and by observing maximal enhancement of the Ca²⁺-ATPase activity and inhibition of the K⁺-EDTA-ATPase activity of myosin. The stoichiometry of the [³H]1,5 IAEDANS bound to myosin indicates the presence of two fast-reacting thiols which correspond to the “SH₁” groups responsible for the catalytic properties of myosin. Moreover, it has been unequivocally demonstrated by gel electrophoresis that the fast-reacting thiol is located on the myosin heavy chain. A single radioactivity-labeled thiol peptide obtained from tryptic digests of myosin labeled with [³H]1,5 IAEDANS or iodo[1-¹⁴C]acetamide indicates strongly that the identical thiol was labeled by both reagents.