Statistical evaluation of ways to analyze nonideal systems by sedimentation equilibrim

Three equations describing sedimentation equilibrium are examined and tested for their ability to analyze data. The testing procedure using simulated data is similar to that described previously (Holladay, L. A., and Sophianopoulos, A. J. (1972) J. Biol. Chem.247, 427–439) and used with another equation. The equations examined here are found to be of much less statistical reliability and of a more restricted range of application than the previously examined equation. The equation described previously, (Holladay, L. A., and Sophianopoulos, A. J. (1972) J. Biol. Chem.247, 427–439) is also used here to examine the conditions necessary to detect isodesmic systems of more than four components. The self-association of lysozyme reported previously (Sophianopoulos, A. J., and Van Holde, K. E. (1964) J. Biol. Chem.239, 2516–2524) is reexamined at pH 8.2, 0.15 ionic strength, and 13°C. The tentative conclusion is that the system is mainly a monomer-dimer, with a small, uncertain amount of tetramer possibly present. Under the above conditions the second virial coefficient, B, is estimated to lie in the range 0–4.4 × 10^?6 mole·dl·g^?2, the dimerization constant. K₂₁, lies in the range 2.3–2.7 × 10^?3m, and the tetramerdimer constant, K₄₂, is in the range 1.5–15 × 10^?3m.     

Trehalose-6-phosphate synthetase activity in extracts of Dictyostelium discoideum.

Trehalose-6-phosphate (T-6-P) synthetase activity in extracts of Dictyostelium discoideum has been reexamined in an effort to resolve discrepancies between the results of previous studies (R. Roth and M. Sussman (1966). Biochim. Biophys. Acta, 122, 225; K. A. Killick and B. E. Wright (1972). J. Biol. Chem., 247, 2967). We find that T-6-P synthetase is not cold sensitive as reported by Killick and Wright (1972), is not present in bacterial-grown vegetative cells (though subject to some modulation by other nutritional conditions), and is not in our hands unmasked or activated by ammonium sulfate fractionation. We conclude that the pattern of T-6-P synthetase accumulation and disappearance during fruiting body construction in D. discoideum is as originally described by R. Roth and M. Sussman (1968). J. Biol. Chem., 243, 5081) and confirmed elsewhere (P. C. Newell et al. (1972). J. Mol. Biol., 63, 373; R. W. Brackenbury et al. (1974). J. Mol. Biol., 90, 529; B. D. Hames and J. M. Ashworth (1974). Biochem. J., 142, 301).     

Characteristics of the induction of aldehyde dehydrogenase in rat liver

The activity of rat liver supernatant aldehyde dehydrogenase is increased by phenobarbital treatment in one selected strain (RR) but not in another strain (rr) of animals derived from randomally bred populations (Deitrich, Collins, and Erwin (1972) J. Biol. Chem., 247, 7232). Before 14 days of age, increased enzyme activity after phenobarbital treatment is minimal but between 30 and 60 days of age there is a maximal increase in activity after phenobarbital treatment. Using animals of this age, it was shown that both cycloheximide and actinomycin D block this response to phenobarbital. Phenobarbital treatment decreases heat stability of crude preparations of the enzyme from RR rats, but increases heat stability of the enzyme from rr animals.     

Acetyl-coenzyme A carboxylase from the developing endosperm of Ricinus communis: II. A two-site kinetic mechanism

The reaction kinetics of acetyl-coenzyme A carboxylase purified from developing castor oil seeds have been examined. On the basis of the substrate interaction and product inhibition results, a hybrid ping-pong mechanism is proposed. This type of mechanism demands that the active site of the enzyme be separated into two functionally distinct catalytic sites. The carboxybiotin intermediate formed at one site by the hydrolysis of ATP swings to the second site where acetyl-CoA is carboxylated to form malonyl-CoA. This hybrid rapid-equilibrium random bi bi uni uni ping-pong mechanism which includes the formation of three abortive complexes, E · HCO₃^? · ADP, E · HCO₃^? · P_i and E · P_i · P_i, is analogous to the hybrid ping-pong mechanism previously described for methylmalonyl-CoA transcarboxylase (D. B. Northrop (1969) J. Biol. Chem., 244, 5808) and pyruvate carboxylase (R. E. Barden, C-H. Fung, M. F. Utter, and M. C. Scrutton (1972) J. Biol. Chem., 247, 1323).     

Isolation and partial characterization of a rat hepatoma heparan sulfate proteoglycan

A proteoglycan was isolated from a Morris rat hepatoma by sequential precipitations with ammonium sulfate and cetyl pyridinium chloride followed by chromatography on Sepharose CL-4B and DEAE-cellulose. The proteoglycan has a molecular weight of about 1.5 × 10⁵ with 40,000 molecular weight glycosaminoglycan side chains, identified as heparan sulfate based on resistance to chondroitinase and susceptibility to nitrous acid treatment. Immunological studies showed that the protein core of this proteoglycan is immunologically distinct from a rat yolk sac tumor chondroitin sulfate proteoglycan (Å. Oldberg, E. G. Hayman, and E. Ruoslahti, 1981,J. Biol. Chem.256, 10847–10852), but resembles a heparan sulfate proteoglycan isolated from a basement membrane-producing mouse tumor (J. R. Hassell, P.M. Robey, H.-J. Barrach, J. Wilczek, S. R. Rennard, and G. R. Martin, 1980, Proc. Nat. Acad. Sci. USA77, 4494–4498).     

Cystathionine β-synthase from human liver: Improved purification scheme and additional characterization of the enzyme in crude and pure form

The previously published procedure (Kraus et al. (1978) J. Biol. Chem.253, 6523–6528) for the purification of cystathionine β-synthase [l-serine hydro-lyase (adding homocysteine) EC 4.2.1.22], a pyridoxal 5′-phosphate-dependent enzyme from human liver has been modified. The new procedure, starting with a liver homogenate “aged” for 7 days at 4 °C, yielded homogeneous enzyme purified over 3000-fold with a much improved yield. “Aging” of the enzyme in crude homogenates yields a form apparently smaller by gel electrophoresis and with significantly increased activity and antigenicity. This species of cystathionine β-synthase does not form stable complexes with other proteins during purification as does the previously employed, freshly used species. An absorption spectrum and an amino acid composition of the pure enzyme were determined; the amino-terminal residue was shown to be methionine. The isoelectric points of holosynthase and aposynthase were estimated to be 5.2 and 5.6, respectively. Rabbit antiserum raised against the pure cystationine β-synthase was characterized using as antigen crude synthase from five different mammalian species as well as the pure human enzyme.     

Evidence for isoaspartyl (deamidated) forms of mouse epidermal growth factor

A variant form of mouse submaxillary gland epidermal growth factor (EGF) was identified by isocratic reversed-phase HPLC of EGF obtained by Bio-Gel P-10 column chromatography ("culture grade"). The variant form was essentially absent in preparations of EGF further purified by chromatography on DEAE-cellulose ("receptor-grade" EGF). The spectral properties and amino acid composition of the variant form (EGF-I) could not be distinguished from those of the intact polypeptide isolated by HPLC (alpha-EGF). Receptor-binding and mitogenic properties of EGF-I were also equivalent to those of alpha-EGF. These data suggested that EGF-I was structurally very similar to EGF. However, the very low yield (less than 4%) obtained by Edman degradation indicated that the N-terminal (Asn1) of the polypeptide was modified. Isoelectric focusing of EGF-I revealed two major immunoreactive bands: one with a pI equivalent to that of alpha-EGF (pI 4.6) and another at pI 4.1. Alkaline treatment of alpha-EGF (0.1 M NH4OH) yielded peak material by HPLC that coeluted with EGF-I; the alkaline-generated EGF-I yielded bands that also focused at pH 4.6 and 4.1. Ammonium hydroxide treatment of [des-Asn1]-EGF (beta-EGF) did not produce conversion to EGF-I. On the basis of these data, we propose that EGF-I was formed by selective deamidation of the N-terminal Asn of intact EGF. This notion is also supported by liquid secondary ion mass spectrometry, which showed that EGF-I was approximately 1.5 mass units greater than alpha-EGF. The heterogeneity observed by isoelectric focusing supports previous studies which have shown that, following deamidation of N-terminal asparagine, a beta-aspartyl shift can occur, which in the present study might yield succinimido-aspartyl1-EGF and beta-aspartyl1-EGF. Low yields observed during Edman degradation indicate that negligible amounts occur as the alpha-aspartyl1-EGF isomer.     

Dextransucrase: studies on donor substrate specificity

Previous studies (D. S. Genghoff and E. J. Hehre, Proc. Soc. Exp. Biol. Med., 1972, 140, 1298–1301) have shown that an α-linked fluorine atom at C-1 of glucose provided sufficient activation to permit this analog to be a donor substrate for dextransucrase. In order to study the specificity at the donor substrate binding site, a series of α-1-fluorosugars have been synthesized. In kinetic experiments, it has been determined that they served as competitive inhibitors of sucrose, the natural substrate. A comparison of the K_i's provided information about the importance of specific changes in the glucose moiety with regard to binding to the enzyme. Similar kinetic studies were carried out with several β-1-fluorosugars, and the corresponding free monosaccharides. These were found to be noncompetitive inhibitors, and to bind poorly. The α-1-fluorosugars were also examined as donor substrates in reactions with known acceptors. With the exception of α-1-fluoroglucose, none of these analogs were active in this capacity.     

Monomeric structure of glutamyl-tRNA synthetase in Escherichia coli.

An investigation of the subunit structure of glutamyl-tRNA synthetase (EC 6.1.1.17) from Escherichia coli indicates that this enzyme is a monomer. The enzyme purified to apparent homogeneity is a single polypeptide chain with a molecular weight of 62,000 ± 3,000 and K^Glu_m ? 50 μM in the aminoacylation reaction. Analytical gel electrophoretic procedures were used to determine the molecular weight of species exhibiting glutamyl-tRNA synthetase activity in freshly prepared extracts of several strains of E. coli, which had been grown under various nutritional conditions and harvested at different stages of growth. In all cases, glutamyl-tRNA synthetase activity was associated with a protein having about the same molecular weight and K^Glu_m as the purified enzyme. Thus, no evidence of an oligomeric form of glutamyl-tRNA synthetase with a greater affinity for l-glutamate was obtained, in contrast to a previous report of J. Lapointe and D. Söll (J. Biol. Chem.247, 4966–4974, 1972).     

Large-scale purification and characterization of calmodulin from ram testis its metal-ion-dependent conformers

Calmodulin was isolated in large quantities from ram testis by a simple procedure involving sequentially ammonium sulfate fractionation, heat treatment, anion exchange chromatography on DEAE-cellulose and gel filtration on Sephacryl S-200. Divalent cations (Mg²⁺ and/or Ca²⁺) were present throughout the purification which was entirely performed in the absence of chelators. The final yield was approx. 90 mg per kg testis. Ram testis calmodulin appears to be essentially identical to the brain homologous protein by the following criteria: ultraviolet absorption spectrum, amino acid composition showing a single residue of ?-N-trimethyl lysine, and tryptic peptide maps obtained by high performance liquid chromatography. Turkey gizzard myosin light-chain kinase, the activation of which is extremely specific for calmodulin (Walsh, M.P., Vallet, B., Cavadore, J.C. and Demaille, J.G. (1980) J. Biol. Chem. 255, 335–337), was indeed activated by ram testis calmodulin in the presence of calcium. The isolated protein migrated at different rates upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, depending on the absence or presence of divalent metals which probably induce different conformations. The relative migration rates were Ca²⁺ > Mn²⁺ > Mg²⁺ > EDTA. In the presenceof divalent metals, the observed doublet may be ascribed to the equilibrium between ion-free and ion-saturated forms, which exhibited different Stokes radii, as already suggested (Grab, D.J., Berzins, K., Cohen, R.S. and Siekevitz, P. (1979) J. Biol. Chem. 254, 8690–8696).     

A simple preparation of beta-trypsin based on a calorimetric study of the thermal stabilities of alpha- and beta-trypsin

Bovine trypsin preparations contain, in addition to the single chain form of the enzyme, an active two-chain autolysis product (Schroeder, D. D., and Shaw, E., J. Biol. Chem. (1968), 243, 2943–2949). Differential scanning calorimetric (DSC) studies showed that the single chain form, β-trypsin, is more stable to thermal denaturation than the two-chain form, α-trypsin. Rate constants and activation energies for the thermal denaturation of β-trypsin are 5 × 10^?5 sec^?1 and 69 kcal/mole and of α-trypsin are 5 × 10^?3 sec^?1 and 38 kcal/mole at pH 4.4 and 48 °C. Preparation of pure β-trypsin can be greatly simplified by prior thermal denaturation of the α form. At least 75% of the α form is denatured by heating a 10–15% solution of commercial crystalline trypsin for 30–45 min at 48 °C, pH 4.4, 0.02 m Ca²⁺. The native β-trypsin is then easily isolated from the denatured α-trypsin by batchwise adsorption onto ovoinhibitor-agarose at pH 8. After elution at pH 2, dialysis, and lyophilization an average preparation contained approximately 85% β-trypsin, 10% α-trypsin, and 5% inactive material. Benzamidine was used during the isolation to decrease the rate of conversion of β- to α-trypsin. Because the separation of active β-trypsin from heat-denatured α-trypsin is relatively easy, the total preparation time has been reduced to 1 day.     

Synthesis of the glycosaminoglycan-protein linkage tetraosyl peptide moieties of betaglycan, which serve as a hexosamine acceptor for enzymatic glycosyl transfer

Betaglycan, also known as TGF-β type III receptor, is a membrane-anchored proteoglycan, which has two glycosaminoglycan (GAG) attachment sites (López-Casillas, F.; Payne, H. M.; Andres, J. L.; Massagué, J. J.Cell Biol.1994, 124, 557-568). Chondroitin sulfate (CS) or heparan sulfate (HS) can attach to the first site, Ser⁵³⁵, whereas only CS attaches to the second, Ser⁵⁴⁶. Although the mechanism behind the assembly of CS and HS is not fully understood, it has been reported that the assembly of HS requires not only a cluster of acidic residues but also hydrophobic residues located near the Ser-Gly attachment sites (Esko, J. D. Zhang, L. Curr. Opin. Struct. Biol.1996, 6, 663-670). To further understand the effects of amino acids close to the Ser residues of the GAG-attachment sites on the glycosyltransferases, two tetraosyl peptides derived from the CS attachment sites of betaglycan, GlcA-Gal-Gal-Xyl-SerGlyAspAsnGly (1) and GlcA-Gal-Gal-Xyl-SerGlyAspAsnGlyPheProGly (2), were synthesized, and used as donor substrates for β1,4-N-acetylgalactosaminyltransferase-I (β4GalNAcT-I) and α1,4-N-acetylglucosaminyltransferase-I (α4GlcNAcT-I). Both the chemically synthesized linkage region tetrasaccharides were far better acceptors for β4GalNAcT-I than for α4GlcNAcT-I in vitro, although they also showed appreciable acceptor activity for α4GlcNAcT-I.     

Rapid purification of a phospholipase-free α-bungarotoxin: Maintenance of cation barriers of acetylcholine receptor membranes upon preincubation with purified toxin

The purification of highly homogeneous, phospholipase-free α-bungarotoxin (α-Bgt) from the venom of the elapid Bungarus multicinctus or from commercial samples of α-Bgt is described. The method combines a conventional procedure for the purification of α-Bgt [D. Mebs, K. Narita, S. Iwanaga, Y. Samejima, and C. Y. Lee (1972) Hoppe-Seyler's Z. Physiol. Chem.353, 243–262] with high-resolution gel-filtration and cation-exchange chromatography steps to remove membrane-damaging, contaminating phospholipase activity. The procedure also removes contaminating radioactive peptides from commercial preparations of ¹²⁵I-α-Bgt. Apparent homogeneity of the purified α-Bgt (referred to as fraction D in the text), as well as the absence of contaminating phospholipase A₂ activity, is assessed by (i) polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, (ii) gel-filtration and cation-exchange high-performance liquid chromatography, (iii) direct measurements of phospholipase A₂ activity under conditions where very low enzymatic levels should be detected, (iv) lack of interference with the passive cation permeability properties of acetylcholine receptor membranes, (v) competitive inhibition of ¹²⁵I-α-Bgt binding to the acetylcholine receptor membranes, and (vi) amino acid analysis and end-group (C- and N-terminus) determination. α-Bgt preparations subjected to these criteria do not exert the increase in membrane passive permeability to cations detected with other laboratory or commercial samples of α-Bgt. Availability of the new α-Bgt preparation allows for an assessment of the inertness of α-Bgt on lipid membrane properties while preventing cholinergic ligand binding to nicotinic acetylcholine receptor-rich membranes. These conditions are necessary for experiments requiring maintenance of the physical and phospholipid integrity of membranes.     

Improved Method for the Purification of Biologically Active Transcobalamin II

Transcobalamin II (TC II) was purified about 300, 000-fold from Cohn fraction III using a modification of the procedure described by Allen and Majerus (J. Biol. Chem. 247, 7709–7717 (1972)). The simplified method incorporated isoelectric precipitation of the TC II into the purification scheme which permitted the elimination of two colimn chromatographic steps originally reported by the above worke-s. The final preparation had 26.7 jjg of vitamin B, ～ (B₁₂) bound per mg of protein and an A₂₈₀/A₃₆₁ ratio of 2.05, both of which are in good agreement with the reported values. The purified TC II was biologically active with respect to its ability to facilitate penetration of B₁₂ into lleLa cells in tissue culture.     

Immunoaffinity chromatography of diadenosine 5',5'-P1,P4-tetraphosphate phosphorylase from Saccharomyces cerevisiae

Diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) phosphorylase has been isolated previously using classical protein isolation techniques [A. Guranowski and S. Blanquet (1985) J. Biol. Chem. 260, 3542-3547]. A protein A-Sepharose immunoaffinity column was prepared to simplify the purification procedure. The immunoaffinity column was prepared using specific polyclonal antibodies to Ap4A phosphorylase covalently coupled to protein A-Sepharose with dimethyl pimelimidate by a modification of the procedure of C. Schneider et al. [(1982) J. Biol. Chem. 257, 10,766-10,769]. The specific activity of the immunoaffinity-purified enzyme showed an increase equivalent to the specific activity obtained by chromatography on DEAE-cellulose and hydroxyapatite columns.     

Epidermal growth factor and transforming growth factor-alpha induce differential processing of the epidermal growth factor receptor

The capacity of epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha) to induce internalization and degradation of the EGF receptor was compared in NIH-3T3 cells expressing the human EGF receptor. This study was initiated following the observation that TGF-alpha was much less efficient relative to EGF in generating a Mr = 125,000 amino-terminally truncated degradation product from the mature EGF receptor (EGF-dependent generation of this degradation product is described in S.J. Decker, J. Biol. Chem., 264:17641-17644). Pulse-chase experiments revealed that EGF generally stimulated EGF receptor degradation to a greater extent than TGF-alpha. Both ligands induced EGF receptor internalization to similar degrees. However, recovery of [125I]-EGF binding following incubation with EGF or TGF-alpha was much faster for TGF-alpha treated cells. Recovery of [125I]-EGF binding after TGF-alpha treatment did not appear to require protein synthesis. Tyrosine phosphorylation of EGF receptor from cells treated with TGF-alpha decreased more rapidly following removal of TGF-alpha compared to cells treated similarly with EGF. These data suggest that EGF routes the EGF receptor directly to a degradative pathway, whereas TGF-alpha allows receptor recycling prior to degradation, and that tyrosine phosphorylation could play a role in this differential receptor processing.     

The epsilon subunit as an ATPase inhibitor of the F1-ATPase in Escherichia coli

The isolation of protein ATPase inhibitor was attempted directly from Escherichia coli membrane extracts to examine the possible presence of a Pullman-Monroy-type inhibitor [M. E. Pullman and G. C. Monroy (1963) J. Biol. Chem. 238, 3762-3769] distinct from the epsilon subunit of E. coli ATPase. Purification to homogeneity was achieved in a sequence of steps involving trichloracetic acid precipitation, DEAE-cellulose, Sephadex G75 chromatography, and a terminal isoelectric focusing step. An inhibitory protein was obtained and was identified by its physicochemical and inhibitory properties as the epsilon subunit of E. coli ATPase. The other inhibitory fraction observed in the purification procedure consisted of aggregated epsilon subunits.     

Determination of erythrocyte surface sialic acid residues by a new colorimetric method.

The 3-methyl-2-benzothiazolone hydrazone method has been applied to the determination of erythrocyte membrane sialic acid residues. This method requires a mild oxidation of sialic acid which results in the formation of analogs. Their separation by chromatography, after labeling, allows the choice of the best conditions for this oxidation. The concomitant liberated formaldehyde is determined. This method requires no prior release of sialic acid as opposed to the periodate-thiobarbituric method of Warren (1959, J. Biol. Chem., 234, 1971–1975). These two methods have been compared.     

NADH-dependent O-deethylation of p-nitrophenetole with rabbit liver microsomes.

A previous paper in this series (C. K. Mathews, (1972) J. Biol. Chem.247, 7430) showed that deoxynucleoside triphosphate pools expand manyfold when DNA synthesis is blocked genetically in infection by bacteriophage T4. This paper describes a more detailed analysis of this phenomenon. The key approach involves labeling with thymine or thymidine under conditions of infection where both phage and host bear mutations that inactivate thymidylate synthetase. Principal findings include the following: (1) Nucleotides in the expanded pools are derived in roughly equal measure from breakdown of host cell DNA and from nucleotide synthesis de novo after infection. (2) Thymidine diphosphate pool expansion is comparable, in rate and extent, to thymidine triphosphate pool expansion, but thymidine monophosphate pools accumulate much less. (3) The rate of expansion of the total thymine nucleotide pool following temperature upshift in infection by a temperature-sensitive gene 45 mutant is approximately equal to the rate of thymine incorporation into DNA immediately preceding the upshift. (4) Similarly, when DNA synthesis is restored by a downshift, the total thymine nucleotide pool drains at a rate commensurate with that of thymine incorporation into DNA. (5) Under these latter conditions the dTTP pool begins to drain earlier than the dTDP pool, suggesting that dTTP is the more proximal DNA precursor in this system.     

Synthesis of L-fucose 2-, 3-, and 4-Sulphates

Treatment of L-fucose with an excess of pyridine-sulphur trioxide gave an equilibrium mixture of mono-, di-,and tri-sulphates. L-Fucose was sulphated under optimal conditions for monosulphate formation, and the monoester fraction was isolated by chromatography on DEAE-cellulose. The isomeric L-fucose 2-, 3-, and 4-sulphates (1-3) were separated on a DEAE-cellulose column by elution with borate buffer. The structures of 1-3 were established by electrophoresis, colour tests, periodate oxidation, and, for the 2-isomer, by comparison with a specimen of 1 that had been definitively synthesised via methyl 3,4-O-isopropylidene-α-L-fucopyranoside (6) and methyl α-L-fucopyranoside 2-(barium sulphate) (5). The latter was rapidly hydrolysed in hot, dilute acetic acid to 1 and methyl α-L-fucopyranoside (4).