Determination of metal-metal distances in E. coli glutamine synthetase by EPR.

This paper reports the first determination of the distance between the two metal ions (per subunit) of $\text{E. coli}$ glutamine synthetase. When Mn(II) is bound at the n₁ metal ion site its EPR spectrum is diminished in intensity but not broadened as Cr(III)-ATP or Cr(III)-ADP is bound to the enzyme. A paramagnetic spin-spin interaction is responsible for this phenomenon and a metal-metal distance of ～7 Å is calculated for enzyme - Mn(II) - Cr(III)-ATP and ～6Å for enzyme - Mn(II) - Cr(III)-ADP. The metal-metal distance changes slightly when substrates or inhibitors are also bound to the enzyme demonstrating induced conformational changes in the protein at the metal ion sites.     

Evidence for methionine sulfoximine as a transition-state analog for glutamine synthetase from NMR and EPR data.

Manganese(II)bound at the “tight” metal ion site of unadenylylated glutamine synthetase (E. coli W) has two rapidly exchanging first coordination sphere water molecules. The solvation number was evaluated from a study of the frequency dependence of $\text{1}\text{pT}{}_{\mathrm{<mtext>1p</mtext>}}$, the paramagnetic contribution to the longitudinal relaxation rate of solvent protons. The number of rapidly exchanging water molecules is reduced to one in the presence of saturating L-glutamate and to ～0.2 when L-methionine SR-sulfoximine (MSOX) is present. MSOX is a linear competitive inhibitor (K_I=3μM) of glutamine synthetase when L-Glu is the substrate. The dissociation constant of MSOX measured by following the 18 fold decrease in $\text{1}\text{pT}{}_{\mathrm{1p}}$ (at 48 MHz) is 30μM and is lowered to ～9μM in the presence of ADP. The high affinity of MSOX for the enzyme suggests that this compound mimicks the “transition-state” for the glutamine synthetase reaction. Further evidence for this postulate is found from the dramatic sharpening of the epr spectrum of enzyme-bound Mn(II) in the presence of MSOX and MSOX plus ADP. The intense change in the epr spectrum arises from reduced solvent accessibility to bound Mn(II) and conformational changes produced by binding MSOX and ADP. The suggestion is made from these data that L-Glu and MSOX bind near or directly to the Mn(II) at the “tight” metal ion site in glutamine synthetase isolated from E. coli W.     

Molecular organization in bacterial cell membranes: IV. Isolation by preparative electrophoresis in sodium dodecylsulphate and properties of the two major polypeptide groups of a “soluble” fraction from Streptomyces albus membranes

Two polypeptide fractions have been purified from a “soluble” fraction of n-butanol-extracted Streptomyces albus membranes by preparative electrophoresis in sodium dodecylsulphate. They accounted for approx. 80% of the protein of the fraction (i.e. 20% of the total membrane protein). The ultraviolet spectrum of Group 1 (relative mobility 1.0) revealed the presence of nucleotide material, while that of Group 3 (relative mobility 0.65±0.05) showed the presence of a possibly aggregated protein-like material. About 100 and 30% of the protein contents (Lowry method) of Groups 3 and 1, respectively, were recovered as amino acid residues. These results confirm the protein nature of both fractions and suggest an overestimation of the protein value in Group 1. Both polypeptide groups can be classified as “extrinsic” membrane proteins on the basis of their similar amino acid composition (Vanderkooi, G. and Capaldi, R. A. (1972) Ann. N.Y. Acad. Sci. 195, 135–138). Three N-terminal amino acids were found in each fraction: one common (alanine), methionine, leucine or isoleucine (Group 3) and glutamic acid, lysine (Group 1). The sedimentation coefficients calculated were 2.46 S for Group 3 and 1.54 S for Group 1. Analysis of the isolated groups by gel electrophoresis under non-dissociating conditions or with Triton X-100, gave aggregate-like patterns.Sodium dodecylsulphate electrophoresis revealed an anomalous staining behaviour of Group 3 depending upon the dissociating conditions. The whole “soluble” fraction bound 0.40 mg dodecylsulphate /mg protein (0.55 mg detergent/mg protein corrected for overestimation). After dialysis, the fraction retained 10% of the bound dodecylsulphate. Circular dichroism of the isolated groups after exhaustive dialysis showed similar spectra, although of lower dichroism, to those obtained by other authors on soluble enzymes treated with sodium dodecylsulphate. Strong acid conditions were required to change the CD spectra of the polypeptides.     

Interactions of inhibitors at the coenzyme binding site of 6-phosphogluconate dehydrogenase.

Nicotinamide mononucleotide adenylyltransferase (EC 2.7.7.1) was purified 200-fold from chicken erythrocyte nuclei. An important feature of the purification procedure is the preliminary preparation of chromatin and extraction of the enzyme from insoluble chromatin into 0.3 m NaCl. Active enzyme in a partially purified preparation has an isoelectric point of 5.5 and a molecular weight of approximately 300,000. The most highly purified enzyme migrates as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a broad pH optimum from 6–9 and is most active at 55 °C. The activation energy for the enzyme-catalyzed reaction is 8.0 kcal/mol. The purified enzyme is able to use either nicotinate mononucleotide or nicotinamide mononucleotide as a substrate. The isolation procedure is applicable to partial purification of enzyme activity from erythrocytes of closely related birds, including pheasant, goose, and turkey. Immunochemical studies of the enzyme are reported in an accompanying article.     

Ligand-induced alterations in the reactivity of sulfhydryl groups and the structure of bovine liver glutamate dehydrogenase

The fluorogenic probe 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) was employed as an environmentally sensitive reporter label for free sulfhydryl groups of bovine liver glutamate dehydrogenase. A maximum of six -SH groups per subunit was titrated in Tris and borate buffers (pH 7.8) in 2 h but there was no reaction in the presence of phosphate buffer. The rate and extent of -SH reactivity was changed significantly by one of the substrates and some allosteric effectors. Adenosine nucleotides, NADH, and α-ketoglutarate promoted conformational alterations in glutamate dehydrogenase such that -SH groups were rendered virtually unreactive in [enzyme-ADP], [enzyme-NADH], and [enzyme-α-ketoglutarate]binary complexes. GTP, a negative allosteric modulator, showed no effect on -SH exposure. Measurements of protein circular dichroism spectra and catalytic activity in conjunction with -SH reactivity demonstrated a direct relationship between structural stability, biological activity, and ligand-induced conformational changes. The ligands that strongly protected the enzyme from reaction with NBD-C1 concomitantly maintained its structural and functional integrity.     

Microviscosity of the lipid domains of normal and hypercholesterolemic very low density lipoprotein.

Very low density lipoprotein (VLDL) has been isolated from normal (n) and dietary-induced hypercholesterolemic (hc) rabbits. Incorporation of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene into the lipid domains of both n VLDL and hc VLDL allowed assessment of the fluidity characteristics of these particles, utilizing fluorescence polarization techniques. Over the temperature range of 5° – 45°, the lipid region of n VLDL consists of an invariant phase, characterized by a microviscosity, η, at 30° of 0.6 ± 0.2 poise and a fusion activation energy, ΔE, of 7.6 ± 1.5 kcal/mole. The lipid region of hc VLDL, over the same temperature range, also is invariant and is characterized by a value of η at 30° of 4.6 ± 0.3 poise, and a ΔE of 7.8 ± 1.5 kcal/mole. Thus, large differences in the fluidit of the lipid in n VLDL and hc VLDL are evident, most probably due to the greatly increased content of cholesterol esters in hc VLDL, compared to n VLDL.     

Motility of the N-terminal tail of phosphorylase b as revealed by crosslinking.

There are lysyl-ε-NH₂ groups within about 3.5 Å distance across the intersubunit contact area of rabbit muscle phosphorylase $\text{b}$, as shown by cross-linking with malonic diimidate. These include the lysines of N-terminal region as revealed by limited tryptic digestion, but the contribution of the tail lysines to overall formation of covalent dimers is small. The fine structure of dimer band on dodecylsulfate-gelelectrophoretograms of crosslinked phosphorylases suggests that the tail retains its freedom in the phosphorylase $\text{b}$-AMP complex. Amidination induces the dissociation of phosphorylase $\text{b}$ dimer, which is slow relative to crosslinking.     

A thermodynamic description of the self-association of flavin mononucleotide.

Systematic heat of dilution studies of the self-association of flavin mononucleotide (FMN) have been conducted as a function of ionic strength (0.05 – 2.0 m) and pH (5–9) in aqueous solution. The data are adequately described by the expression $\text{Q}{}_{\mathrm{T}}\text{= ΔH ? (}\text{ΔH}\text{K}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(}\text{Q}{}_{\mathrm{T}}\text{c}{}_{\mathrm{T}}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for an isodesmic self-association. Q_T is the molar heat of dilution, ΔH and K are the derived enthalpy and equilibrium constants for the process FMN + (FMN)_i?1 ? (FMN)_i, and c_T is the concentration of FMN expressed in monomer units. Typical values derived for the various thermodynamic parameters at 25 °C are ΔG = ?3.56 kcal mol^?1, ΔH = ?3.72 kcal mol^?1, and ΔS = ?0.54 cal (mol · deg)^?1. These data, plus nuclear magnetic resonance evidence (Yagi, K., Ohishi, N., Takai, A., Kawano, K., and Kyogoku, Y., 1976, Biochemistry15, 2877–2880) argue in favor of an open-ended association of flavin molecules. The signs of the various thermodynamic parameters suggest that both hydrophobic and surface energy forces contribute significantly to the association, while the lack of any significant ionic strength dependence indicates the lack of any ionic centers in the association.     

Reactions of the sulfhydryl groups of alanyl-tRNA synthetase

The six sulfhydryl groups in each subunit of the alanyl-tRNA synthetase of Escherichia coli react with sulfhydryl reagents with at least four different rates. One reacts very rapidly with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), and a second reacts somewhat less rapidly with this reagent. These two groups are required for transfer activity, which is lost in proportion to the extent of derivatization. Two other groups react more slowly, with a consequent loss of exchange activity. The remaining two sulfhydryl groups do not react with DTNB until the protein is denatured. The inactivations are reversed by dithiothreitol. Two sulfhydryl groups react with N-ethylmaleimide (NEM) and with a spin-label derivative of NEM. These reactions resemble the modification of two sulfhydryl groups with DTNB, in that they also inactivate the transfer reaction but not the ATP:PP_i exchange. The two spin labels are incorporated at similar rates but are in very different environments, one highly exposed and one highly immobilized. These groups do not interact with Mn²⁺, which is bound to the enzyme in the absence of ATP.     

PMR relaxation times of micelles of the nonionic surfactant Triton X-100 and mixed micelles with phospholipids.

Previous pmr studies at 220 MHz have led to the suggestion that phosphatidylcholine and the nonionic surfactant Trition-X-100 form mixed micellar structures at high molar ratios of trition to phosphalipid. These mixed micelles provide one form of the phospholipid which the enzyme phospholipase A2 can utilize as substrate. Spin-lattice relaxation times (T1) and spin-spin relaxation times (T2) obtained from line widths for resolvable protons in Triton X-100 micelles and mixed micelles with egg phosphatidycholine and dipalmitoyl phosphatidylcholine are reported. They suggest that the structure of the mixed micelles is generally similar to that of pure Triton X-100 micelles. The T1 values for the phsopholipid in the mixed micelles are found to be similar to those reported for phospholipid in sonicated vesicle preparations which are used as membrane models, but the lines are somewhat sharper suggesting the possibility of less anisotropic motion in the mixed micelles than in the vesicles.     

Binding of cytochrome c to the cytochrome bc1 complex (complex III) and its subunits cytochrome c1 and b1.

Cytochrome $\text{c}$₁, the electron donor for cytochrome $\text{c}$, is a subunit of the mitochondrial cytochrome $\text{bc}$₁ complex (complex III, cytochrome $\text{c}$ reductase). To test if cytochrome $\text{c}$₁ is the cytochrome $\text{c}$-binding subunit of the $\text{bc}$₁ complex, binding of cytochrome $\text{c}$ to the complex and to isolated cytochrome $\text{c}$₁ was compared by a gel-filtration method under non-equilibrium conditions (a $\text{bc}$₁ complex lacking the Rieske ironsulfur protein was used; von Jagow et al. (1977) Biochim. Biophys. Acta $\text{462}$, 549–558). The approximate stoichiometries and binding affinities were found to be very similar. Binding of cytochrome $\text{c}$ to isolated cytochrome $\text{b}$ which is another subunit of the reductase was not detectable by the gel-filtration method. Further, the same lysine residues of cytochrome $\text{c}$ were shielded towards chemical acetylation in the complexes $\text{c}\text{:}\text{c}$₁ and $\text{c}\text{:}\text{bc}$₁. From this we conclude that the same surface area of cytochrome $\text{c}$ is in direct contact with cytochrome $\text{bc}$₁ and with cytochrome $\text{c}$₁ in the respective complexes and that therefore cytochrome $\text{c}$ is most probably the structural ligand for cytochrome $\text{c}$ in mitochondrial cytochrome $\text{c}$ reductase.     

The reversible phosphorylation of isocitrate dehydrogenase of Salmonella typhimurium

The 46,000 dalton phosphoprotein in Salmonella typhimurium is isocitrate dehydrogenase, an enzyme at the branch point between the glyoxylate and Krebs cycle pathways. The enzyme is phosphorylated by a kinase which is controlled by growth conditions; and it is dephosphorylated by a phosphatase. Acetate, ethanol, α-methylglucoside, and deoxyglucose cause an activation of the phosphorylation reaction in intact cells. A number of other compounds are found to affect the kinase and phosphatase activities. The reversible phosphorylation of isocitrate dehydrogenase plays a major role in the control of the Krebs cycle and glyoxylate pathways.     

Reduction and subsequent oxidation of a cytochrome b of human neutrophils after stimulation with phorbol myristate acetate.

The level of nerve growth factor-like immunoreactivity in the lower limb muscles, the site where the dystrophic mice are effected, of both normal and dystrophic mice was studied by solid-phase radioimmunoassay. Nerve growth factor-like immunoreactivity levels of the homozygous dystrophic mice were about two times lower than those of the heterozygous dystrophic mice at 10–11 weeks and 7–8 weeks of age. The levels in 4–5 week old homozygous mice were too low for detection and remarkable differences between the homozygous and the heterozygous mice were observed at this age. These differences in the level of nerve growth factor-like immunoreactivity suggest that the factor may have some relation to the disease.     

Proton nuclear magnetic resonance studies of the manganese (II) binding site of concanavalin A. Effect of saccharides on the solvent relaxation rates

The longitudinal relaxation rate ($\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$) of water protons was studied in solutions of Mn(II)-concanavalin A at a number of frequencies. These relaxation rates were lowered in the presence of a variety of saccharides which have affinities for concanavalin A which range over two orders of magnitude. A good correlation was found in which saccharides which bind tightly have the greatest effect and saccharides which bind weakly or not at all have little effect on the $\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$ values. The temperature dependence of the proton relaxation rates showed that the lowering of these rates in the presence of saccharides was most likely due to a change in the exchange rate of solvent interacting with protein-bound Mn(II), $\text{1}\text{T}{}_{\mathrm{<mtext>m</mtext>}}$.An analysis of the temperature and frequency dependence of the $\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$ and $\text{1}\text{T}{}_{\mathrm{<mtext>2</mtext><mtext>p</mtext>}}$ (transverse) solvent proton relaxation rates resulted in evaluation of a number of parameters for solvent water molecules interacting in the first coordination sphere of Mn(II) bound to concanavalin A. The ratio of the number of water molecules (q) to the Mn(II)-proton distance (r) obtained from a computer fit of the data over a limited temperature range is in accord with the findings of Koenig et al. ((1973) Proc. Nat. Acad. Sci.70, 475) and Meirovitch and Kalb ((1973) Biochim. Biophys. Acta303, 258). However, our studies of $\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$ and $\text{1}\text{T}{}_{\mathrm{<mtext>2</mtext><mtext>p</mtext>}}$ of water over a more extensive temperature range are best fit with the following conclusions: at low temperatures (<20 °C), the data are consistent with an outer-sphere relaxation process. At higher temperatures (> 30 °C), the water molecule in the inner coordination sphere of the bound Mn(II) begins exchanging more rapidly and contributes to the relaxation processes ($\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$ and $\text{1}\text{T}{}_{\mathrm{<mtext>2</mtext><mtext>p</mtext>}}$). The relaxation time of protons in the inner coordination shell, T_1M, contributes over the entire temperature range and produces a frequency dependence in the relaxivity data from 6 to 100 MH_z since the contributions to the correlation times are in the range 10^?9-10^?8 sec.     

Vacuum ultraviolet circular dichroism spectrum of beta-turn in solution.

The vacuum ultraviolet circular dichroism spectrum of an isolated 4 → 1 hydrogen bonded β-turn is reported. The observed spectrum of N-acetyl-Pro-Gly-Leu-OH at ? 40°C in trifluoroethanol is in good agreement with the theoretically calculated CD spectrum of the β-turn conformation. This spectrum, particularly the presence of a strong negative band around 180 nm and a large ratio $\text{[θ]}{}_{201}\text{[θ]}{}_{225}$, can be taken as a characteristic feature of the isolated β-turn conformation. These CD spectral features can thus be used to distinguish the β-turn conformation from the β-structure in solution.     

The mechanism of activation of rabbit plasminogen by urokinase. Lack of a preactivation peptide

We have obtained direct evidence which we interpret to prove that an amino terminal peptide need not be released from rabbit plasminogen prior to its conversion to plasmin by urokinase. The single chain plasminogen molecule possesses an amino terminal amino acid sequence of NH₂-glu-pro-leu-asp-asp. When this plasminogen is activated to plasmin by urokinase in the presence of the Kunitz bovine trypsin-plasmin-kallikrein inhibitor (BTI), a two chain disulfide linked molecule of plasmin is obtained. The heavy chain of this plasmin is directly derived from the original amino terminus of plasminogen since it possesses the identical amino terminal sequence as does native plasminogen. When the same plasminogen activation is carried out in the absence of BTI, the heavy chain of the plasmin obtained has a molecular weight of 6,000–8,000 less than the heavy chain of the plasmin obtained in the presence of this inhibitor. In addition, the heavy chain of this latter plasmin has an amino terminal sequence which differs from the original native plasminogen. These data show, in agreement with others, that the activation of plasminogen by urokinase is accompanied by the loss of an amino terminal peptide from plasminogen but also show, in contrast to the human plasminogen system, that cleavage of the internal peptide bond, leading to plasmin formation, can occur without cleavage of the amino terminal peptide.     

Expression of fetal thymidine kinase in human cobalamin or folate deficient lymphocytes.

In extracts of peripheral blood lymphocytes of cobalamin or folate deficient patients thymidine kinase activity is increased three fold and exhibits properties of the fetal isoenzyme. Appropriate vitamin therapy results in reduction of this activity to normal levels and change from fetal to adult isoenzyme. The occurrence in cobalamin or folate deficiency of fetal thymidine kinase activity in non proliferating human lymphocytes is unique and may reflect events in the deficient marrow lymphoid progenitor cells.