Block copolymers of amino acids. I. Synthesis and structure of copolymers of L-alanine or L-phenylalanine with D,L-lysine-d7 or D,L-lysine

Water-soluble block copolymers of the type (A)_m-(B)_n-(A)_p, where (A)_m,p was either poly(D ,L -lysine-α,β,β,γ,γ,δ,δ-d₇) or poly(D ,L -lysine) and (B)_n was either poly(L -alanine) or poly(L -phenylalanine), were synthesized for conformational studies by proton magnetic resonance spectroscopy. Analytical determination of the amount of the initiator fragment (n-hexylamine) at the C-terminus of the copolymers was used to obtain the number-average degrees of polymerization, DP _n, and thereby, together with the amino acid composition, to establish the covalent structures of the polymers. The values of DP _n were found to be much lower than those deduced from sedimentation equilibrium or form viscosity measurements. These deviations, which also are thought to have arisen in similar studies reported in the literature, are attributable to intermolecular aggregation; the relation of such aggregation to covalent structure (and its effect on the polymerization reaction) is discussed in terms of the conditions and mechanism of synthesis of block copolymers of amino acids.     

Raman spectra of copoly(D,L-alanines)

Raman spectra in the region 1000–150 cm^?1 were measured for copoly(D ,L -alanines) with the D -residue contents, 3, 7, 10, and 20%, and compared with the spectrum of the α-helical poly-L -alanine. The 532- and 378-cm^?1 peaks were assigned to the L -residues with a right-handed α-helix-like local conformation or to the D -residues with a left-handed α-helix-like local conformation. From the intensity of the latter peak the contents of these local conformations were estimated as a function of the D -residue contents for the copolymers. The 264-cm^?1 peak, which has been assigned to the breathing vibration of the α-helical poly-L -alanine, shows a marked decrease in its intensity upon the introduction of the D residues. This result suggests that the overall deformation vibration of the α-helix arises from rather long sequences of the L - and D -alanine residues with the α-helical conformation and that the intensity of this vibration depends on the content of these sequences in the copolymers.     

Achromobacter cycloclastes nitrite reductase. The function of copper, amino acid composition, and ESR spectra.

1. Dialysis against cyanide at pH 7 of Achromobacter cycloclastes nitrite reductase [EC 1.7.99.3] of a dissimilatory type led to the removal of about 50% of the copper from the enzyme molecule, with a concomitant decrease of the enzymatic activities. It was inferred that enzyme-bound copper atoms play an essential role in the catalytic activities of the enzyme. 2. The amino acid composition of the enzyme was determined after acid hydrolysis. 3. ESR spectra of the frozen solution and lyophilized powder of the nitrite reductase predominantly showed the presence of two kinds of copper: Type 1 Cu2+, which had narrow and sharp hyperfine splitting, and Type 2 Cu2+, which had broader hyperfine splitting. The bond between the oxidized enzyme and nitrite seems to be ionic.     

Dimerisation mutants of Lac repressor. II. A single amino acid substitution, D278L, changes the specificity of dimerisation

Assembly of the lactose repressor tetramer involves two subunit interfaces, the C-terminal heptad repeats, and the monomer-monomer interface. Dimerisation between two monomers of Lac repressor of Escherichia coli lacking the two C-terminal heptad repeats occurs through the interactions between three alpha-helices of each monomer, which form a highly hydrophobic interface. Residues possibly involved in specific dimer formation are known from X-ray studies and from the phenotypes of more than 4000 single amino acid substitutions. During the examination of numerous mutants within the dimerisation interface of Lac repressor, we found that substitution of one amino acid, D278 to leucine, is sufficient to change the specificity of dimerisation. Analysis of this single substitution indicates that D278L mutant Lac repressor represses like wild-type. However, it no longer forms heterodimers with wild-type Lac repressor.     

Low-frequency Raman spectra of lysozyme.

New techniques in laser Raman spectroscopy are used to obtain spectra of aqueous solutions of lysozylme for frequency shifts as small as 5 cm^?1. In addition, Raman measurements are made on two crystalline forms of hen egg white lysozyme. The spectra obtained from the solution and from the crystal are found to be similar for frequencies above 100 cm^?1. However, a low-frequency band at 25 cm^?1 observed in crystalline lysozyme is not found in the solution, indicating that this band cannot be attributed to an internal molecular vibration.     

pH-Dependent Raman spectra and thermal melting profiles for polycytidylic acid

The Raman spectrum of polycytidylic acid was investigated in the pH range of 6.6–4.1. The thermal melting temperatures and the nature of the thermal melting profiles change in this range as monitored by the three Raman band envelopes, which include the 780-, 805-cm^?1 bands, the 1190-, 1285-cm^?1 bands, and the 1527-cm^?1 band. By coupling these data with the theory of Raman scattering intensity and quantitative pH profiles for cytidine, it is shown that the band envelopes studied exhibit specific, yet different information regarding the thermal melting process. The band envelopes at 1170–1310 and 1527 cm^?1, which are a sensitive function of both the extent of protonation and base stacking (hypochromic), reveal T_m values which agree with values derived from uv melting profiles. The 760–830-cm^?1 envelope, which is not directly sensitive to cytosine residue protonation, but includes information associated with base stacking (the 780-cm^?1 band) and the nature of the phosphodiester backbone (the frequency-dependent 805-cm^?1 component), exhibits T_m values which deviate from the values obtained from the other bands. The observed differences are pH-dependent and correlate well with the extent of deprotonation that takes place in the denaturation process. Details of the spectrum of neutral and protonated poly(C) from pH 7 to 4.1 are discussed and related to the nature of the thermal denaturation process.     

Far-infrared spectra of sequential copolymers of amino acids with alkyl group side chains

Far-infrared spectra were measured for the sequential copolymers of amino acids with alkyl group side chains. The analysis of the spectra showed that (L -Ala-L -Ala-Gly)_n, (L -Ala-Gly)_n, (L -Ala-Gly-Gly)_n, (L -Val-L -Ala-L -Ala)_n, and (L -Val-L -Ala)_n, have the antiparallel pleated sheet structures and that the backbone conformations of (L -Val-L -Val-L -Ala)_n and (L -Val-L -Val-Gly)_n are the same as that of poly-L -valine. The far-infrared bands characteristic of the antiparallel pleated sheet structure were assigned on the basis of the result of the normal coordinate analysis of poly-L -alanine with this structure. The intersheet and interchain spacings of the sequential copolymers with the antiparallel pleated sheet structure were determined from the x-ray powder-diffraction patterns of these samples.     

Effects of D and L amino acid residues in linear peptides on carbon-13 nuclear magnetic resonance parameters.

The 13C spectra of the linear tripeptidyl diastereoisomers, Gly-Gly-Leu, Gly-Gly-D-Leu, Leu-Gly-Gly, D-Leu-Gly-Gly, Ala3, Ala-Ala-D-Ala, Ala-D-Ala-Ala, Val3, and Val-Val-D-Val are very similar or even identical at pH meter readings of 1.0, 7.0 and 12.0 in D2O. The spectra of Pro-Leu-Gly-NH2 and Pro-D-Leu-Gly-NH2 likewise show only minor differences in 13C chemical shifts (less than 0.4 p.p.m.) under similar conditions. This contrasts significantly with previous findings comparing 13C chemical shifts of cyclo(Pro-Leu) and cyclo(Pro-D-Leu) where major differences in chemical shifts were observed for both residues due to differences in conformational constraints present in these cyclic proline-containing peptides. The least-squares fit of spin-lattice relaxation times (T1) for Pro-Leu-Gly-NH2 and Pro-D-Leu-Gly-NH2 show that it is not possible to fit all the T1 values to a unique and rigid structure whether folded or extended. The glycyl residue undergoes enhanced motion when compared with the prolyl and leucyl residues. Internal motion must be postulated within the proline ring and for the CH3 groups of leucine.     

Resonance Raman spectra of anionic semiquinoid form of a flavoenzyme, D-amino acid oxidase

Resonance Raman (RR) spectra of the complex of anionic semiquinoid D-amino acid oxidase (DAO) with picolinate in H2O and D2O were observed in the 300-1,750 cm-1 region. RR spectra were also measured for the complex of the semiquinoid enzyme reconstituted with isotopically labeled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]-FAD. On the basis of the isotope effects, tentative assignments of the observed bands of the anionic semiquinoid flavin were made. The spectra differ from those of oxidized, neutral semiquinoid, and anionic reduced flavins previously reported. The 1,602 cm-1 band was not shifted for any FAD labeled in ring II and/or ring III and was assigned to a ring I mode. The 1,516 cm-1 band underwent an isotopic shift upon [4a-13C]- or [4,10a-13C2]-labeling. The band was assigned to the mode containing C(4a)-C(10a) stretching. The 1,331 and 1,292 cm-1 bands shifted upon [4a-13C]- or [5-15N]-labeling and were assigned to the modes containing C(4a)-N(5) stretching. The 1,217 and 1,188 cm-1 bands were assigned to the skeletal vibrations of ring III coupled with the N(3)-H bending mode. The RR spectrum of the complex of anionic semiquinoid DAO with alpha-iminopropionate or N-methyl-alpha-iminopropionate was essentially identical with that of the complex with picolinate.     

Bovine cathepsins S and L: isolation and amino acid sequences.

The purification procedure of cathepsin S includes acid activation of spleen homogenate, incubation at 37 degrees C, precipitation with (NH4)2SO4 in H2O/tert-butanol medium, gel chromatography, chromatofocusing, covalent chromatography and cation chromatography of FPLC system. Cathepsin S has a M(r) of about 24,000 Da with pI of 6.5 and 6.8. The mixture of both forms gave a single sequence. Cathepsin L was purified from bovine kidney by acid treatment and incubation of 37 degrees C, precipitation by (NH4)2SO4, two ion exchange chromatographies on CM-Sephadex, gel chromatography and ion exchange chromatography on FPLC system. Cathepsin L exists in multiple forms with pI 5.3-5.7 and M(r) of about 29,000 Da. N-terminal amino acid sequence confirms that cathepsin L and cathepsin S are different enzymes.     

Resonance Raman spectra of whole mitochondria.

The resonance Raman spectra of reduced cytochromes b and c and cytochrome oxidase in whole mitochondria have been recorded without any instrument modifications. The contributions of the individual cytochromes have been identified by comparison with the characteristic features observed in partially purified preparations including: (i) the strong dependence of the intensity patterns on excitation wavelength relative to the peak positions of the alpha, beta, and gamma absorption bands of the cytochromes; and (ii) the presence of marker bands for heme type. Since the Raman spectra can be used as an intrinsic indicator of interaction between hemes, the ability to record spectra in intact mitochondria opens the possibility to study heme-heme interactions in the functioning membrane in situ.     

Regulation of amino acid transport in L6 myoblasts. II. Different chemical properties of transport after amino acid deprivation

The mechanism of stimulation of amino acid transport system A caused by amino acid deprivation in L6 cells was investigated. In cells loaded with alpha-aminoisobutyric acid (AIB), amino acid deprivation increased the rate of proline uptake only after the intracellular [AIB] dropped below 7 mM. Efflux of proline was not sensitive to the presence of proline in the outer medium (with or without external Na+), suggesting that efflux through system A (and possibly uptake) is not susceptible to transinhibition. Transport (stimulated uptake) into amino acid-deprived cells and that into amino acid-supplemented cells differed in several chemical properties: 1) In the former group, transport was higher at lower pH values than in the latter, and the optimum pH values were 7.5 and 7.8, respectively. 2) Unlike proline uptake in supplemented cells, uptake in deprived cells was inhibited by 50% with N-ethylmaleimide (1 mM) or by 50 microM p-chloromercuribenzoate (PCMBS). Inhibition by PCMBS was not due to collapse of the Na+ gradient. The mercurial inhibited only the deprivation-induced stimulation of transport, bringing the rate of proline uptake to the "basal" uptake level observed in amino acid-supplemented cells. Proline uptake was not stimulated by a second deprivation following treatment with PCMBS and a supplementation-deprivation cycle. However, in untreated cells, or by reversing mercaptide formation with dithiotreitol, the second deprivation stimulated transport. Deprivation at 4 degrees C did not elicit stimulation of proline uptake. Cycloheximide prevented the stimulation and decreased the rate of proline uptake in deprived cells more efficiently than in supplemented cells. Actinomycin D prevented stimulation when added at the onset of deprivation. The above data indicate that stimulation of transport by deprivation is protein synthesis-dependent and that the stimulated transport had chemical properties distinct from the "basal" transport in supplemented cells. The evidence presented is consistent with a model of activation of a finite pool of transporters upon deprivation, the chemical characteristics of which differ from those of the "basal" transport system.