Biophysical studies of an NAD(P)<Superscript>+</Superscript>-dependent aldehyde dehydrogenase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Aldehyde dehydrogenase (ALDH) catalyzes the conversion of aldehydes to the corresponding acids by means of an NAD(P)⁺-dependent virtually irreversible reaction. In this investigation, the biophysical properties of a recombinant Bacillus licheniformis ALDH (BlALDH) were characterized in detail by analytical ultracentrifuge (AUC) and various spectroscopic techniques. The oligomeric state of BlALDH in solution was determined to be tetrameric by AUC. Far-UV circular dichroism analysis revealed that the secondary structures of BlALDH were not altered in the presence of acetone and ethanol, whereas SDS had a detrimental effect on the folding of the enzyme. Thermal unfolding of this enzyme was found to be highly irreversible. The native enzyme started to unfold beyond ~0.2 M guanidine hydrochloride (GdnHCl) and reached an unfolded intermediate, [GdnHCl]_{05, N-U}, at 0.93 M. BlALDH was active at concentrations of urea below 2 M, but it experienced an irreversible unfolding under 8 M denaturant. Taken together, this study provides a foundation for the future structural investigation of BlALDH, a typical member of ALDH superfamily enzymes.     

Effect of temperature and guanidine hydrochloride on ferrocytochrome<Emphasis Type="Italic"> c</Emphasis> at neutral pH

Thermally denatured horse heart ferrocytochrome c (ferrocyt c) has been characterized using absorption spectroscopy, differential scanning calorimetry (DSC) and viscometry at pH 7.0. DSC experiments have yielded the transition temperature of denaturant-free ferrocyt c unfolding as 100.6±0.3 °C, indicating an extremely high stability of the protein. The presence of guanidine hydrochloride (GdnHCl) facilitated estimation of the structural features of thermally unfolded ferrocyt c. The stability of the protein, expressed by G _D at 25 °C, is 59±5 kJ mol^–1 (DSC) and 65±6 kJ mol^–1 (absorption spectroscopy). An absorption spectrum of ferrocyt c demonstrates that the heme occurs in the high-spin state at extreme denaturing conditions (94 °C, 6.6 M GdnHCl). Absorption spectroscopy, using heme as a probe, shows that thermal denaturation of ferrocyt c occurs as a transition from a native low-spin (Met80/His18) to a high-spin disordered state with involvement of non-native, low-spin (bis-His) species.Abbreviations CD circular dichroism - cyt c cytochrome c - DSC differential scanning calorimetry - ferricyt c ferricytochrome c - ferrocyt c ferrocytochrome c - GdnHCl guanidine hydrochloride - NHE normal hydrogen electrode     

Biophysical Characterization of a Recombinant α-Amylase from Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. strain TS-23

Environmental variables can significantly influence the folding and stability of a protein molecule. In the present study, the biophysical properties of a truncated Bacillus sp. TS-23 α-amylase (BACΔNC) were characterized in detail by glutaraldehyde cross-linking, analytical ultracentrifugation, and various spectroscopic techniques. With cross-linking experiment and analytical ultracentrifuge, we demonstrated that the oligomeric state of BACΔNC in solution is monomeric. Far-UV circular dichroism analysis revealed that the secondary structures of BACΔNC were significantly altered in the presence of various metal ions and SDS, whereas acetone and ethanol had no detrimental effect on folding of the enzyme. BACΔNC was inactive and unstable at extreme pH conditions. Thermal unfolding of the enzyme was found to be highly irreversible. The native enzyme started to unfold beyond ~0.2 M guanidine hydrochloride (GdnHCl) and reached an unfolded intermediate, [GdnHCl]_{0.5, N–U}, at 1.14 M. BACΔNC was active at the concentrations of urea below 6 M, but it experienced an irreversible unfolding by >8 M denaturant. Taken together, this work lays a foundation for the future structural studies with Bacillus sp. TS-23 α-amylase, a typical member of glycoside hydrolases family 13.     

Conformational Properties of Polyglutamine Sequences in Guanidine Hydrochloride Solutions

Two sets of iso-1-cytochrome c variants have been prepared with N-terminal insertions of pure polyglutamine, i.e., PolyQ variants, or polyglutamine interrupted with lysine every sixth residue, i.e., Gln-rich variants. The polymer properties of these pure polyGln or Gln-rich sequences have been evaluated using equilibrium and kinetic His-heme loop formation methods for loop sizes ranging from 22 to 46 in 1.5, 3.0, and 6.0 M guanidine hydrochloride (GdnHCl). In 6.0 M GdnHCl, the scaling exponent, ν₃, for the pure polyGln sequences, is ∼1.7—significantly less than ν₃ ≈ 2.15 for the Gln-rich sequences. The stability of the His-heme loops becomes progressively greater for the pure polyGln sequences relative to the Gln-rich sequences as GdnHCl concentration decreases from 6.0 to 1.5 M. Thus, the context of the sequence effects the polymer properties of Gln repeats even in denaturing concentrations of GdnHCl. Comparison of data for the Gln-rich variants with previous results for Gly-rich and Ala-rich variants shows that ν₃ ∼ 2.2 for the Gln-rich, Gly-rich, and Ala-rich sequences in 6.0 M GdnHCl, whereas ν₃ remains unchanged at 3.0 M GdnHCl concentration for the Gln-rich and Ala-rich sequences but decreases to ∼1.7 for the Gly-rich sequences. Thus, the polymer properties of Gln-rich and Ala-rich sequences are less sensitive to solvent quality in denaturing solutions of GdnHCl than Gly-rich sequences. Evaluation of Flory’s characteristic ratio, C_n, for the Gln-rich and Ala-rich sequences relative to the Gly-rich sequences shows that Gln-rich sequences are stiffer than Ala-rich sequences at both 3.0 and 6.0 M GdnHCl.     

Structure of native proteoglycan aggregates from chick limb-bud chondrocytes

Laser light-scattering has been used to investigate the size of native proteoglycan aggregates (PGA-aA1) from day-8 chick limb-bud chondrocyte cultures isolated under associative extraction and purification conditions in 0.4M guanidinium chloride (GdnHCl) solution. Dynamic light-scattering measurements yielded a hydrodynamic radius, R_s, of 244 ± 10 nm for PGA-aA1 in 0.4M GdnHCl, and a weight-average molecular weight (M _w) of 150 ± 50 × 10⁶ was obtained from a Zimm plot. Disaggregation in 4.0M GdnHCl aqueous solution yielded proteoglycan subunits (PGS) with R_s = 39 ± 2 nm, M _w = 1.6 ± 0.3 × 10⁶, which reassembled in 0.4M GdnHCl to form “reconstituted native” aggregates (PGA-raA1) with R_s = 121 ± 6 nm, M _w = 17 ± 3 × 10⁶. A second specimen of PGA-aA1 had R_s = 192 ± 10 nm, M _w = 100 ± 10 × 10⁶. The latter value was estimated from an empirical relationship between M _w and R_s. After dissociation, this specimen reassembled to form PGA-raA1 with R_s = 85 ± 5 nm, M _w = 12 ± 1 × 10⁶. These data are compared with those for a specimen of reconstituted aggregate (PGA-A1) that had been extracted under dissociative conditions and then reaggregated by dialysis to 0.4M GdnHCl aqueous solution, for which R_s = 138 ± 9 nm, M _w = 45 ± 8 × 10⁶. From these values, we have calculated the weight-average number of subunits per aggregate N_w: 111 for PGA-aA1 and 12 for raA1 (70 and 7 for the second PGA-aA1 and PGA-raA1 specimen, respectively) as compared to 32 for PGA-A1. The numbers of subunits per aggregate were also determined from electron micrographs of spread specimens. The latter results show the same trends as those obtained by light scattering, but lead in each case to lower numbers of subunits per aggregate. These data demonstrate conclusively that PGA samples exhibit a higher degree of aggregation in solution than visualized in typical electron microscopy (EM) preparations, probably due to disaggregation during EM specimen preparation. Since N_w determined both by light scattering (LS) and by EM are larger for native versus reconstituted aggregate samples, our data point to a more compact aggregation of subunits along the hyaluronic acid (HA) chains in the former.     

Characterization of N-terminal amino group–heme ligation emerging upon guanidine hydrochloric acid induced unfolding of <Emphasis Type="Italic">Hydrogenobacter thermophilus</Emphasis> ferricytochrome <Emphasis Type="Italic">c</Emphasis><Subscript>552</Subscript>

Nonnative heme coordination structures emerging upon guanidine hydrochloric acid (GdnHCl) induced unfolding of Hydrogenobacter thermophilus ferricytochrome c ₅₅₂ were characterized by means of paramagnetic NMR. The heme coordination structure possessing the N-terminal amino group of the peptide chain in place of axial Met (His–N_term form) was determined in the presence of GdnHCl concentrations in excess of 1.5 M at neutral pH. The stability of the His–N_term form at pH 7.0 was found to be comparable with that of the bis-His form which has been recognized as a major nonnative heme coordination structure in cytochrome c folding/unfolding. Consequently, in addition to the bis-His form, the His–N_term form is a substantial intermediate which affects the pathway and kinetics of the folding/unfolding of cytochromes c, of which the N-terminal amino groups are not acetylated. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Entropic stabilization of myoglobin by subdenaturing concentrations of guanidine hydrochloride

To find out the changes in the internal dynamics and function of proteins as a consequence of their binding interactions with guanidine hydrochloride (GdnHCl), laser flash photolysis and optical absorption methods have been used to study the dynamic events in the horse myoglobin–CO complex (MbCO) in the presence of subdenaturing concentrations of GdnHCl at pH 7, 22 °C. The rate coefficients for geminate rebinding and bimolecular rebinding (k _on) were measured by laser photolysis of CO in MbCO, and the CO dissociation rate (k _off) was determined by the CO replacement method using hexacyanoferrate ion or NO. Starting from the native-state condition, the values of k _on and k _off decrease by approximately 1.4 (±0.1)-fold in the presence of 0.1–0.3 M GdnHCl, and then increase at higher concentrations of the denaturant. This has been taken as evidence for internal motional constraints and increased stability of the protein in the subdenaturing region giving rise to gated entry of the photolyzed CO from the solvent. The rate for geminate rebinding does not show any decrease in the rate versus GdnHCl concentration plots. The values for the activation enthalpy for the CO dissociation reaction and the entropy loss relative to the native-state entropy, both measured as a function of GdnHCl concentration, indicate that the protein is indeed stabilized under subdenaturing conditions. Analyses of thermal unfolding transitions of myoglobin in the presence of different concentrations of GdnHCl indicate that the stability of this protein extracted from the linear free energy model is approximately 3–4 kcal mol⁻¹ less than the true stability. The results indicate the appropriateness of the denaturant binding model for the analysis of GdnHCl-induced unfolding data, and provide a value of 7.9 kcal mol⁻¹ as the true stability of the protein. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Unfolding Studies of <Emphasis Type="Italic">Escherichia coli</Emphasis> Maltodextrin Glucosidase Monitored by Fluorescence Spectroscopy

Equilibrium unfolding of a 69-kDa monomeric Escherichia coli maltodextrin glucosidase (MalZ) was studied using intrinsic and extrinsic fluorescence spectroscopy. The unfolding transition of MalZ followed a three-state process, involving the formation of a stable intermediate state having more exposed hydrophobic surface. It was found that the protein structure can be easily perturbed by low concentration of guanidium hydrochloride (GdnHCl) and, at a GdnHCl concentration of 2 M, MalZ was denatured completely. The active site of the protein also has been proved to be sensitive to a low concentration of GdnHCl since MalZ deactivated at 0.5 M GdnHCl completely. The surface hydrophobicity and ANS-binding site of the protein have been determined to be 150.7 and 0.24, respectively. Perhaps the formation of the stable unfolding intermediate, having higher surface hydrophobicity, may be one of the reasons for aggregation of MalZ and its recognition by chaperonin GroEL during the assisted folding pathway.     

Unfolding analysis of the mature and unprocessed forms of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> γ-glutamyltranspeptidase

Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) undergoes an autocatalytic process to generate 44.9 and 21.7 kDa subunits; however, a mutant protein (T399A) loses completely the processing ability and mainly exists as a precursor. For a comprehensive understanding of their structural features, the biophysical properties of these two proteins were investigated by circular dichroism and fluorescence spectroscopy. Tryptophan fluorescence and circular dichroism spectra were nearly identical for BlGGT and T399A, but unfolding analyses revealed that these two proteins had a different sensitivity towards temperature- and guanidine hydrochloride (GdnHCl)-induced denaturation. BlGGT and the unprocessed T399A displayed T _m values of 61.4°C and 68.1°C, respectively, and thermal unfolding of both proteins was found to be highly irreversible. Fluorescence quenching analysis showed that T399A had a dynamic quenching constant similar to that of the wild-type enzyme. BlGGT started to unfold beyond ∼2.14 M GdnHCl and reached an unfolded intermediate, [GdnHCl]_{0.5, N − U}, at 2.85 M, corresponding to free energy change ( DG_H2O )\left( {{\Delta }G_{\rm{H}_{2}{O}} } \right) of 12.34 kcal mol^− 1, whereas the midpoint of the denaturation curve for T399A was approximately 3.94 M, corresponding to a DG_H2O\Delta G_{\rm{H}_{2}{O}} of 4.45 kcal mol^− 1. Taken together, it can be concluded that the structural stability of BlGGT is superior to that of T399A.     

Structural thermodynamics of a random coil protein in guanidine hydrochloride

An important problem in protein folding is to understand the relationship between the structure of a denatured ensemble and its thermodynamics. Using 0 – 6M GdnHCl at fixed pH, we evaluated dimensional changes of an extensively denatured ensemble along with a thermodynamic parameter (Δυ) that monitors the proton inventory of the ensemble. Reduced and carboxyamidated ribonuclease A (RCAM) is a member of a class of disulfide‐free RNase A molecules believed to be random coils (extensively denatured) in aqueous solution. Because GdnHCl interacts more favorably with the protein than water does, this denaturant is observed to increase the Stokes radius of the random coil, with the greatest Stokes radius change occurring in the 0 – 1.5M GdnHCl range. Measurement of the degree of protonation (proton inventory) of the ensemble as a function of GdnHCl at the fixed pH shows that the thermodynamic character of the ensemble also changes markedly in the 0 – 1.5M GdnHCl range, but with little or no change beyond 1.5M GdnHCl. To obtain denaturant‐independent ΔG°_N–D values, the linear extrapolation method (LEM) requires the thermodynamic character of the native and denatured ensembles to be invariant in the transition zone. The results reported here indicate that proteins with a transition midpoint in the 0 – 1.5M GdnHCl range will not give denaturant‐concentration independent ΔG°_N–D values. Such LEM‐derived ΔG°_N–D quantities are a property of the protein and the denaturant, a condition that considerably limits their value in understanding structural energetics. Proteins 2000;41:44–49. © 2000 Wiley‐Liss, Inc.     

A thermostable leucine aminopeptidase from<Emphasis Type="Italic"> Bacillus kaustophilus</Emphasis> CCRC 11223

Two degenerate primers established from the consensus sequences of bacterial leucine aminopeptidases (LAP) were used to amplify a 360-bp gene fragment from the chromosomal DNA of thermophilic Bacillus kaustophilus CCRC 11223 and the amplified fragment was successfully used as a probe to clone a leucine aminopeptidase (lap) gene from a genomic library of the strain. The gene consists of an open reading frame (ORF) of 1,494 bp and encodes a protein of 497 amino acid residues with a calculated molecular mass of 53.7 kDa. The complete amino acid sequence of the cloned enzyme showed greater than 30% identity with prokaryotic and eukaryotic LAPs. Phylogenetic analysis showed that B. kaustophilus LAP is closely related to the enzyme from Bacillus subtilis and is grouped with the M17 family. His₆-tagged LAP was generated in Escherichia coli by cloning the coding region into pQE-30 and the recombinant enzyme was purified by nickel-chelate chromatography. The pH and temperature optima for the purified enzyme were 8 and 65°C, respectively, and 50% of its activity remained after incubation at 60°C for 32 min. The enzyme preferentially hydrolyzed l-leucine-p-nitroanilide (l-Leu-p-NA) followed by Cys derivative.Communicated by G. Antranikian     

LC3-associated phagocytosis initiated by integrin ITGAM-ITGB2/Mac-1 enhances immunity to Listeria monocytogenes

The macroautophagic/autophagic machinery cannot only target cell-endogenous components but also intracellular pathogenic bacteria such as Listeria monocytogenes. Listeria are targeted both by canonical autophagy and by a noncanonical form of autophagy referred to as LC3-associated phagocytosis (LAP). The molecular mechanisms involved and whether these processes contribute to anti-listerial immunity or rather provide Listeria with a replicative niche for persistent infection, however, remained unknown. Recently, using an in vivo mouse infection model, we have been able to demonstrate that Listeria in tissue macrophages are targeted exclusively by LAP. Furthermore, our data show that LAP is required for killing of Listeria by macrophages and thereby contributes to anti-listerial immunity of mice, whereas canonical autophagy is completely dispensable. Moreover, we have elucidated the molecular mechanisms that trigger LAP of Listeria and identified the integrin ITGAM-ITGB2/Mac-1/CR3/integrin α_Mß₂ as the receptor that initiates LAP in response to Listeria infection.     

Overexpression,Purification, and Characterization of the Recombinant Leucine Aminopeptidase II of <Emphasis Type="Italic">Bacillus stearothermophilus</Emphasis>

For expression of Bacillus stearothermophilus NCIB 8924 leucine aminopeptidase II (LAP II) in Escherichia coli regulated by a T5 promoter, the gene was amplified by polymerase chain reaction and cloned into expression vector pQE-32 to generate pQE-LAPII. The His₆-tagged enzyme was overexpressed in IPTG-induced E. coli M15 (pQE-LAPII) as a soluble protein and was purified to homogeneity by nickel-chelate chromatography to a specific activity of 425 U/mg protein with a final yield of 76%. The subunit molecular mass of the purified protein was estimated to be 44.5 kDa by SDS-PAGE. The temperature and pH optima for the purified protein were 60°C and 8.0, respectively. Under optimal condition, the purified enzyme showed a marked preference for Leu-p-nitroanilide, followed by Arg- and Lys-derivatives. The His₆-tagged enzyme was stimulated by Co²⁺ ions, but was strongly inhibited by Cu²⁺ and Hg²⁺ and by the chelating agents, DTT and EDTA. The EDTA-treated enzyme could be reactivated with Co²⁺ ions, indicating that it is a cobalt-dependent exopeptidase. Taking the biochemical characteristics together, we found that the recombinant LAP II exhibits no important differences from those properties described for the native enzyme. Received: 16 August 2002 / Accepted: 4 September 2002     

A New Method for the Characterization of Strain-Specific Conformational Stability of Protease-Sensitive and Protease-Resistant PrPSc

Although proteinacious in nature, prions exist as strains with specific self-perpetuating biological properties. Prion strains are thought to be associated with different conformers of PrP^Sc, a disease-associated isoform of the host-encoded cellular protein (PrP^C). Molecular strain typing approaches have been developed which rely on the characterization of protease-resistant PrP^Sc. However, PrP^Sc is composed not only of protease-resistant but also of protease-sensitive isoforms. The aim of this work was to develop a protocol for the molecular characterization of both, protease-resistant and protease-sensitive PrP^Sc aggregates. We first set up experimental conditions which allowed the most advantageous separation of PrP^C and PrP^Sc by means of differential centrifugation. The conformational solubility and stability assay (CSSA) was then developed by measuring PrP^Sc solubility as a function of increased exposure to GdnHCl. Brain homogenates from voles infected with human and sheep prion isolates were analysed by CSSA and showed strain-specific conformational stabilities, with mean [GdnHCl]_1/2 values ranging from 1.6 M for MM2 sCJD to 2.1 for scrapie and to 2.8 M for MM1/MV1 sCJD and E200K gCJD. Interestingly, the rank order of [GdnHCl]_1/2 values observed in the human and sheep isolates used as inocula closely matched those found following transmission in voles, being MM1 sCJD the most resistant (3.3 M), followed by sheep scrapie (2.2 M) and by MM2 sCJD (1.6 M). In order to test the ability of CSSA to characterise protease-sensitive PrP^Sc, we analysed sheep isolates of Nor98 and compared them to classical scrapie isolates. In Nor98, insoluble PrP^Sc aggregates were mainly protease-sensitive and showed a conformational stability much lower than in classical scrapie. Our results show that CSSA is able to reveal strain-specified PrP^Sc conformational stabilities of protease-resistant and protease-sensitive PrP^Sc and that it is a valuable tool for strain typing in natural hosts, such as humans and sheep.     

Effect of copper exposure on GST activity and on the expression of four GSTs under oxidative stress condition in the monogonont rotifer, Brachionus koreanus

Glutathione S-transferases (GSTs; EC 2.5.1.18) are major enzymes that function in Phase II detoxification reactions by catalyzing the conjugation of reduced glutathione through cysteine thiol. In this study, we cloned and sequenced four GST genes from the monogonont rotifer Brachionus koreanus. The domain regions of four Bk-GSTs showed a high similarity to those of other species. In addition, to evaluate the potential of GST genes as an early warning signal for oxidative stress, we exposed sublethal concentrations of copper (Cu) to B. koreanus and measured glutathione (GSH) contents and several antioxidant enzymes such as glutathione S-transferase (GST), glutathione peroxidase (GPx; EC 1.11.1.9), and glutathione reductase (GR; EC 1.8.1.7). The reactive oxygen species (ROS) at 12 h and 24 h after copper exposure increased significantly. GSH contents however did not increase significantly and even it decreased at 0.24 mg/L at 12 h. The activities of several antioxidant enzymes, particularly GPx and GR, showed a dramatic increase in 0.24 mg/L of CuCl₂. Messenger RNAs of each Bk-GST showed different patterns of modulations according to GST types, and particularly, Bk-GST-omega, Bk-GST-sigma, and Bk-GST zeta genes were highly sensitive to Cu. These results indicate that Bk-GSTs, functioning as one of the enzymatic defense mechanisms particularly in the early stage of oxidative stress response, were induced by Cu exposure. This also suggests that these genes and related enzymes have a potential as biomarkers for a more sensitive initial stress response.     

Effects of guanidine hydrochloride on the conformation and enzyme activity of streptomycin adenylyltransferase monitored by circular dichroism and fluorescence spectroscopy

Equilibrium denaturation of streptomycin adenylyltransferase (SMATase) has been studied by CD spectroscopy, fluorescence emission spectroscopy, and binding of the hydrophobic dye 1-anilino-8-naphthalene sulfonic acid (ANS). Far-UV CD spectra show retention of 90% native-like secondary structure at 0.5 M guanidine hydrochloride (GdnHCl). The mean residue ellipticities at 222 nm and enzyme activity plotted against GdnHCl concentration showed loss of about 50 and 75% of secondary structure and 35 and 60% of activity at 0.75 and 1.5 M GdnHCl, respectively. At 6 M GdnHCl, there was loss of secondary structure and activity leading to the formation of GdnHCl-induced unfolded state as evidenced by CD and fluorescence spectroscopy as well as by measuring enzymatic activity. The denaturant-mediated decrease in fluorescence intensity and 5 nm red shift of λ_max point to gradual unfolding of SMATase when GdnHCl is added up from 0.5 M to a maximum of 6 M. Decreasing of ANS binding and red shift (∼5 nm) were observed in this state compared to the native folded state, indicating the partial destruction of surface hydrophobic patches of the protein molecule on denaturation. Disruption of disulfide bonds in the protein resulted in sharp decrease in surface hydrophobicity of the protein, indicating that the surface hydrophobic patches are held by disulfide bonds even in the GdnHCl denatured state. Acrylamide and potassium iodide quenching of the intrinsic tryptophan fluorescence of SMATase showed that the native protein is in folded conformation with majority of the tryptophan residues exposed to the solvent, and about 20% of them are in negatively charged environment. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 11, pp. 1514–1523.     

Hydrodynamic properties of proteoglycan subunit from bovine nasal cartilage. Self-association behavior and interaction with hyaluronate studied by laser light scattering

Measurements of translational diffusion coefficients by quasielastic laser light scattering, sedimentation coefficients, and intrinsic viscosities at zero shear of proteoglycan subunit fraction A1-D1-D1 isolated from bovine nasal septa are reported. Molecular weights and hydrodynamic dimensions are compared with those expected on the basis of structural models previously proposed. Comparison of the concentration dependence of the diffusion coefficient in the presence of NaCl and GdnHCl leads to the conclusion that significant self-association behaviour of subunit occurs in the absence of GdnHCl. In the absence of added salt, anomalous nonlinear concentration dependence of D_t estimated from wide-angle light-scattering experiments is observed. In addition, D_t apparently becomes angle dependent. These results are interpreted in terms of the perturbation of normal translational diffusion of the monomer by strong repulsive intermolecular interactions due to the combined effects of long-range electrostatic forces and macromolecular congestion at higher concentrations. By carrying out experiments at small scattering angles, it is possible to determine D for proteoglycan subunit in the absence of supporting electrolyte. Titration of a dilute solution of subunit with hyaluronic acid results in a sigmoidal behaviour of the Stokes radius, indicating the formation of complexes of higher molecular weight results from the noncovalent association of proteoglycan subunits with hyaluronate. Observation of D_t appears to provide a useful method for studying the proteoglycan subunit–hyaluronate interactions.     

Subtilase from Beauveria sp.: conformational and functional investigation of unusual stability

Retention of total activity of the subtilisin-like serine protease from Beauveria sp. MTCC 5184 (Bprot) in the vicinity of (1) 3 M GdnHCl for 12 h, (2) 50 % methanol and dimethyl sulfoxide each for 24 h, and (3) proteolytic enzymes (trypsin, chymotrypsin, and proteinase K) for 48 h led to expect the enzyme to be a kinetically stable protein. Also, the structure of the protein was stable at pH 2.0. Biophysical characterization and conformational transitions were monitored using steady-state and time-resolved fluorescence, FTIR, and CD spectroscopy. Single tryptophan in the protein exists as two conformers, in hydrophobic and polar environment. The secondary structure of Bprot was stable in 3 M GdnHCl as seen in far-UV CD spectra. The active fraction of Bprot obtained from size-exclusion chromatography in the presence of GdnHCl (1.0–3.0 M) eluted at reduced retention time. The peak area of inactive or denatured protein with the same retention time as that of native protein increased with increasing concentration of denaturant (1.0–4.0 M GdnHCl). However, the kinetics of GdnHCl-induced unfolding as studied from intrinsic fluorescence revealed k _unf of native protein to be 5.407 × 10^?5 s^?1 and a half-life of 3.56 h. The enzyme is thermodynamically stable in spite of being resistant to the denaturant, which could be due to the effect of GdnHCl imparting rigidity to the active fraction and simultaneously unfolding the partially unfolded protein that exists in equilibrium with the folded active protein. Thermal and pH denaturation of Bprot exhibited interesting structural transitions.     

Viscoelastic properties of human tracheobronchial mucin in aqueous solution

Human tracheobronchial mucin isolated from cystic fibrosis patients (CF HTBM) was purified using a combination of gel filtration and density gradient centrifugation. The resulting mucin was fractionated to reduce polydispersity and to facilitate studies of the molecular weight dependence of mucin viscoelasticity in concentrated solution. The viscoelastic properties of CF HTBM were examined in distilled water, 0.1M salt solutions and chaotropic solvents. In controlled strain experiments (strain ≥ 5%) with increasing mucin concentration, a crossover from sol to gel behavior is observed. The gel strength, as measured by the magnitude of the storage modulus at comparable mucin concentrations, is greatest for distilled water, intermediate for 0.1M NaCl, and lowest far 6M GdnHCl. In distilled water, high molecular weight mucin undergoes a sol-gel transition at ～ 12 mg/mL, and shows evidence of a plateau modulus at higher concentrations. The storage and loss moduli of concentrated high molecular weight fractions in 6M GdnHCl exhibit a power law dependence on frequency typical of weak gels near the sol–gel transition at 20 mg/mL. Similar rheology is observed in 0.1M NaCl and 0.091M NaCl/3 mM CaCl₂, but with evidence for additional weak associations at low frequency. The power law exponent in these systems is 0.70 ± 0.02, in good agreement with prediction for networks formed by a percolation mechanism. Low molecular weight fractions in these solvents exhibit a fluid-like viscoelastic response. However, low molecular weight mucin in distilled water shows a strain-dependent increase in elasticity at low frequency indicative of weak intermolecular associations. Comparison of the rheological behavior of CF HTBM with our earlier studies of ovine submaxillary mucin lends support to the idea that carbohydrate side-chain interactions are important in the gelation mechanism of mucins. © 1995 John Wiley & Sons, Inc.     

Production of L-leucine aminopeptidase by selected Streptomyces isolates

Aims: To screen various Streptomyces cultures producing l ‐leucine aminopeptidase (LAP). Methods and Results: Twenty‐one Streptomyces strains were screened for LAP production. The best three producers were found to be Streptomyces mobaraensis NRRL B‐3729, Streptomyces gedanensis IFO 13427, and Streptomyces platensis NRRL 2364. pH optima of the three enzymes were in the range of 8·0–8·5 and the temperature optima varied between 50 and 65°C. LAP of S. mobaraensis was stable at 60°C and pH 8·5 for 60 min. Metal ion salts, CoCl₂.6H₂O and ZnSO₄.7H₂O in 0·7 mmol l^?1 concentration enhanced the relative enzyme activity in all three enzymes. Molecular mass of LAP of S. mobaraensis was found to be approx. 37 kDa. Conclusions: Streptomyces mobaraensis NRRL B‐3729, S. gedanensis IFO 13427, and S. platensis NRRL 2364 were found to be good producers of extracellular LAP. The approx. 37 kDa enzyme of S. mobaraensis is considerably thermostable. Significance and Impact of the Study: A good number of Streptomyces were screened and the ability of the aminopeptidases to release a particular N‐terminal amino acid along with its good thermal stability makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate.