INFLUENCE OF THE CONCENTRATION OF ELECTROLYTES ON THE ELECTRIFICATION AND THE RATE OF DIFFUSION OF WATER THROUGH COLLODION MEMBRANES

1. When a watery solution is separated from pure water by a collodion membrane, the initial rate of diffusion of water into the solution is influenced in an entirely different way by solutions of electrolytes and of non-electrolytes. Solutions of non-electrolytes, e.g. sugars, influence the initial rate of diffusion of water through the membrane approximately in direct proportion to their concentration, and this. influence begins to show itself under the conditions of our experiments when the concentration of the sugar solution is above M/64 or M/32. We call this effect of the concentration of the solute on the initial rate of diffusion of water into the solution the gas pressure effect. 2. Solutions of electrolytes show the gas pressure effect upon the initial rate of diffusion also, but it commences at a somewhat higher concentration than M/64; namely, at M/16 or more (according to the nature of the electrolyte). 3. Solutions of electrolytes of a lower concentration than M/16 or M/8 have a specific influence on the initial rate of diffusion of water through a collodion membrane from pure solvent into solution which is not found in the case of the solutions of non-electrolytes and which is due to the fact that the particles of water diffuse in this case through the membrane in an electrified condition, the sign of the charge depending upon the nature of the electrolyte in solution, according to two rules given in a preceding paper. 4. In these lower concentrations the curves representing the influence of the concentration of the electrolyte on the initial rate of diffusion of water into the solution rise at first steeply with an increase in the concentration, until a maximum is reached at a concentration of M/256 or above. A further increase in concentration causes a drop-in the curve and this drop increases with a further increase of concentration until that concentration of the solute is reached in which the gas pressure effect begins to prevail; i.e., above M/16. Within a range of concentrations between M/256 and M/16 or more (according to the nature of the electrolyte) we notice the reverse of what we should expect on the basis of van''t Hoff''s law; namely, that the attraction of a solution of an electrolyte for water diminishes with an increase in concentration. 5. We wish to make no definite assumption concerning the origin of the electrification of water and concerning the mechanism whereby ions influence the rate of diffusion of water particles through collodion membranes from pure solvent to solution. It will facilitate, however, the presentation of our results if it be permitted to present them in terms of attraction and repulsion of the charged particles of water by the ions. With this reservation we may say that in the lowest concentrations attraction of the electrified water particles by the ions with the opposite charge prevails over the repulsion of the electrified water particles by the ions with the same sign of charge as that of the water; while beyond a certain critical concentration the repelling action of the ion with the same sign of charge as that of the water particles upon the latter increases more rapidly with increasing concentration of the solute than the attractive action of the ion with the opposite charge. 6. It is shown that negative osmosis, i.e. the diminution of the volume of the solution of acids and of alkalies when separated by collodion membranes from pure water, occurs in the same range of concentrations in which the drop in the curves of neutral salts occurs, and that it is due to the same cause; namely, the repulsion of the electrified particles of water by the ion with the same sign of charge as that of the water. This conclusion is supported by the fact that negative osmosis becomes pronounced when the ion with the same sign of charge as that of the electrified particles of water carries more than one charge.     

The effect of norepinephrine and dibutyryl cyclic adenosine monophosphate on cation transport in duck erythrocytes

Freshly prepared duck erythrocytes, incubated either in plasma or an isotonic synthetic medium containing norepinephrine ([K] of both media ∼ 2.5 mM), maintain water and electrolyte composition in the steady state (upper steady state) for at least 90 min. If incubated in the synthetic medium without norepinephrine or in plasma to which a β-adrenergic blocking agent (propranolol) is added, the cells lose both water and electrolyte (predominantly KCl) until a new steady state is reached (lower steady state). Reaccumulation of water and electrolyte from isotonic solutions toward the upper steady-state levels requires the addition of norepinephrine and KCl. Reaccumulation is maximal when the concentration of K and norepinephrine in the medium is 15 mM and 10^-7 M, respectively. Dibutyryl cyclic-AMP (10^-2 M) mimics norepinephrine in lower steady-state cells. Although an analogous effect in upper steady-state cells was not established with certainty, it is proposed that the catecholamine-induced net changes in water and electrolyte movement in duck erythrocytes are a consequence of stimulation of the activity of a membrane-bound adenyl cyclase system.     

The effect of neutral salts on the conformational transition of poly-alpha-amino acids. I. Potentiometric titration and optical rotatory dispersion studies

The effect of salts on the coil-to-helix transition of poly-α-amino acids was investigated by optical rotatory dispersion and potentiometric titration techniques. Both charge-dependent and charge-independent contributions to the free energy were considered. The free energy of formation ΔF° of the uncharged α-helix from the uncharged random coil for poly-L -glutamic acid (PGA) decreases very rapidly in the limit of zero added salt concentration. This effect probably depends on the uncertainty affecting the choice of the extrapolation of the apparent pK for the random coil at low ionic strength. Above 0.1 M salt, where the free energy determination becomes meaningful, the anions and cations investigated do not affect the value of ΔF°, with the exception of Li⁺. Our data support the point of view that this cation binds to the peptide group. A class of salts produces an increase of the helical content of poly-L -ornithine (PO) both at low and high degree of ionization. This effect appears to be anion dependent. It is shown that (1) no change of ΔF° is involved; (2) recent theories of polyelectrolyte solutions cannot account for our results. We suggest that a true site binding of the anions to the charged amino groups occurs. The role of electrostatic binding in determining the conformational stability of proteins in the presence of some anions is stressed, and a general treatment for the electrostatic binding equilibria is outlined.     

The palladium(II) promoted hydrolysis of the methyl esters of glycyl-L-leucine,glycyl-L-alanine and L-alanylglycine

The palladium(II)-promoted hydrolysis of the methyl esters of glycyl-L-leucine, glycyl-L-alanine and L-alanylglycine have been studied at 25 °C and I=0.1 M in the pH range 4–5. At a 1:1 metal to ligand ratio the peptide esters act as tridentate ligands, donation occurring via the terminal amino group, the deprotonated amide nitrogen, and the carbonyl group of the ester. Due to the high Lewis acidity of Pd(II) rapid hydrolysis of the ester function by water and hydroxide ion occurs. Rate constants k_OH and k_H2O have been obtained for base hydrolysis and water hydrolysis of the coordinated peptide esters at 25 °C. The rate constants for base hydrolysis are 3.4 X 10⁶ M⁻¹ s⁻¹ (L-alaglyOMe), 6.4 X 10⁶ M⁻¹ s⁻¹ (gly-L-alaOMe) and 2.3 X 10⁷ M⁻¹ s⁻¹ (gly-L-leuOMe). Base hydrolysis of the coordinated peptide esters is at least 10⁶ times that of the free unprotonated ligand. Activation parameters have been obtained for both water and base hydrolysis of the Pd(II) complex of methyl L-alanylglycinate and possible mechanisms for the hydrolyses are considered.     

The solution behavior of poly-L-proline: I. Light scattering studies in water and concentrated aqueous neutral salt solutions

The solution bebavior of poly-L -proline Form II has been studied in water and aqueous salt solutions by both elastic and quasi-elastic light -scattering techniques. The results of this study suggest that polyproline Form II can exist in water at 24 °C as an associated polymer complex and that certain salts which do not appear to affect the helix integrity, e.g., guanidinium-HCl, resutl in dissociation of the aggregate. Other neutral salts, of the variety effective in mediating unfolding of the Form II helix (e.g., 4M NaClO₄) also induce aggregate dissociation, but 4M CaCl₂ results in enhanced aggregation of polyproline. Kinetic experiments indicate that a time of 20 hours is necessary for the completion of the “large” to “small” transformation (at 22°C) which is induced by the addition of 4M NaClO₄. Thus it appears that neutral salts additives in aqueous solutions of polyproline influence both the state of aggregation and the conformation of this polymer.     

Quasielastic light scattering,electrophoresis, and fluorescence studies of lysozyme-poly(2-acrylamido-methylpropylsulfate) complexes

Complexation between lysozyme and sodium poly(2-acrylamido-2-methylpropanesulfonate) (PAMPS) was studied by quasielastic light scattering, electrophoretic light scattering, fluorescence, and turbidimetry in electrolyte solution. These techniques show that complexation occurs at pH 9.6 in an ionic strength buffer of 0.25M NaCl + 25 mM Na₂B₄O₇. At constant lysozyme concentration (C_pro). The structure of the complex depends on the polymer concentration. At low polymer concentration (relative to C_pro), an intrapolymer complex is formed. This intrapolymer complex aggregates to an interpolymer species upon increase in polymer concentration. Complex formation was also studied by fluorescence using pyrene-labeled PAMPS (Py-PAMPS). Energy transfer from singlet-excited tryptophan residues in lysozyme to the pyrene label occurs when the complex is formed. Fluorescence and turbidity data indicate that lysozyme interacts with Py-PAMPS preferentially at pyrene sites, which leads to static quenching of tryptophan fluorescence via energy transfer to the pyrene label. © 1995 John Wiley & Sons, Inc.     

Characterization of the transition state of lysozyme unfolding. II. Effects of the intrachain crosslinking and the inhibitor binding on the transition state

The reversible refolding of a lysozyme derivative containing an extra crosslink between Glu 35 and Trp 108 was observed at pH 3.7 in solutions of 4.5M LiBr and 4.SM 1-PrOH. The rate constants of unfolding and folding for crosslinked lysozyme were compared with those for intact lysozyme. In LiBr solutions, the crosslinking greatly increases k_f but only slightly decreases k_uf. This means that the crosslinking restricts possible conformations of the U state to some conformations on the folding pathway, whereas the possible conformations of the transition state are little restricted. Although the crosslinking produces an apparently different effect on the rate constants in a PrOH solution, this could be explained by assuming that the crosslinking counteracts the effect of PrOH on the transition state. These observations suggest that the Glu 35 and Trp 108 of intact lysozyme already come into contact with each other in the course of refolding to the transition state. Kinetics of the unfolding and folding reactions of intact lysozyme were measured in the presence of 8mAf (NAG)₃. The apparent folding rate (k) was nearly equal to k_f, while the apparent unfolding rate (k) decreased sixfold. This means that the inhibitor binding stabilizes the native conformation but makes no contribution to the stabilization of the transition state. Specific interactions between (NAG)₃ and the cleft of lysozyme become available only at the last stage of folding.     

Thermodynamic parameters of helix-coil transition in polypeptide chains. II. Poly-L-lysine

The helix–coil transitions for poly-L -lysine (PL) were investigated by the methods of spectropolarimetry, viscometry and potentiometric titration in 0.2M NaCl at different temperatures as well as in 0.2MNaBr, 1MKCl, and in mixtures of 0.2MNaCl or NaBr with methanol at room temperature. The enthalpy and entropy differences between the helical and coillike states of uncharged PL molecules in 0.2.M NaCl were determined from the potentiometric titration curves. The cooperativity parameters σ for PL in different solvents were determined by two methods (from the sharpness of the transition and from the dependence of the intrinsic viscosity on the helical content in the transition region). In 0.2MNaCl σ has a value of (2.3 ± 0.5) × 10^?4 and does not depend on temperature, i.e., the cooperativity of the helix-coil transition, as for PGA, is mainly of an entropy origin (the initiating of the helical region is accompanied by the entropy decrease ΔS_i = ?12 eu/mole of helical regions). A comparison of the obtained results for PGA and PL with the molecular theories of the helix-coil transitions shows that the role of dipole-dipole interactions of nonneighboring peptide groups is greatly overestimated in these theories, leading to a considerable enthalpy contribution to the free energy of initiating helical regions which is not observed in the experiment.     

Modifications of myelin basic protein which occur during its isolation.

Abstract— Chromatography of myelin basic protein (BP) on carboxymethylcellulose gives a pattern of multiple components, of which three are major. Component 1 is considered the unmodified species of BP while component 2 has been found to be modified primarily by deamidation and component 3 by phosphorylation (Chou et al., 1976). ³ 3 The numbering system for the components is that used by DEIBLER & MARTENSON (1973) for guinea pig BP and is preferred over the reverse system of numbering used by CHOU et al. (1976); i.e. components 1, 2 and 3 of DEIBLER & MARTENSON (1973) are the same as components 6, 5 and 4 of CHOU et al. (1976), respectively.
In contrast to BP prepared from tissue delipidated in the standard fashion in chloroform–methanol (CM powder), BP prepared from tissue delipidated first in acetone and then in chloroform–methanol (ACM powder) gave an elution pattern on carboxymethylcellulose characterized by a decrease in component 1 and an increase in the earlier eluting, less basic components. Studies with radiolabelled component 1 showed that this difference in elution patterns was due to the partial conversion of component 1 to less basic components during the extraction of ACM powder at neutral pH. The components derived from component 1 (D2, D3 and D4) were then isolated and subjected to tryptic peptide map analyses and determination of their carboxy-terminal arginine content and content of phosphorus. None of the derived components contained phosphorus but tryptic peptide map analyses did show the presence of two minor peptides, T14M₂ and T20M, previously found in component 2 from CM powder and considered to be the deamidation products of their parent peptides T14 and T20 (Chou et al., 1976). In addition components D3 and D4 were shown to have lost appreciable arginine from their carboxy-termini. Since none of the efforts to reduce enzyme activity in vitro had any appreciable effect on components 2 and 3 it was concluded that phosphorylation probably occurs exclusively in vivo, that deamidation occurs both in vivo and in vitro and that loss of carboxy-terminal arginine occurs exclusively in vitro.     

Soil moisture and potassium affect the performance of symbiotic nitrogen fixation in faba bean and common bean

Potassium (K) is reported to improve plant's resistance against environmental stress. A frequently experienced stress for plants in the tropics is water shortage. It is not known if sufficient K supply would help plants to partially overcome the effects of water stress, especially that of symbiotic nitrogen fixation which is often rather low in the tropics when compared to that of temperate regions. Thus, the impact of three levels of fertilizer potassium (0.1, 0.8 and 3.0 mM K) on symbiotic nitrogen fixation was evaluated with two legumes under high (field capacity to 25% depletion) and low (less than 50% of field capacity) water regimes. Plants were grown in single pots in silica sand under controlled conditions with 1.5 mM N (¹⁵N enriched NH₄NO₃). The species were faba bean (Vicia faba L.), a temperate, amide producing legume and common bean (Phaseolus vulgaris L.), a tropical, ureide producing species. In both species, 0.1 mM K was insufficient for nodulation at both moisture regimes, although plant growth was observed. The supply of 0.8 or 3.0 mM K allowed nodulation and subsequent nitrogen fixation which appeared to be adequate for respective plant growth. High potassium supply had a positive effect on nitrogen fixation, on shoot and root growth and on water potential in both water regimes. Where nodulation occurred, variations caused by either K or water supply had no consequences on the percentage of nitrogen derived from the symbiosis. The present data indicate that K can apparently alleviate water shortage to a certain extent. Moreover it is shown that the symbiotic system in both faba bean and common bean is less tolerant to limiting K supply than plants themselves. However, as long as nodulation occurs, N assimilation from the symbiotic source is not selectively affected by K as opposed to N assimilation from fertilizer.     

Establishment of an in Vitro Model of the Human Placental Barrier by Placenta Slice Culture and Ussing Chamber

Aggregation occurs through hydrophobic interactions when a polypeptide chain refolds in non-native states or when genetic variants of biologically active proteins assume inappropriate conformations, as observed in the case of dysfunctional serpins. Here, using the molecular chaperone BiP from bovine liver microsomes, we characterized the hydrophobic nature of the peptide segment which is considered to be a site required for aggregation among a non-inhibitory serpin ovalbumin in a heat-denatured state. Screening of the peptide scan for binding of BiP showed that BiP-binding sites are mostly buried in the folded ovalbumin. When ovalbumin was heat-denatured, the denatured protein was recognized by the antibody that reacts with the hydrophobic surface of the amino-terminal segment of ovalbumin. This antibody significantly suppressed the binding of BiP to denatured ovalbumin. BiP also bound the immobilized peptide in an ATP-dependent manner and the peptide stimulated the ATPase activity of BiP with a K _m of 165 μM and a V _max of 0.4 nmol/min per milligram. Measurement of surface plasmon resonance showed that the peptide had a K _d of 0.52 μM by BiP, lower than that for RCMLA (K _d=1.1 μM) and even lower than that of the peptide P10K, PLSRTLSVAAKK, (K _d=21 μM). These results demonstrate that the aggregation-prone site on heat-denatured ovalbumin has almost the same hydrophobic nature of interacting with the molecular chaperone BiP as the conventionally known peptides that bind to the Escherichia coli chaperone DnaK.     

The peptide hormone ghrelin binds to membrane-mimetics via its octanoyl chain and an adjacent phenylalanine

The peptide hormone ghrelin, which is the natural ligand of the membrane-bound growth hormone secretagogue receptor (GHS-R), regulates overall body and cell growth, energy homeostasis, carbohydrate, protein and lipid metabolism and water electrolyte balance. It contains an O-acyl linked octanoyl group on Ser3 and is the only peptide known to contain such a modification. Using solution state NMR spectroscopy and ultrafiltration we found that human ghrelin binds to membrane-mimetic environments via its octanoyl group as well as the aromatic moiety of Phe4. Relaxation enhancements in a paramagnetic environment reveal that both the octanoyl group on Ser3 and the aromatic group on Phe4 are inserted deep into the hydrophobic core of phosphocholine assemblies while the remaining peptide is freely mobile in solution. In contrast, no binding was observed for des-octanoyl ghrelin. Thus, the octanoyl chain, together with the Phe4 aromatic group of ghrelin, functions as a membrane anchor. Our results are in parallel with the previous finding that a bulky hydrophobic group on Ser3 and Phe4 of ghrelin are necessary for its function and thus indicate that membrane-binding is essential for ghrelin function.     

Circular dichroism of corticotropin, fragment 1-24, and model compounds. An assessment of the contributions of the peptide chromophore and armoatic residues.

The far-ultraviolet circular dichroic spectrum of the 39-residue peptide hormone porcine corticotropin and the biologically active fragment corticotropin 1–24 is negative from 250 nm to 195 nm in water, but in 6M guanidinium chloride a positive band appears at about 225 nm. The temperature and guanidinium chloride dependence of this spectral transition indicates the absence of any stable ordered secondary structure in corticotropin and the spectrum is seen to be in only partial agreement with results using the model peptide chromophore, Ala-Ala-Ala. Using oligopeptides containing aromatic amino acid residues sandwiched between glycyl residues, it is shown that the shape and intensity of the corticotropin 225 nm positive band which appears in 6M guanidinium chloride is in agreement with the far-ultraviolet transitions of the aromatic chromophores in the hormone. Curve resolution of the near-ultraviolet circular dichroic spectrum of corticotropin and comparison of the rotational strengths of the phenylalanyl and tyrosyl bands reveals no evidence for increased rotational freedom in 6M guanidinium hydrochloride. Spectral changes are observed, however, in the transitions arising from the single tryptophan. This study suggests that corticotropin in aqueous solution may serve as a better model for the circular dichroic spectrum of the aperiodic regions in globular proteins than either synthetic homopolypeptides or reference proteins for which spectral and X-ray diffraction data are available.     

INFLUENCE OF THE CONCENTRATION OF ELECTROLYTES ON SOME PHYSICAL PROPERTIES OF COLLOIDS AND OF CRYSTALLOIDS

1. When a 1 per cent solution of a metal gelatinate, e.g. Na gelatinate, of pH = 8.4 is separated from distilled water by a collodion membrane, water will diffuse into the solution with a certain rate which can be measured by the rise of the level of the liquid in a manometer. When to such a solution alkali or neutral salt is added the initial rate with which water will diffuse into the solution is diminished and the more so the more alkali or salt is added. This depressing effect of the addition of alkali and neutral salt is greater when the cation of the electrolyte added is bivalent than when it is monovalent. This seems to indicate that the depressing effect is due to the cation of the electrolyte added. 2. When a neutral M/256 solution of a salt with monovalent cation (e.g. Na₂SO₄ or K₄Fe(CN)₆, etc.) is separated from distilled water by a collodion membrane, water will diffuse into the solution with a certain initial rate. When to such a solution alkali or neutral salt is added, the initial rate with which water will diffuse into the solution is diminished and the more so the more alkali or salt is added. The depressing effect of the addition of alkali or neutral salt is greater when the cation of the electrolyte added is bivalent than when it is monovalent. This seems to indicate that the depressing effect is due to the cation of the electrolyte added. The membranes used in these experiments were not treated with gelatin. 3. It can be shown that water diffuses through the collodion membrane in the form of positively charged particles under the conditions mentioned in (1) and (2). In the case of diffusion of water into a neutral solution of a salt with monovalent or bivalent cation the effect of the addition of electrolyte on the rate of diffusion can be explained on the basis of the influence of the ions on the electrification and the rate of diffusion of electrified particles of water. Since the influence of the addition of electrolyte seems to be the same in the case of solutions of metal gelatinate, the question arises whether this influence of the addition of electrolyte cannot also be explained in the same way, and, if this be true, the further question can be raised whether this depressing effect necessarily depends upon the colloidal character of the gelatin solution, or whether we are not dealing in both cases with the same property of matter; namely, the influence of ions on the electrification and rate of diffusion of water through a membrane. 4. It can be shown that the curve representing the influence of the concentration of electrolyte on the initial rate of diffusion of water from solvent into the solution through the membrane is similar to the curve representing the permanent osmotic pressure of the gelatin solution. The question which has been raised in (3) should then apply also to the influence of the concentration of ions upon the osmotic pressure and perhaps other physical properties of gelatin which depend in a similar way upon the concentration of electrolyte added; e.g., swelling. 5. When a 1 per cent solution of a gelatin-acid salt, e.g. gelatin chloride, of pH 3.4 is separated from distilled water by a collodion membrane, water will diffuse into the solution with a certain rate. When to such a solution acid or neutral salt is added—taking care in the latter case that the pH is not altered—the initial rate with which water will diffuse into the solution is diminished and the more so the more acid or salt is added. Water diffuses into a gelatin chloride solution through a collodion membrane in the form of negatively charged particles. 6. When we replace the gelatin-acid salt by a crystalloidal salt, which causes the water to diffuse through the collodion membrane in the form of negatively charged particles, e.g. M/512 Al₂Cl₆, we find that the addition of acid or of neutral salt will diminish the initial rate with which water diffuses into the M/512 solution of Al₂Cl₆, in a similar way as it does in the case of a solution of a gelatin-acid salt.     

PROTOPLASMIC POTENTIALS IN HALICYSTIS : III. THE EFFECTS OF AMMONIA

The nature and origin of the large "protoplasmic" potential in Halicystis must be studied by altering conditions, not only in external solutions, but in the sap and the protoplasm itself. Such interior alteration caused by the penetration of ammonia is described. Concentrations of NH₄Cl in the sea water were varied from 0.00001 M to above 0.01 M. At pH 8.1 there is little effect below 0.0005 M NH₄Cl. At about 0.001 M a sudden reversal of the potential difference across the protoplasm occurs, from about 68 mv. outside positive to 30 to 40 mv. outside negative. At this threshold value the time curve is characteristically S-shaped, with a slow beginning, a rapid reversal, and then an irregularly wavering negative value. There are characteristic cusps at the first application of the NH₄Cl, also immediately after the reversal. The application of higher NH₄Cl concentrations causes a more rapid reversal, and also a somewhat higher negative value. Conversely the reduction of NH₄Cl concentrations causes recovery of the normal positive potential, but the threshold for recovery is at a lower concentration than for the original reversal. A temporary overshooting or increase of the positive potential usually occurs on recovery. The reversals may be repeated many times on the same cell without injury. The plot of P.D. against the log of ammonium ion concentration is not the straight line characteristic of ionic concentration effects, but has a break of 100 mv. or more at the threshold value. Further evidence that the potential is not greatly influenced by ammonium ions is obtained by altering the pH of the sea water. At pH 5, no reversal occurs with 0.1 M NH₄Cl, while at pH 10.3, the NH₄Cl threshold is 0.0001 M or less. This indicates that the reversal is due to undissociated ammonia. The penetration of NH₃ into the cells increases both the internal ammonia and the pH. The actual concentration of ammonium salt in the sap is again shown to have little effect on the _P.D. The pH is therefore the governing factor. But assuming that NH₃ enters the cells until it is in equilibrium between sap and sea water, no sudden break of pH should occur, pH being instead directly proportional to log NH₃ for any constant (NH₄) concentration. Experimentally, a linear relation is found between the pH of the sap and the log NH₃ in sea water. The sudden change of P.D. must therefore be ascribed to some system in the cell upon which the pH change operates. The pH value of the sap at the NH₃ threshold is between 6.0 and 6.5 which corresponds well with the pH value found to cause reversal of P.D. by direct perfusion of solutions in the vacuole.     

Analysis of expression and chitin‐binding activity of the wing disc cuticle protein BmWCP4 in the silkworm,Bombyx mori

The insect exoskeleton is mainly composed of chitin filaments linked by cuticle proteins. When insects molt, the cuticle of the exoskeleton is renewed by degrading the old chitin and cuticle proteins and synthesizing new ones. In this study, chitin‐binding activity of the wing disc cuticle protein BmWCP4 in Bombyx mori was studied. Sequence analysis showed that the protein had a conservative hydrophilic “R&R” chitin‐binding domain (CBD). Western blotting showed that BmWCP4 was predominately expressed in the wing disc‐containing epidermis during the late wandering and early pupal stages. The immunohistochemistry result showed that the BmWCP4 was mainly present in the wing disc tissues containing wing bud and trachea blast during day 2 of wandering stage. Recombinant full‐length BmWCP4 protein, “R&R” CBD peptide (CBD), non‐CBD peptide (BmWCP4‐CBD^?), four single site‐directed mutated peptides (M₁, M₂, M₃ and M₄) and four‐sites‐mutated peptide (M_F) were generated and purified, respectively, for in vitro chitin‐binding assay. The results indicated that both the full‐length protein and the “R&R” CBD peptide could bind with chitin, whereas the BmWCP4‐CBD^? could not bind with chitin. The single residue mutants M₁, M₂, M₃ and M₄ reduced but did not completely abolish the chitin‐binding activity, while four‐sites‐mutated protein M_F completely lost the chitin‐binding activity. These data indicate that BmWCP4 protein plays a critical role by binding to the chitin filaments in the wing during larva‐to‐pupa transformation. The conserved aromatic amino acids are critical in the interaction between chitin and the cuticle protein.     

Linear relaxation analysis of the mechanochemical transformation of collagen fibers

The isometric tensile stress generation observed when collagen fibers are immersed in aqueous solutions of lithium bromide ranging in molar concentration up to 7 was studied at 23°C. The reverse process, namely, isometric stress relaxation of the fiber occurring by subsequent immersion in distilled water, was also studied. We find that the data in the region of LiBr concentration up to about 2.5 moles/liter are adequately represented by a superposition integral where σ(t) is the time-dependent stress generated by the collagen fiber held at fixed length, c(t) is the history of LiBr molar concentration, and K(t) is the isometric contractility function, expressed as stress per unit salt concentration. We conclude that, within a limited range of salt concentration, a collagen fiber in a LiBr bath behaves as if it were a linear, time-invariant system defined mechanochemically by a single function K(t) which depends on the structural characteristics of the fiber while being independent of salt concentration. An analysis is presented of isometric mechanochemical data obtained under conditions of equilibrium by other workers who studied the behavior of collagen fibers in aqueous solutions either of urea, LiBr, or KCNS. The analysis shows that these independent (equilibrium) data confirm the linarity of the relation between isometric contractile stress and salt concentration on which our superposition integral representation is based. We also find that the asymptotic (infinite-time) value of the isometric stress is linearly related to the chemical potential of the salt as well, in agreement with the equilibrium thermodynamic treatment of mechanochemical processes by Katchalsky and Oplatka.     

Influence of salinity on the biological and biochemical activity of a calciorthird soil

Irrigation of agricultural land with saline waters can lead to soil degradation. In this paper the effect of the irrigation with water containing different concentrations of NaCl or Na₂SO₄ (0.1 M, 0.3 M, 0.6 M, 0.8 M, 1 M, and 1.3 M) on the biological and biochemical characteristics of a calciorthid soil was studied. In general, the increase of scil electrical conductivity caused by the addition of saline solutions had a negative effect on soil's biological and biochemical fertility, (that related directly with the soil's microbiological activity) this effect being more noticeable with NaCl than with Na₂SO₄. Soil microbial respiration was inhibited as much as 57% by a 1.3 M solution of NaCl. The activity of hydrolases such as protease, -glucosidase and phosphatase was more negatively affected by salinity than that of oxidoreductases (dehydrogenase and catalase). Soil NO₃ ^- content decreased with salinity while NH₄ ⁺ content increased. Carbohydrate content, which is closely connected with soil aggregate stability, was also negatively affected by salinity.     

Static and dynamic quenching of protein fluorescence. II. lysozyme.

The fluorescence parameters—lifetime, relative quantum yield, wavelength of maximum fluorescence intensity, half-width, and polarization—of 0.01% lysozyme were measured at 15°C in aqueous solution, in glycerol–water mixtures (0–90% v/v glycerol), in aqueous urea (0–8M) solutions, and in aqueous guanidine hydrochloride (0–6.4M) solutions. The changes in the static and dynamic quenching of lysozyme fluorescence, monitored by the quantum yield and lifetime measurements, were correlated with the other fluorescence parameters and compared with our earlier results with bovine serum albumin. The results were interpreted in terms of induced conformational changes. The various perturbants altered the fluorescence parameters of lysozyme and bovine serum albumin very differently. The differences were shown to be entirely consistent with our earlier conclusion that bovine serum albumin fluorophores are nonsurface residues and with the conclusion of others that lysozyme fluorophores are surface residues. Unlike their effects on bovine serum albumin, urea and guanidine hydrochloride affect lysozyme structure quite differently, both in nature and degree. We have suggested that the affect of urea on lysozyme fluorescence is an indirect result of reduction in the size of the cleft brought about by the structure-breaking action of urea on water in the cleft. 4M Urea is sufficient for this reaction. Large decreases in the polarization of the fluorescence of lysozyme in the 0.8–1.6M and 3.2–4.8M guanidine hydrochloride ranges demonstrated two guanidine hydrochloride-induced conformation changes. A red shift of the fluorescence maximum to 354 nm indicated that the second transition completely exposes all fluorescing tryptophan residues of lysozyme to mobile solvent water. However, even 6.4M guanidine hydrochloride did not completely unravel the lysozyme molecule at 15°C, as evidenced by its failure to cause any of the tyrosine residues to become fluorescent.     

The Inhibition of Cow-Liver Catalase by 3-Nitropropionate

The cow-liver, catalase was inhibited by 3-nitropropionate (3-NPA) at every pH tested. The 50% inhibition degree, ?, of 3-NPA under experimental condition was 8.7 × 10^?4 M, which was almost that of phenol (1.0 × 10^?3 M). Among 3-NPA, 1-nitropropane, and propionate, 3-NPA had the most effective inhibitory effect. The maximum absorption spectrum of free catalase in the Soret region was not shifted clearly by an addition of 3-NPA, but the intensity of it was decreased. The inhibition type of 3-NPA was riot definitely classified as the cyanide type. Results indicate that this inhibition essentially be due to reaction between the prosthetic group and anion form of nitro group in 3-NPA, and that the inhibitory action of nitro group may be stimulated by carboxylic group. Finally, this inhibition could be thought as a partial toxic cause in vivo.