Kimio Oikawa

Circular dichroism studies on concanavalin A

Abstract Optical rotatory dispersion and circular dichroism studies on native and demetallized concanavalin A indicate that this protein consists essentially of a mixture of β-form and random coil. The induction of α-helix in the native protein is accomplished through the use of 2-chloroethanol. The sequential addition of transition metal, Ca2+ and α-methyl-D-glucopyranoside to solutions of demetallized protein results in small but reproducible alterations in the aromatic CD spectrum, the largest changes being noted with calcium addition. Aromatic CD difference spectral studies suggest that a single transition may be involved in the binding of metal and α-MG to the protein; the wavelength position of this transition implicates tyrosine residues. At the same time, no significant variations occur in the secondary structure of the protein as reflected by the constancy of the far ultraviolet CD spectrum with ligand complexation. Exploration of the CD aromatic fine structure as a function of pH indicates that both tyrosine and tryptophan chromophores are involved.

The effect of terbium on the structure of actin and myosin subfragment 1 as measured by circular dichroism

Substitution of Ca2+ by La3+ or Tb3* produced a pronounced inhibitory effect on the ATPase activity of myosin S-l alone, or in combination with F-actin [ 11. To gain further insight into the possible mechanism of action of En3” a CD investigation was carried out, wherein the effect of Tb3+ on the secondary structure of Gand F-actin, myosin S-l, as well as comb~at~ons of these proteins, was established. The results suggest that Tb3’ induces structural decreases in all these proteins and interactions between myosin S-l and actin seem to be weakened in the presence of this lanthanide.

Equilibrium dialysis binding studies of 1,N6-ethenoadenosine diphosphate (εADP) to myosin, heavy meromyosin, and subfragment one

Abstract The binding of the fluorescent analog of adenosine diphosphate (ADP) ∗ , 1,N 6 -ethenoadenosine diphosphate (eADP) to myosin and its subfragments, heavy meromyosin (HMM) and subfragment one (S1), has been studied under analagous conditions to those previously used in comparable studies on the binding of ADP to these molecules. The results indicate that there are two binding sites for eADP on myosin and HMM, and one site on S1. The dissociation constants for all had an identical value, within experimental error, of 2.0 ( ± .5) × 10 −5 M −1 . This is identical to the values found by Young (J. Biol. Chem., 242 , 2790 (1967)) for ADP. In addition, the kinetics of hydrolysis of eATP versus ATP by S1 were studied. Values of V max and K m were 25 μM phosphate sec −1 (gm protein) −1 and 5 × 10 −5 M −1 for ATP, and 80 μN phosphate sec −1 (gm protein) −1 and 45 × 10 −5 M −1 for eATP. The results indicate that the increased V max that occurs when eATP is used as a substitute for ATP is not due to either an increased binding affinity of ATP for myosin and its subfragments, nor due to a decreased binding affinity of eATP versus ADP. This in turn suggests that the increase in V max may be due to an increased hydrolytic rate of eATP vs ATP in the enzyme substrate complex.

Structural Properties of the Myelin-Associated Glycoprotein Ectodomain

Abstract: Myelin‐associated glycoprotein (MAG) has been proposed to mediate adhesive interactions during myelin development. We have used the baculovirus expression system to produce a truncated form of this molecule [soluble extracellular domain of MAG (sMAG)] consisting of the complete extracellular ectodomain. Spectroscopic studies indicate a high β‐sheet content, consistent with the prediction of Ig‐like structure. Hydrodynamic studies indicate an asymmetric monomer, with a Stokes radius of 4.1–4.6 nm, a sedimentation coefficient of 3.6S, and a frictional ratio of ∼1.6. We postulate that the outer two Ig‐like domains form a unit that folds back over the rest of the molecule. Fluorescence quenching studies indicate that sMAG interacts with divalent cations and may have a functional lectin domain.

Comparative calcium binding and conformational studies of turkey and rabbit skeletal troponin C

Troponin C from turkey skeletal muscle has been compared with its chicken counterpart in terms of amino acid composition and fragmentation patterns and with rabbit TN‐C by Ca2+ binding and conformational response to Ca2+ as monitored by CD and fluorescence. Cyanogen bromide and tryptic digestion mixtures of chicken and turkey TN‐C have been separated by reversed‐phase HPLC. The similarity of the elution profiles, along with the almost identical amino acid compositional data, suggest that the sequences are essentially equivalent. Both turkey and rabbit TN‐C bound 2 mol Ca2+/mol protein at pH 5.3, while at pH 6.8, this figure was raised to 4 mol/mol protein. Circular dichroism and fluorescence measurements indicated that the conformations of the two proteins responded in a very similar manner to the presence of Ca2+

INFLUENCE OF SOLVENT COMPOSITION ON CARDIAC AND SKELETAL MYOSIN A AS DETERMINED BY OPTICAL ROTATORY DISPERSION MEASUREMENTS.

Abstract Optical rotatory studies in the ultraviolet region in 0.5 M KCl solutions of cardiac and skeletal myosin indicate that the helical content of these proteins, based on the amplitude of the negative Cotton effect at 233 mμ, is in good agreement with values obtained from the slope term of Moffit-type treatment, based on measurements in the visible spectrum. The addition of 50–67 volumes per cent ethylene glycol to the aqueous system causes a reduction in helical content, as evidenced by the decreased amplitude of the protein conformational trough at 233 mμ. The addition of 10 −3 M ATP to solutions of cardiac and skeletal myosin results in no change in helical content as determined by optical rotatory measurements in both the visible and ultraviolet regions of the spectrum. Comparison of values of helical content of aqueous solutions of cardiac and skeletal myosins by two methods of optical rotational analysis suggests that the secondary structure of these proteins is very much alike.

Circular dichroism studies on chemically modified derivatives of concanavalin A

Earlier observations [ 1,2] revealed that native Con A at pH 5.2 and 7.0 possessed a definite fine structure in the CD spectral region of aromatic absorption, i.e., 250-320 nm. The involvement of both tyrosine and tryptophan chromophores in the generation of these ellipticity bands was suggested, although no attempt was made to assign a particular origin to any of the bands. The rationale behind this study was to prepare derivatives of Con A in which a number of tyrosine and tryptophan residues were independently chemically modified. lt was felt that the resulting CD spectra should then lend themselves to a more detailed view as to the origin of the various bands noted in native Con A. This approach has been used with considerable success in studies by Gorbunoff of several common protein systems [3,4].

Modulation of substrate selectivity in plasma lipid transfer protein reaction over structural variation of lipid particle.

The modulation of substrate selectivity of human plasma LTP reaction is the subject of the present investigation. The moderate selectivity by a factor of 5 to 6 was observed in the LTP-catalyzed transfer of cholesteryl ester over triacylglycerol between plasma lipoproteins. On the other hand, the transfer of cholesteryl ester by LTP was highly selective over the negligible transfer of triacylglycerol, by a factor of 60 to 500, between the microemulsions with LDL size, regardless of the activators such as human and pig apolipoprotein (apo) A-I, human apo C-III and apo E that bound to the surface of the emulsion in equilibrium. The presence of free cholesterol in these microemulsions reduced slightly the rate of cholesteryl ester transfer but had no effect on triacylglycerol transfer. Other surface-active reagents such as cholic acid, Triton X-100 and Tween-20, did not have an effect on the triacylglycerol transfer either. Triacylglycerol transfer by LTP became measurable between such lipid particles as prepared by co-sonication of lipid with pig apo A-I and isolated as the mixed-microemulsions in the density of LDL and HDL. In these conditions, the substrate selectivity for cholesteryl ester over triacylglycerol was a factor of 6 to 16 mimicking the ratio in plasma lipoproteins. The conformation of pig apo A-I estimated by circular dichroism showed that its apparent helical content was further more induced when apo A-I was integrated into the mixed-microemulsion by co-sonication than the lipid-bound apo A-I in equilibrium. Apo A-I, thus integrated into lipid particles, was highly resistant to the denaturation by guanidine hydrochloride while the lipid-bound apo A-I in equilibrium was denatured as readily as the lipid-free protein. Thus, triacylglycerol transfer by LTP was induced by structural modulation of substrate-carrying lipid particles such as higher integration of apolipoproteins.

Circular dichroism studies on native fetuin and some of its derivatives

Abstract Circular dichroism studies on native fetuin suggest that the molecule possesses a low α-helical content in agreement with earlier optical rotatory dispersion studies on this protein. Addition of sodium dodecyl sulfate to the native protein increases the apparent helical content. Neuraminidase-treated fetuin possesses a circular dichroism spectrum similar to the native protein. The disulfide bridges in fetuin stabilize the native structure, since both oxidation and reduction of these moieties at pH 8.0 result in the increased presence of unordered sections in the polypeptide chain. Over the pH range 8.0 to 4.5, the reduced, carboxymethylated and oxidized derivatives all undergo a random coil a β transition. The native protein shows a distinct but complicated fine structure in the 250–300-mμ region, which reflects asymmetrical interactions involving tyrosine, tryptophan and disulfide bridges. When the molecule unfolds on denaturation with 8 M urea, or upon oxidation or reduction of the disulfide bridges, the aromatic side chains are separated from their close association with the centers of optical asymmetry, and the circular dichroism dichroic spectra in this region disappear or are diminished in intensity.

The effects of terbium and lanthanum on the biological activity of some representative muscle protein systems

Calcium plays a significant regulatory role in many biological processes, often by interaction with proteins. Substitution of Ca2+ in these metal-protein complexes, by members of the rare-earth or lanthanide series results in derivatives which are useful probes of the chemical and structural nature of Ca’+ binding sites (reviewed 111). However, these ions have been used in physicochemical studies such as fluorescence, without close attention to biological replaceability. As pointed out [l] substitution of Ca2+ by Ln3+ can either inhibit, activate or have essentially no effect on a particular system. Since Ln3+ substitution is becoming very popular in the field of muscle biochemistry we have studied the effects of La3* and Tb3’ on the ATPase activity of the myosin S-l enzyme system alone, and in combination with F-a&in. We show that these ions have a pronounced inhibitory effect on this enzymic reaction, central to muscle contraction. These studies suggest that a bio-assay should be employed to ascertain the degree of preservation of the intact, native, active structure, before using current sophisticated physical techniques. Interpretation of the resulting data might be much simpler and more relevant to the biological system under study.