Molecular weight estimation of proteins by electrophoresis in linear polyacrylamide gradient gels in the absence of denaturing agents.

It was shown that in linear polyacrylamide gradient gels migration distance of a given protein increases as a function of the square root of the time of electrophoresis. The linearity between these two parameters is demonstrated by the statistical analysis of experimental data obtained with proteins of different shapes and a wide range of electrophoretic mobility. The slopes of the regression lines calculated by this method can be utilized to determine the molecular weight of a nondenatured protein. In fact, there is a linear relationship between the log of the molecular weights and the log of the slopes for proteins with M_rs between 20,000 and 950,000.     

Osmotic Pressure of Aqueous Polyethylene Glycols : Relationship between Molecular Weight and Vapor Pressure Deficit

Osmotic pressures (II) of aqueous solutions of polyethylene glycols (PEGs) of average relative molecular weight (M_r) between 200 and 10,000 were measured using vapor pressure deficit osmometry. The relationships between molarity and II were described with high precision by second order polynomials for each of the PEGs studied. In contrast to previous reports, equivalent weights of different polymers in solution did not generate the same II; low M_r PEGs generated a higher II than the higher M_r PEGs. The effect of PEGs upon II represents an interaction between concentration and M_r.     

Peptide mapping of fat cell membrane substrates for cholera toxin-catalyzed ADP-ribosylation

Incubating rat fat cell membranes with [³²P]NAD⁺ and cholera toxin results in ADP-ribosylation of three distinct components with approximate molecular weights of 42 000, 46 000 and 48 000. Partial proteolytic peptide maps of the M_r = 46 000 and 48 0000 toxin-specific substrates generated by elastase, α-chymotypsin, or Staphylococcus aureus V-8 protease were nearly identical, while those of the M_r = 42 000 target lacked several peptides common to both of the larger molecular weight targets. In addition, peptide maps generated from the M_r = 42 000 target displayed a number of peptides which were absent from the maps generated from either the M_r = 46 000 or 48 000 targets. These data suggest that the M_r = 46 000 and 48 000 substrates are closely related proteins, however the relationship between the M_r = 42 000 toxin-specific substrate and the larger peptides remains to be established. The relative patterns of fat cell membrane labelling by cholera toxin in the presence of [³²P]NAD⁺     

Thermostable phenylalanine dehydrogenase from a mesophilic Microbacterium sp. strain DM 86-1

Bacteria that produced NAD⁺-dependent phenylalanine dehydrogenase (EC 1.4.1.20) were selected among l-methionine utilizers isolated from soil. A bacterial strain showing phenylalanine dehydrogenase activity was chosen and classified in the genus Microbacterium. Phenylalanine dehydrogenase was purified from the crude extract of Microbacterium sp. strain DM 86-1 (TPU 3592) to homogeneity as judged by SDS-polyacrylamide disc gel electrophoresis. The enzyme has an isoelectric point of 5.8 and a relative molecular weight (M _r) of approximately 330,000. The enzyme is composed of eight identical subunits with an M _r of approximately 41,000. The apparent K _m values for l-phenylalanine and NAD⁺ were calculated to be 0.10 mM and 0.20 mM, respectively. No loss of the enzyme activity was observed upon incubation at 55° C for 10 min. Received: 30 July 1997 / Accepted: 4 November 1997     

Muscle Contraction : (Outline Studies in Biology) by C.R. Bagshaw Chapman & Hall; London,New York, 1982 79 pages. £2.75

On incubation of B. subtilis RM125(arg15 leuA8 r_M^? m_M^?) with DNA from alkalophilic Bacillus, the transformants (Arg⁺Leu^? or Leu^?Arg⁺) appeared at pH 10. The transformants were able to grow even at pH 7. Alkalophilic Bacillus was resistant to bacteriophages π105D1C2·1012 grown on B. subtilis 1012(r^-m_M⁺) and π105D1C2·ISMR4 grown on B. subtilis ISMR4r_M⁺r_R⁺m_M⁺m_R⁺), but the recipient B. subtilis and the transformant(Arg⁺Leu^?) were susceptible to both the of the bacteriophages. The results indicate that the transformant is a B. subtilis derivative and that alkalophilicity of alkalophilic Bacillus was transferred to B. subtilis.     

The use of liquid polyacrylamide in electrophoresis: III. Properties of liquid polyacrylamide in the presence of cellulose acetate

Solutions of uncross-linked liquid polyacrylamide (LPA) stabilized by cellulose acetate are shown to represent efficient molecular sieves for RNA and for sodium dodecyl sulfate-protein complexes. Endosmosis and evaporation have to be considered to obtain corrected estimates of mobilities of the macro-ions. The efficiency of molecular sieving is strongly influenced by the chain length of the single polymer molecules. This can be shown both in terms of the steepness of the relationship between logarithm of molecular weight and mobility and in the relationship between log (K_R) and log molecular weight. Comparable chain length effects are found in cross-linked polyacrylamide, suggesting a common mechanism of action of the two electrophoretic sieving media. The correlation of polymer viscosity and of the impedance towards migration of macro-ions, as demonstrated by LPA-electrophoresis, suggests the participation of hydrodynamic interactions in the molecular sieving process.     

Structural polypeptides and products of late genes of bacteriophage Mu: Characterization and functional aspects

This paper describes the identification and functional role of late gene products of bacteriophage Mu, including an analysis of the structural proteins of the Mu virion.In vitro reconstitution of infectious phage particles has shown that four genes (E, D, I, J) control the formation of phage heads and that a cluster of eight genes (K, L, M, N, P, Q, R, S) controls the formation of phage tails.Sodium dodecyl sulphate/polyacrylamide gel electrophoresis of Mu polypeptides synthesized in Escherichia coli minicells infected by Mu phages carrying amber mutations in various late genes has resulted in the identification of the products of gene C (15.5 × 10³M_r); H (64 × 10³M_r); F (54 × 10³M_r); G (16 × 10³M_r); L (55 × 10³M_r); N (60 × 10³M_r); P (43 × 10³M_r) and S (56 × 10³M_r). Minicells infected with λpMu hybrid phages and deletion mutants of Mu were used to identify polypeptides encoded by the V-β region of the Mu genome. These are the products of genes V, W or R (41.5 × 10³M_r, and 45 × 10³M_r); U (20.5 × 10³M_r) and of genes located in the β region (24 × 10³M_r (gpgin) and 37 × 10³M_r (possibly gpmom)).Analytical separation of the proteins of the Mu virion revealed that it consists of a major head polypeptide with a molecular weight of 33 × 10³, a second head polypeptide of 54 × 10³ (gpF) and two major tail polypeptides with molecular weights of 55 × 10³ and 12.5 × 10³ (gpL and gpY, respectively). In addition, there are five minor components of the tail (including gpN, gpS and gpU) and approximately seven minor components of the head structure of the virion (including gpH).     

Coagulation factor V exists uncomplexed in bovine plasma

Bovine coagulation factor V has been examined immunochemically to ascertain whether the coagulant polypeptide (h) with M_r = 290 000–330 000 is complexed in plasma with a second immunochemically distinct polypeptide (I₂) of M_r = 400 000. Antiserum containing antibodies to h and l₂ detects the l₂ polypeptide eluting earlier than the h chain on gel filtration of plasma with either added calcium or EDTA, consistent with the behavior of a higher molecular weight noninteracting species. An immobilized monospecific antibody to l₂ removes only the l₂ polypeptide from a purified factor V preparation containing both h and l₂. Moreover, while a monospecific antibody to the h chain was able to precipitate purified radioactively labelled h chain alone or mixed with plasma, the l₂ antibody was unable to precipitate radioactively labelled h chain even after attempted recombination of the h chain with l₂ present in plasma. These studies indicate that the l₂ polypeptide is not complexed to the h chain in a purified system or in plasma and reinforce the conclusion that factor V is a single polypeptide chain uncomplexed in plasma.     

Precision of sodium dodecyl sulfate-polyacrylamide-gel electrophoresis for the molecular weight estimation of a membrane glycoprotein: studies on bovine rhodopsin.

Criteria for assessing the precision and accuracy of methods for estimation of molecular weight for proteins using sodium dodecyl sulfate-polyacrylamide-gel electrophoresis have been applied to rhodopsin from bovine visual cell outer segment membranes. Various methods of preparing this hydrophobic protein for electrophoresis differ in their ability to solubilize and disaggregate polypeptide constituents of the outer segment membrane, with resultant variations in the pattern of protein bands and the apparent molecular weight of rhodopsin. Even with optimal solubilization and disaggregation, the behavior of rhodopsin relative to a series of standard proteins is such that the apparent molecular weight decreases systematically from 40,400 to 34,500 as the acrylamide concentration increases from 4 to 10%. As demonstrated by Ferguson plots of logR_f vs gel concentration and split gel experiments, this discrepancy is explained by the fact that the extrapolated R_f for zero gel concentration (Y₀) for rhodopsin is significantly lower than the Y₀'s for the soluble proteins used as molecular weight standards. In such cases, a possibly more reliable molecular weight estimate is obtained by plotting the retardation coefficient (K_R) vs molecular weight. This method yields a value of 29,500 ± 1000 for bovine rhodopsin if only the errors in measurement of R_f are considered and a quadratic relationship between K_R and molecular weight is used. Using weighted linear regression for K_R vs molecular weight, we obtain a molecular weight estimate of 32,700 ± 5000 when the uncertainty in the calibration curve is considered. Because of uncertainties regarding the detergent-binding properties of rhodopsin and the relationship of its Stokes radius to its molecular weight by comparison with the soluble protein standards, these values must be viewed with caution.     

Characterization and immunocytochemical demonstration of glucocorticoid receptor using antisera specific to transformed receptor

Cytosolic and nuclear forms of the glucocorticoid receptor were characterized using immunochemical techniques. Antibodies were raised in rabbits to an M_r 58,000 fragment of the transformed (DNA-binding) glucocorticoid receptor purified from rat liver cytosol by DNA-cellulose chromatography and polyacrylamide gel electrophoresis. Antibodies reacted with the transformed receptor form in a radioimmunoassay for glucocorticoid receptor. Western blot analysis of antibody reactivity revealed a single M_r 185,000 receptor form in rat liver cytosol but a smaller M_r 85,000 form in nucleosol, indicating the M_r 85,000 form is the transformed receptor. Furthermore, western blot analysis indicates that the M_r 185,000 receptor undergoes proteolysis during receptor purification and in vitro transformation processes by generating immunochemically similar proteins of smaller molecular weights. An identical M_r 185,000 glucocorticoid receptor was detected in cytosols of four rat tissues; liver, brain, adrenal medulla, and thymus. The glucocorticoid receptor was localized to the cytoplasm and nucleus of rat adrenal medulla cells by immunohistochemistry, demonstrating the existence in vivo of the transformed receptor and translocation of the receptor from cytoplasm to nucleus.     

Analysis of changes of insect-plant ratio-improvement of key-factor analysis for evaluating bottom-up effects in insect population dynamics

A revised key-factor analysis was presented for analyzing the temporal changes in the ratio of insect absolute number to plant resource. Ten data sets for 5 insect species were then analyzed. In this key-factor analysis, the key factor is defined as the factor contributing highly to between-year variation inR _r , the log rate of the inter-year change of the insect-plant ratio. The yearly change of plant resource was handled as a separate factor, expressed byr _pl , log ratio of plant resource in yearn to plant resource in yearn+1. The following was revealed: 1) In 7 of the 10 data sets examined,r _pl influenced variations ofR _r ; in particular in 3 casesr _pl was the main key factor. 2) Generation-to-generation fluctuations of absolute insect densities showed density dependence in 4 cases, while those of insect-plant ratios, in 8 cases. 3) The Royama model or a linear model, explained well the relationship between log insect-plant ratio (X _r ) andR _r and the relationship betweenX _r and log yearly change rate of absolute insect density (R _abs ). However, in the 7 cases in whichr _pl was a critical factor for variations ofR _r , with, increase ofX _r ,R _r showed a steeper, decrease around the equilibrium point (the point for whichR _r is 0) thanR _abs . This occurred becauser _pl tended to be negatively correlated withX _r . Consequently, in two casesX _r fluctuated cyclicly or chaotically although without the changes in plant resource, fluctuations ofX _r would be damped oscillations approaching equilibrium.     

Relationship between the Molecular Weight and the Color Intensity of Color Components of Melanoidin from the Glycine-Xylose System

The molecular weights of color components (designated as P1, P2, P3, P4, P5, P6, P7 and P8 in order of elution from a DEAE-cellulose column) isolated by the conversion of color components of melanoidin produced from the glycine-xylose system in an oxidative browning were studied in relation to the color intensity. The molecular weights of P5, P6, P7 and P8 estimated by gel filtration on Sephadex G–50, G–75 and G–150 using dextran as a standard were approximately 2,140, 3,550, 5,600 and 14,200, respectively. The molecular weights of P1, P2, P3 and P4 could not be estimated by the gel filtration method because of their low values.

On the other hand, a linear relationship between Kd, the distribution coefficient in dextran gel, and E₄₅₀ (E) of the color components was observed. Thus, there is considered to be a linear relationship between log E and log molecular weight (M). The correlation coefficient between log E and log M was calculated to be from 0.96 to 0.98 in the visible wavelength region. Therefore, the equation, E=k × M^α was adopted. The value, E=2.15 × M^0.29 was obtained from melanoidin prepared from the glycine-xylose system. The molecular weights of P1, P2, P3 and P4 were calculated from the equation to be 290, 360, 700 and 1,200, respectively. The equation, E=k × M^α, was demonstrated to be reasonably applicable to the melanoidin from Glu-, Lys-, Gly₂-, Gly-Leu-, and Gly₃-xylose systems. It is concluded that the polymerization of the structural unit in melanoidin occurs in an oxidative browning and that their color tone is darkened and the color in melanoidin is increased by polymerization according to the equation, E=k × M^α.     

Liver protein phosphatases: studies of the presumptive native forms of phosphorylase phosphatase activity in liver extracts and their dissociation to a catalytic subunit of Mr 35,000.

Several aspects of the properties of phosphorylase phosphatase in crude rat liver extracts were investigated. Treatment of tissue extracts with either trypsin, ethanol, or urea was found to dissociate phosphorylase phosphatase activity to a form of M_r 35,000. The M_r 35,000 enzyme form was derived from three native enzyme forms. The major cytosolic form of phosphorylase phosphatase had a molecular weight of 260,000 as determined by gel filtration and was dissociated to a M_r 35,000 form by treatment with either ethanol or urea. Treatment of the M_r 260,000 form with trypsin led to its conversion to M_r 225,000 and a M_r 35,000 form. A minor cytosolic form of M_r 200,000 was also present. This minor activity was latent until activated by trypsin treatment and was converted to a M_r 35,000 form by such treatment. The third form was found to chromatograph as a form of molecular weight greater than 500,000 on gel filtration and, like the M_r 200,000 form, was only detected after activation with trypsin. Subsequent to this treatment, it too behaved as a M_r 35,000 enzyme. Although a single major enzyme form was present in the cytosol, multiple molecular weight forms could be generated in crude extracts simply by the use of vigorous mechanical homogenization procedures. This suggested that artifactual forms of enzyme may readily be produced, possibly by proteolytic cleavage of the native enzyme.     

Structural studies of a french bean storage protein: phaseolin

Molecular weights and sedimentation coefficients have been measured for different oligomeric forms of phaseolin, the major storage protein in seeds of Phaseolus vulgaris L. The results indicate that phaseolin is a trimer ($\text{M}{}_{\mathrm{r}}\text{= 150000}$) at neutral pH which aggregates further to a dodecamer form ($\text{M}{}_{\mathrm{r}}\text{= 596000}$) at pH 4.5. The subunit size is in good agreement with the recently determined sequence molecular weight, if allowance is made for bound oligosaccharide and phytic acid moieties. The trimeric nature at neutral pH has been confirmed by chemical crosslinking studies using dimethylsuberimidate and dithiobis(succinimidylpropionate). Analyses of optical rotatory dispersion and circular dichroism data have been used to examine the corformation of phaseolin. In common with other seed globulins, a low proportion of α-helix ($\text{～ 10\%}$) coupled with a high level of β-sheet ($\text{～50\%}$) is predicted. These data are compared with a structural analysis based on the amino acid sequence of a phaseolin subunit polypeptide. The predicted level of α-helix is increased ($\text{～20\%}$) when phaseolin is heated in sodium dodecyl sulphate, but not when the detergent is added at room temperature.     

Studies on the structure of the calcium-dependent adenosine triphosphatase from rabbit skeletal muscle sarcoplasmic reticulum

The linear arrangement of the three fragments of Ca²⁺-ATPase from rabbit skeletal muscle sarcoplasmic reticulum with molecular weights of 20,000, 30,000, and 45,000 obtained by limited tryptic hydrolysis was determined by locating the NH₂-terminal acetylated methionyl residue of the original peptide in the M_r = 20,000 fragment. Since both the M_r = 20,000 and 30,000 polypeptides originate from a M_r = 55,000 fragment which is distinct from the M_r = 45,000 polypeptide, the sequence of these three fragments was determined to be 20,000, 30,000, and 45,000. The M_r = 20,000 fragment was further cleaved by cyanogen bromide to yield a M_r = 7,000 COOH-terminal fragment which is relatively hydrophilic. The NH₂-terminal portion is rich in glutamyl residues. The COOH-terminus of the M_r = 30,000 fragment was determined by both digestion with carboxypeptidases and cyanogen bromide cleavage. Using the partial amino acid sequence of the Ca²⁺-ATPase, it was deduced that the active site phosphoaspartyl residue is 154 amino acids from the COOH-terminus of the M_r = 30,000 fragment and hence approximately 35,000 M_r from the NH₂-terminus of the original Ca²⁺-ATPase molecule. Furthermore, it was shown that the two tryptic cleavages of the Ca²⁺-ATPase generating these three large fragments were both single hydrolyses of arginylalanine peptide bonds.     

Accumulation and excretion of pesticides used as insecticides or fungicides in agricultural products by the willow shiner Gnathopogon caerulescens

The average bioconcentration factors (BCF) in the whole body of willow shiner (Gnathopogon caerulescens) after 24–336 hr exposure were 810 for chlorpyriphosmethyl, 802 for vamidothion, 110 for edifenphos, 25 for ethoprophos, 83 for bendocarb, 39 for pirimicarb and 114 for methyl parathion. The correlations between a logarithm of 48 hr-Lc₅₀ to carp (log Lc₅₀ and the parameters (a logarithm of n-octanol-water partition coefficiens (log P_ow) and log BCF in willow shiner) were investigated for the pesticides studied here and already reported. The correlation factor (r) was −0.136 (N = 21) for log Lc₅₀ vs log P_ow and 0.039 (N = 24) for log Lc ₅₀ vs log BCF. The excretion rate constants (k) from the whole body of the fish were 0.01 hr⁻¹ for chlorpyriphosmethyl, 0.03 hr⁻¹ for vamidothion, 0.11 hr⁻¹ for edifenphos, 0.27 hr⁻¹ for ethoprophos, 0.18 hr⁻¹ for bendiocarb, 0.01 hr⁻¹ for pirimicarb and 0.08 hr⁻¹ for methyl parathion. The correlation between log k and log BCF was investigated for 22 pesticides already reported and studied here. The r value was not so high (−0.537, N = 22) but higher (−0.672, N = 2) in the case of excluding simetryne.     

The molecular transformation of rabbit testis proacrosin into acrosin.

Partially purified rabbit testis proacrosin formed only one acrosin of 73,000 ± 3000 apparent molecular weight (M_r) during the early phase of “autoactivation” at pH 8. Complete “autoactivation” then converted this acrosin to a 38,000 ± 3000 M_r, acrosin. These results suggest the existence of a proacrosin dimer (73,000) or a dimer of proacrosin and an acrosomal membrane protein which were converted first to an acrosin dimer (73,000 M_r) or to an acrosin-membrane protein dimer and then to the acrosin monomer (38,000). The formation of a 73,000 ± 3000 M_r acrosin from a 73,000 ± 3000 proacrosin is explainable by assuming that either a small activation pertide(s) is released or none at all.     

Three-dimensional reconstruction and averaging of 50 S ribosomal subunits of Escherichia coli from electron micrographs

The thermal stability and melting kinetics of the α-helical conformation within several regions of the rabbit myosin rod have been investigated. Cyanogen bromide cleavage of long myosin subfragment-2 produced one coiled-coil α-helical fragment corresponding to short subfragment-2 with molecular weight 90,000 (M_r = 45,000) and two fragments from the hinge region with molecular weights of 32,000 to 34,000 (M_r = 16,000 to 17,000) and 24,000 to 26,000 (M_r = 12,000 to 13,000). Optical rotation melting experiments and temperature-jump kinetic studies of long subfragment-2 and its cyanogen bromide fragments show that the hinge and the short subfragment-2 domains melt as quasi-independent co-operative units. The α-helical structure within the hinge has an appreciably lower thermal stability than the flanking short subfragment-2 and light meromyosin regions of the myosin rod. Two relaxation processes for helix-melting, one in the submillisecond range (τ_f) and the other in the millisecond range (τ_s), are observed in the light meromyosin and short subfragment-2 regions of the rod, but melting in the hinge domain is dominated by the fast (τ_f) process. Results suggest that the hinge domain of the subfragment-2 link may be the locus of force generation in a cycling cross-bridge.     

Isolation and immunological studies of high and low molecular weight cysteine proteinase inhibitors in bovine serum

Two kinds of cysteine proteinase inhibitor (M_r 145 000 and M_r 15 500) were purified from bovine serum. These purified inhibitors showed a single band on SDS-polyacrylamide gel electrophoresis, respectively. The isoelectric point of the high molecular weight inhibitor was found to be 4.4 and that of the low molecular weight inhibitor was 8.6. The high molecular weight inhibitor inhibited papain and cathepsin H, but had little activity against cathepsin B. While the low molecular weight inhibitor was a strong inhibitor of papain and cathepsin H and showed a weak inhibition of cathepsin B. These two inhibitors showed different immunological reactivities.     

Molecular weights of protein multimers from polyacrylamide gel electrophoresis

A nonlinear relationship between polyacrylamide gel electrophoresis (PAGE) retardation coefficients (K_R) and molecular weights has been observed during analysis of several multimeric proteins. Although the deviation from linearity over a wide range of molecular weights is slight, it can lead to significant errors in the estimation of the sizes of individual multimers. Two alternative methods of analysis of PAGE results are compared and demonstrated to yield linear relationships for multimeric proteins having molecular weights as high as 900,000.