Cross partition and determination of net charge of the isoenzymes of enolase

Enolase from bakers' yeast was separated into three isoenzymes by countercurrent distribution. The isoenzymes were partitioned in aqueous polymer two-phase systems containing positively charged trimethylamino poly(ethylene glycol) or negatively charged poly(ethylene glycol) sulphonate. The plots of the partition coefficient of each isoenzyme versus pH in the two biphasic systems intersect at pH equal to the isoelectric point. From slopes of the plots, the net charge of the isoenzymes at pH 6.57 was determined to be +2, −3, and −8 respectively.     

Purification of three distinct enolase isoenzymes from yeast

A method for the rapid isolation of yeast enolases, yielding three distinct isoenzymes, has been devised. In the first step anionic proteins were precipitated with polyethyleneimine, whereas hydrophobic enolase isoenzymes remained in the supernatant. Secondly, the supernatant was 45% saturated with ammonium sulfate and bound to phenyl-Sepharose CL-4B. Decreasing ammonium sulfate and simultaneously increasing ethylene glycol concentrations were used for elution. Finally, enolase isoenzymes were separated by chromatofocusing. The purified isoenzymes gave single bands after isoelectric focusing.     

Various functional characteristics of enolase isoenzymes in mammals

Considerable stability of beta beta- (muscular) and gamma gamma- (neurospecific) forms to the denaturing influence of neutral salts of alkali metals is shown on purified isoenzymes (alpha alpha-, beta beta-, gamma gamma-forms) of beef brain and rabbit muscle. The rate of influence of these salts on the enzymic activity of the mentioned isoenzymes corresponds to the "lyotropic" series. Km of alpha alpha- and gamma gamma-isoenzymes are determined for 2-phosphoglycerate and phosphoenolpyruvate. It is found that alpha alpha-isoenzyme has larger affinity to phosphoenolpyruvate than gamma gamma-isoenzyme and it appears to play an essential role in participation of the given isoenzymes in the glycolysis and glyconeogenesis processes.     

Cell surface charge. Correlation of partition with electrophoresis

Critical mixtures of aqueous solutions os polymers separate into two or more immiscible phases. Particulate materials distribute in such phase systems generally between one bulk phase and the interface between bulk phases. The distribution is described by a simple partition law, and is qunatitatively determined by, inter alia, the nature of the particle surface, particularly net electrical charge. The partition behaviour of various cells, native or modified by treatment with trypsin, neurominidase or maleic anhydride, correlate strongly with electrophoretic mobility. Partition behaviour and electrophoretic mobility are both dependent upon cell surface charge. Thus, in appropriate conditions, changes in surface charge may be registered as changes in partition.     

Content of neuron-specific and non-neuron-specific enolase isoenzymes in human brain structures

The distribution of neuron-specific (NSE) and non-neuronal (NNE) isoforms of glycolytic enzyme enolase (EC 4.2.I.II.) in the human brain was studied using immunoenzyme assay. The maximum NSE concentration was measured in the frontal and occipital brain cortex, hippocampus, limbic cortex and hypothalamus (12-14 micrograms/mg of water-soluble protein), the minimum level was observed in the brain stem structures (3-6 micrograms/mg). The maximum NNE content was determined in thalamus (34 micrograms/mg). The data can prove useful for the study of enolase isoform distribution in the brain of neurological and psychiatric patients.     

Enolase isoenzymes in adult and developing Xenopus laevis and characterization of a cloned enolase sequence.

As part of a study of glycolysis during early development we have examined the pattern of expression of enolase isoenzymes in Xenopus laevis. In addition, the nucleotide sequence of a cDNA clone coding for the complete amino acid sequence of one enolase gene (ENO1) in X. laevis was determined. X. laevis ENO1 shows highest homology to mammalian non-neuronal enolase. Analysis of enolase isoenzymes in X. laevis by non-denaturing electrophoresis on cellulose acetate strips revealed five isoenzymes. One form was present in all tissues tested, two additional forms were expressed in oocytes, embryos, adult liver and adult brain, and two further forms were restricted to larval and adult muscle. Since enolase is a dimer, three different monomers (gene products) could account for the observed number of isoenzymes. This pattern of enolase isoenzyme expression in X. laevis differs from that of birds and mammals. In birds and mammals the most acidic form is neuron-specific and there is only one major isoenzyme expressed in the liver. RNAase protection experiments showed the presence of ENO1 mRNA in oocytes, liver and muscle, suggesting that it codes for a non-tissue-restricted isoenzyme. ENO1 mRNA concentrations are high in early oocytes, decrease during oogenesis and decrease further after fertilization. Enolase protein, however, is maintained at high concentrations throughout this period.     

Experimental determination of net protein charge, [A]tot, and Ka of nonvolatile buffers in bird plasma.

The quantitative mechanistic acid-base approach to clinical assessment of acid-base status requires species-specific values for [A]tot (the total concentration of nonvolatile buffers in plasma) and Ka (the effective dissociation constant for weak acids in plasma). The aim of this study was to determine [A]tot and Ka values for plasma in domestic pigeons. Plasma from 12 healthy commercial domestic pigeons was tonometered with 20% CO2 at 37 degrees C. Plasma pH, Pco2, and plasma concentrations of strong cations (Na, K, Ca), strong anions (Cl, L-lactate), and nonvolatile buffer ions (total protein, albumin, phosphate) were measured over a pH range of 6.8-7.7. Strong ion difference (SID) (SID5=Na+K+Ca-Cl-lactate) was used to calculate [A]tot and Ka from the measured pH and Pco2 and SID5. Mean (+/-SD) values for bird plasma were as follows: [A]tot=7.76+/-2.15 mmol/l (equivalent to 0.32 mmol/g of total protein, 0.51 mmol/g of albumin, 0.23 mmol/g of total solids); Ka=2.15+/-1.15x10(-7); and pKa=6.67. The net protein charge at normal pH (7.43) was estimated to be 6 meq/l; this value indicates that pigeon plasma has a much lower anion gap value than mammals after adjusting for high mean L-lactate concentrations induced by restraint during blood sampling. This finding indicates that plasma proteins in pigeons have a much lower net anion charge than mammalian plasma protein. An incidental finding was that total protein concentration measured by a multianalyzer system was consistently lower than the value for total solids measured by refractometer.     

Experimental determination of net protein charge and A(tot) and K(a) of nonvolatile buffers in human plasma.

The mechanism for an acid-base disturbance can be determined by using the strong ion approach, which requires species-specific values for the total concentration of plasma nonvolatile buffers (Atot) and the effective dissociation constant for plasma weak acids (Ka). The aim of this study was to experimentally determine Atot and Ka values for human plasma by using in vitro CO2 tonometry. Plasma Pco2 was systematically varied from 25 to 145 Torr at 37 degrees C, thereby altering plasma pH over the physiological range of 6.90-7.55, and plasma pH, Pco2, and concentrations of quantitatively important strong ions (Na+, K+, Ca2+, Mg2+, Cl-, lactate) and buffer ions (total protein, albumin, phosphate) were measured. Strong ion difference was estimated, and nonlinear regression was used to calculate Atot and Ka from the measured pH and Pco2 and estimated strong ion difference; the Atot and Ka values were then validated by using a published data set (Figge J, Rossing TH, and Fencl V, J Lab Clin Med 117: 453-467, 1991). The values (mean +/- SD) were as follows: Atot = 17.2 +/- 3.5 mmol/l (equivalent to 0.224 mmol/g of protein or 0.378 mmol/g of albumin); Ka = 0.80 +/- 0.60 x 10-7; negative log of Ka = 7.10. Mean estimates were obtained for strong ion difference (37 meq/l) and net protein charge (13+.0 meq/l). The experimentally determined values for Atot, Ka, and net protein charge should facilitate the diagnosis and treatment of acid-base disturbances in critically ill humans.     

Direct determination of the stoichiometry of charge transfer complexes.

The stoichiometry of the charge transfer complex between N-acetyl-L-tryptophan and 1-methylnicotinamide chloride has been determined to be precisely 1:1 by direct measurement of the molecular weight of the complex. The result is of interest both in terms of a general method for determining the stoichiometry of charge transfer complexes, and in terms of the probable stoichiometry of specific charge transfer complexes between 1-methylnicotinamide chloride and the exposed tryptophyl side chains of certain proteins. In the latter case, the result provides experimental proof for the assignment of extinction coefficients of specified magnitudes to the homomorphic model complexes which serve as the basis for the interpretation of results with proteins.     

Electrofocusing-induced alteration in the net charge of human macrophage-stimulating protein

Macrophage-stimulating protein is a mammalian serum protein, detected by its capacity to render mouse resident peritoneal macrophages responsive to chemoattractants. The purified protein was shown by column electrofocusing over a period of 48 h to have an isoelectric point of 7.0. In this study we determined the isoelectric point of the native molecule by electrofocusing human serum on a flat bed of prefocused ampholytes. Values in the range of 5.5–6.2 were obtained. A shift in the isoelectric point to 7.0–7.6, without significant alteration in the biological activity of the molecule, occurred when electrofocusing was prolonged to 48 h or was carried out in 6 m urea.     

Strongyloides ratti and Trichinella spiralis: net charge of epicuticle

The intact epicuticles of Strongyloides ratti stage-3 larvae and Trichinella spiralis stage-1 larvae were found to have a surface net negative charge. Ultrastructural studies on S. ratti using cationized ferritin and ruthenium red showed the negative charge to be dense and uniformly distributed over the epicuticular surface. Staining with acetic acid-ferric oxide hydrosol occurred at pH 1.65 and suggests that amino acid carboxyl groups were not responsible for the negative charge property. Alcian blue staining occurred at pH 0.5 and at a critical electrolyte concentration (CEC) of 0.9 M MgCl2, a property similar to that of highly sulfated mucopolysaccharides such as the proteoglycan keratan sulfate. In contrast, T. spiralis larvae failed to stain with alcian blue below pH 5.0 or at a CEC of 0.1 M, suggesting its negative charge is associated with dissociated amino acid carboxyl groups. Attempts to remove the negative charge-bearing components in the epicuticle of S. ratti by detergents, organic solvents, denaturing agents, proteases, uronidases, neuraminidases, and lipases were unsuccessful. The presence of elastin in the S. ratti larval outer cortical layer was indicated by its vulnerability to elastase and its reaction to aldehyde fuchsin-alcian blue stain. These results show that the epicuticle of S. ratti is not a typical cell membrane, although it appears to have ultrastructural similarities. It is suggested that the association of highly sulfated mucopolysaccharides with the epicuticular surface of free-living nematodes such as S. ratti L3 may reflect a greater need to protect against surface desiccation. It is also postulated that the highly negatively charged surface may have anticomplementary and anticoagulation effects.     

Photovoltage kinetics of the acid-blue and acid-purple forms of bacteriorhodopsin: evidence for no net charge transfer.

Time-resolved photovoltage measurements were performed with the acid-blue (bR605A) and acid-purple (bR565A) forms of bacteriorhodopsin (bR) in the time range from 25 ns to 100 s. The bR605A and bR565A pigments were formed by titration with H2SO4 in the absence and presence of 150 mM KCI, respectively. Qualitatively the kinetics of the charge displacement in these two states are similar and consist of two fast phases in one direction (100 ns bandwidth limited and approximately 1 microsecond) followed by a decay in the opposite direction via one component for bR605A (4.4 +/- 0.6 ms) or two components for bR565A (33 +/- 8 microseconds and 3.6 +/- 0.5 ms). The transient photovoltage signal returns exactly to the initial value after several milliseconds, well before the passive discharge of the electrical measuring system at 2 s. We conclude that no net charge transfer occurs in either bR605A or bR565A. The direction of the fast components is opposite that of net proton translocation in bR at pH 7. So, if the charge that moves back and forth is due to a proton, it moves first in the direction of the cytoplasmic side of the membrane (< 1 microsecond) and returns to its initial position via the 4.4 ms (bR605A) or the 33 microseconds and 3.6 ms (bR565A) decay components. The amplitude of the charge motion in both low pH forms is too large to be due to isomerization alone and is comparable to one of the major components in bR at pH 7.2     

Microquantitative determination of lactate dehydrogenase isoenzymes in the myocardium and the conducting system

Summary A newly developed technique was used for the electrophoretic separation of lactate dehydrogenase (LDH) isoenzymes from lyophilized tissue samples in the nanogram range. In this study portions of 10–200 ng from the myocardium and the conducting system of cattle, sheep, pig and man were microdissected and analysed.In the heart tissues of cattle, sheep and pig, the isoforms LDH1, LDH2 and LDH3 were detected in species-specific varying amounts. In all these animals, the conducting system is marked by high LDH1 activity, which is present at a ratio of about 2:1 compared with the myocardium. The values in man, however, differ from these values, but this might be due to post-mortem changes. The findings are discussed with respect to possible aerobic-anaerobic functions.     

High-throughput microplate assay for the determination of drug partition coefficients

Partition coefficients (K(p)) of drugs between the phospholipid bilayer and the aqueous phase provide useful information in quantitative structure-activity relationship studies. Hexadecylphosphocholine (HePC) micelles, composed of a zwitterionic hydrophilic surface and a hydrophobic core, mimic the biomembranes and have several advantages over other lipid structures to assess K(p) values. Their preparation is easy, fast and avoids the use of toxic organic solvents, and the output has fewer spectroscopic interferences. Here, we describe a high-throughput microplate protocol for assessing the K(p) of drugs using HePC micelles as membrane models and derivative spectrophotometry as the detection technique. Moreover, the time-consuming data treatment to assess K(p) values is easily performed by a dedicated Excel routine developed here and described in detail. The K(p) values of nonsteroidal anti-inflammatory drugs (acemetacin, clonixin, diclofenac and indomethacin) were determined to show the simplicity of the method and to validate this protocol, which provides K(p) values (n = 3) of two drugs in ～ 2 h.     

Relative influence of hydrophobicity and net charge in the aggregation of two homologous proteins

A potentially amyloidogenic protein has to be at least partially unfolded to form amyloid aggregates. However, aggregation of the partially or totally unfolded state of a protein is modulated by at least three other factors: hydrophobicity, propensity to form secondary structure, and net charge of the polypeptide chain. We propose to evaluate the relative importance of net charge, as opposed to the other factors, on protein aggregation and amyloidogenicity. For this aim, we have used two homologous proteins that were previously shown to be able to form amyloid fibrils in vitro, the N-terminal domain of HypF from Escherichia coli (HypF-N) and human muscle acylphosphatase (AcP). The aggregation process from an ensemble of partially unfolded conformations is ca. 1000-fold faster for HypF-N than for AcP. This difference can mainly be attributed to a higher hydrophobicity and a lower net charge for HypF-N than for AcP. By using protein engineering methods, we have decreased the net charge of AcP to a value identical to that of wild-type HypF-N and increased the net charge of HypF-N to a value identical to that of wild-type AcP. Amino acid substitutions were selected to minimize changes in hydrophobicity and secondary structure propensities. We were able to estimate that the difference in net charge between the two wild-type proteins contributes to 20-25% of the difference in their aggregation rates. An understanding of the relative influences of these forces in protein aggregation has implications for elucidating the complexity of the aggregation process, for predicting the effect of natural mutations, and for accurate protein design.