Ferguson Plot Analysis Research Articles

Characterization of intracellular deoxyribonucleases using polynucleotide‐polyacrylamide gel electrophoresis

AbstractIntracellular deoxyribonucleases (DNases) in human peripheral blood leukocytes have been identified and characterized by polynucleotide‐polyacrylamide gel electrophoresis (PPAGE). This technique combines the protein separating powers of polyacrylamide gel electrophoresis with the sensitivity of detection characteristic of an enzyme assay. Visualization of nuclease activity is made possible by the inclusion of high molecular weight DNA substrate within the sieving portion of a narrow polyacrylamide gel. No sodium dodecyl sulfate, urea, or other protein denaturants are used in this procedure. However, electrophoresis of the contents of up to 106 cells/gel is accomplished using conditions under which the enzymes are inactive. The positions of DNases are then revealed by incubation of the gel in an appropriate buffer followed by staining for intact DNA. Colorless regions correspond to the presence of enzyme. Used as a pattern recognition tool, PPAGE revealed that leukocytes from normal donors contained lower levels of certain activities than did cells from donors with chronic myelogenous leukemia. The rapid simultaneous characterization of multiple activities is made possible by simply using different buffer compositions in the incubation step. For example, ionic strength and pH optima may be found directly for activities present in unpurified human intracellular contents. The reproducibility of this method permits comparison of individual activities by using Ferguson plot analysis to detect differences or similarities among enzymes. These analyses revealed that DNase activities in human peripheral blood leukocytes fell into a minimum of two families. Modifications of overall effective charge probably account for microheterogeneity.

Synthesis of the myelin proteolipid protein in the developing mouse brain

Mice ranging in age from 16 to 44 days were injected intracerebrally with 3H-leucine, and incorporation into total brain proteolipids and the myelin proteolipid protein was measured. All proteolipids were isolated from whole brain by ether precipitation and separated into their individual components by SDS polyacrylamide gel electrophoresis. Two major proteolipids with apparent molecular weights of 20,700 and 25,400 were observed in these preparations, and their proportion increased over the developmental period examined. A Ferguson plot analysis comparing these proteins with those of isolated myelin showed that the 25,400-dalton proteolipid component from whole brain was the myelin proteolipid protein. Rates of incorporation of 3H-leucine into total brain proteolipids peaked at 22 days of age. Synthesis of the myelin proteolipid protein increased rapidly to a maximum value at 22 days and decreased rather slowly until at 44 days it was about 83% of its maximum rate of synthesis. The data indicate that the developmental pattern of synthesis of the myelin proteolipid protein is unlike that of the myelin basic proteins. Synthesis of the major myelin proteins is developmentally asynchronous in that peak synthesis of the myelin proteolipid appears to occur several days later than the basic proteins. In addition, it maintains its maximum rate of synthesis over a longer period of time than do the basic proteins.

Purification and characterization of the estrogen-induced protein (IP) of the rat uterus

One of the earliest responses of the rat uterus to estrogen is increased synthesis of a specific cytosol protein (IP). This synthesis is detectable within 40 min and is dependent on an even earlier actinomycin D-sensitive function. IP has now been purified to homogeneity as determined by SDS polyacrylamide gel electrophoresis (SDS PAGE). The procedure was complicated by a tendency of the more homogeneous and concentrated material to aggregate. Purification consisted of sequential chromatography, in the presence of 0.1% triton X-100, via DEAE-cellulose (2X), hydroxylapatite and agarose-acrylamide. These were followed by preparative PAGE and finally SDS PAGE. Molecular weight determination by Ferguson plot analysis yielded an apparent molecular weight of 45 000. On final SDS PAGE, the material consisted of two major bands: the 45 000 molecular weight IP band and a band with an estimated molecular weight of 80 000. This second band displayed an elevated synthesis in E 2-stimulated uteri similar to IP and appeared to consist of some form of aggregated IP. Carbohydrate determination on SDS gels using periodic acid-Schiff (PAS) stain was negative. Co-purification of labeled cytosol proteins from uteri of control and E 2-stimulated rats revealed that IP is synthesized to some extent in the unstimulated animal as well as the stimulated.

Structural and enzymological characterization of the homogeneous deoxyribonucleic acid polymerase from Mycoplasma orale

We have purified the DNA polymerase from Mycoplasma orale to homogeneity. The protein structure of the enzyme was declined by sodium dodecyl sulfate gel electrophoresis, which revealed a single band of 116 000 daltons that was coincident with the polymerase activity profile in the final step of DNA--cellulose chromatography, and by two-dimensional gel analysis, which demonstrated a single protein species at pI = 6.8 that was congruent with enzyme activity and contained the same 116 000 polypeptide. although severe enzyme aggregation occurs during nondenaturing gel electrophoresis, a monomer species can be resolved with a Mr of 140 000 by the Ferguson plot analysis. Gel filtration and velocity gradient centrifugation yield a Stokes radius of 4.8 nm and a sedimentation coefficient of 5.6 S, respectively, from which Mr values of 106 000--128 000 can be computed. The different size values suggest that the polymerase molecule is asymmetric. The purified enzyme has a specific activity of approximately 6 x 10(5) units/mg of protein and in completely devoid of exodeoxyribonuclease and endodeoxyribonuclease activities, at exclusion limits of 10(-4)--10(-6%) of the polymerase activity. The mechanism of polymerization is moderately processive, with an average of 14 +/- 4 nucleotides incorporated per binding event, and the "effective template length" on activated DNA is approximately 40 nucleotides.

Adenylate cyclase in polyacrylamide gel electrophoresis: Solubilized but active

An intrinsic “insoluble” membrane-bound enzyme, adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) was analyzed in a detergent solubilized but active form by polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis provided improved resolution of the enzyme from contaminant proteins compared to previously used column chromatographic procedures, and allowed the analysis of much smaller quantities of material. The success of this approach depended on a particular choice of the nature and concentration of the detergent, of pH, of buffer constituents, of temperature, and of gel architecture. Using either the basal enzyme or enzyme pretreated in the membrane with guanyl-5′-yl imidodiphosphate (Gpp(NH)p) in the presence or absence of glucagon, up to 90% of enzymatic activity was recovered. However, the basal enzyme lost sensitivity to stimulation by Gpp(NH)p but not fluoride during electrophoresis. Ferguson plot analysis was used to determine the size and charge parameters of the enzyme, and bandwidth to estimate the degree of polydispersity. Increasing detergent concentrations reduced the effective size and degree of polydispersity of the enzyme, although this also led to progressive inactivation. The size of the enzyme was independent of preactivation by Gpp(NH)p alone or Gpp(NH)p plus glucagon. The “smallest and least polydisperse” size to which the active enzyme could be reduced was 3.6 ± 0.8 to 4.0 ± 0.8 nm (geometric mean radius).

Phosphoglycolate Phosphatase: PURIFICATION AND PROPERTIES

Phosphoglycolate phosphatase (EC 3.1.3.18) was purified 1500-fold from field-grown tobacco leaves by acetone fractionation, DEAE-cellulose and molecular sieve chromatography, and preparative polyacrylamide gel electrophoresis. Preparations were judged 90 to 95% homogeneous by chromatography on DEAE-cellulose, polyacrylamide gel electrophoresis, and by isoelectric focusing. The highest specific activity obtained was 468 umol of phosphate released/min/ mg of protein. The native protein has a molecular weight of 80,500 by Ferguson plot analysis and 86,300 by sedimentation velocity on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gels gave a molecular weight of 20,700, indicating that P-glycolate phosphatase is a tetramer with identical or near identical subunits. The enzyme, freshly purified or in crude homogenates, had a pi of 3.8 to 3.9 pH units by isoelectric focusing. Phosphoglycolate phosphatase from spinach leaves has a molecular weight of 93,000 and, unlike the enzyme from tobacco leaves, it is extremely unstable after DEAE-cellulose chromatography and is inactivated by lipase (EC 3.1.1.3). The phosphatase from both plants was stabilized by the addition of citrate or isocitrate in the buffers. Ribose 5-phosphate is a competitive inhibitor of phosphoglycolate phosphatase at physiological concentrations, while other phosphate esters of the photosynthetic carbon cycle were without effect.

Adaptation at specific loci. I. Natural selection on phosphoglucose isomerase of Colias butterflies: Biochemical and population aspects.

Electrophoretic variants of phosphoglucose isomerase (PGI) in Colias butterflies have been studied from field and laboratory viewpoints. The transmission pattern is that of a dimeric enzyme controlled by one structural gene locus. Populations usually harbor four to six allelic mobility classes. These mobility classes are shared among species complexes, though their frequencies differ widely. Preliminary Ferguson plot analysis of the variants has been carried out. Purified preparations of Colias PGI alleles are more effective in standardizing Ferguson plots than heterologous proteins, such as ferritin. Variation of Ferguson plot parameters is not an infallible guide to electrophoretically "cryptic alleles," as one putative case proved to be due to nonallele-specific effects. S, M, and F mobility classes in two Colias semispecies show the same retardation coefficients in Ferguson plots. Adults early in the flight periods of their nonoverlapping generations show genotype frequencies in Hardy-Weinberg equilibrium, but heterozygote excess develops as the insects age. Simple directional selection and large-scale population mixing are unlikely to be causes of this, although several other selection modes remain possible. Identical-by-descent lines of the four frequent-to-common alleles in C. eurytheme have been set up in culture, and enzyme has been purified from these for study of functional properties. Major differenecs in heat stability and in various kinetic parameters are found among the ten possible genotypes. In some cases, heterosis for kinetic parameters is seen; in other cases, opposing trends in kinetic function and heat stability create potential for net heterosis in function. Possible interpretations of these results in an adaptive metabolic context are discussed, and directions for further work are stated.