Partial purification and characterization of the multiple molecular forms of staphylococcal clotting activity (coagulase).

The clotting activity of Staphylococcus aureus strain 104 was purified 46,000-fold, but absolute purity was not achieved. Carbohydrate content of the purified material was not more than 5%. Elution of clotting activity from denaturing and nondenaturing polyacrylamide gels revealed the presence of four distinct molecular forms. Molecular weights of the forms were approximately 31,500, 34,800, 44,800, and 56,800 as determined by gel filtration in 8 M urea, by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis, and by calculation with determined values for the Stokes radius and sedimentation coefficient. Molecular weights determined on sodium dodecyl sulfate-urea gels were found to decrease as the gel concentration increased, suggesting that the amount of sodium dodecyl sulfate bound was less than normal. Estimated frictional ratios for the forms showed that they differ in shape from one another and that they are all highly asymmetrical. Each of the forms had an isoelectric point between pH 5.44 and 5.47 when focused in 6% polyacrylamide gels for 9 h; however, prolonged focusing altered the isoelectric point of the forms to within the range of pH 4.35 to 4.65. The multiple clotting forms were not artifacts of the purification procedure and did not appear to be products of the proteolytic degradation of a larger protein.     

In situ staining procedure for the detection of ornithine transcarbamylase activity in polyacrylamide gels

Ornithine transcarbamylase deficiency is a human genetic disease potentially susceptible to gene therapy. A murine model system exists for the disease in the sparse-fur (spf) mouse. Before gene therapy studies can be performed it is necessary to have practical methods which could detect successful gene transfer. Therefore we have developed an in situ staining procedure for the detection of ornithine transcarbamylase activity in polyacrylamide gels. Following electrophoretic separation under nondenaturing conditions inorganic phosphate cleaved from carbamyl phosphate in gels as a result of enzymatic activity was precipitated as phosphomolybdic acid and visualized by reduction with ascorbic acid. Results from the procedure correlated with ornithine transcarbamylase activity as measured by solution assay for citrulline, the other product of the reaction. This procedure readily distinguished mutant forms of ornithine transcarbamylase as exemplified by the murine spf mutation and resolved ornithine transcarbamylases of all animals tested into multiple forms. The procedure further distinguished ornithine transcarbamylases of animals of several different genera while yielding virtually identical patterns of the enzyme from species within the same genus. This procedure also suggested that the human enzyme was more labile than murine ornithine transcarbamylase; direct thermolability studies confirmed this finding.     

Electrophoretic analysis of the estrogen receptor. Relationship of multiple receptor forms to the molybdate-stabilized form

The origin of and relationships among multiple forms of the estrogen receptor from rat uteri were investigated using electrophoretic and conventional hydrodynamic methods of analysis. Evidence is presented that the molybdate-stabilized, multimeric receptor (Stokes radius approximately 70A; S20,w approximately 9.5 S; Mr approximately 290,000) corresponds to an acidic form of the receptor that has relatively high electrophoretic mobility. This discrete form, which appears to represent the untransformed state that does not bind to DNA, was converted to a number of derived forms by exposure to conditions that result in receptor transformation and/or subunit dissociation. In crude cytosol, transformation always generated receptor forms that were excluded from polyacrylamide gels, and it was shown that these are large heterogeneous aggregates. This explains previous failed attempts to analyze the receptor by polyacrylamide gel electrophoresis. Transformation of partially purified, molybdate-stabilized receptor never led to aggregate formation, but resulted instead in the generation of two relatively basic estrogen-binding species of low electrophoretic mobility. These components may represent the free or dissociated estrogen-binding subunits. Together, the results suggest a model for the molybdate-stabilized receptor wherein at least one of its components is an acidic, nonestrogen-binding subunit.     

Human pancreatic alpha-amylase: phenotypic codominance and new electrophoretic variants.

The genetic heterogeneity of human pancreatic alpha-amylase (alpha-1,4-glucan 4-glucanohydrolase, E.C. 3.2.1.1) has been better defined through the development of an asparagine buffered electrophoretic gel system. Three alleles had been identified for the pancreatic amylase locus, AMY2, with two variant alleles as autosomal dominant traits on Tris HCl buffered sheet gels. The asparagine buffered sheet gel now allows the differentiation of the genotypes AMY2B/AMY2B,AMY2B/AMY2A, and AMY2B/AMY2C, thus classifying these three alleles as codominants. Asparagine buffered polyacrylamide gels and thin layer polyacrylamide isoelectric focusing aided in the identification of three new pancreatic amylase variants: AMY2D,AMY2E, and AMY2F. AMY2E has been identified only in AMY2B and AMY2E individuals. This allele is proposed as a quantitative activity variant with essentially the same electrophoretic mobility as AMY2A. The other new autosomal variants have each been identified in single white families. AMY2D is dominant and AMY2F is a codominant trait as shown on thin layer polyacrylamide isoelectric focusing gels.     

Cytosolic aspartate aminotransferases from different chicken tissues: purification and characterization of their multiple forms

Cytosolic aspartate aminotransferases from chicken heart, liver, spleen, skeletal muscle and breast muscle differed in number of their molecular forms, detected by polyacrylamide gel electrophoresis and specific staining. The number of molecular forms varied from tissue to tissue but the electrophoretic mobilities of a given form in all tissues were analogous. Within a single tissue most of the enzyme activity was present as the lowest-running band (alpha form) and the rest was distributed in minor bands termed (B,tau, alpha and epsilon forms). We report a method for the purification of cytosolic aspartate aminotransferases from various chicken tissues. The procedure can be carried out in one week and allows the obtention of isolated molecular forms of the enzyme, independently of the tissue under study. Separation of multiple forms was also achieved by chromatofocusing. The isoelectric points determined by this method for a given form in all five tissues were analogous and differed from those of the molecular forms of the enzyme from other origins. An Mr of 100,000 was obtained for all molecular forms of the five chicken tissues studied.     

Improved zymographic detection of bovine acrosin.

Acrosomal materials extracted from bovine spermatozoa contain a trypsin-like proteinase termed acrosin (1–3) (EC 3.4.21.10). The presence of multiple molecular forms of this spermatozoal enzyme has been demonstrated both by gel filtration (2) and by polyacrylamide gel electrophoresis (1,3).The enzymic reactions of proteinases can be detected in electrophoretic patterns by incorporating proteinaceous substrates in the electrophoretic media (4) or by using specific amino acid derivatives of β-naphthylamine (1,5,6). Andary and Dabich (6) recently reported that the former method was improved by diffusing the proteinaceous substrate into the gel during a 1-hr incubation period following electrophoresis. The zymogram then required an additional incubation of the gel in buffer solution for 2–12 hr before the transparent zones of proteinase activity were detectable. Incubation periods of less than 1 hr are normally required for the zymographic staining methods that use synthetic arginine derivatives of β-napthylamine to detect acrosin activity. These systems do, however, suffer from a lack of sensitivity and fading of the diazonium-coupled product (1). An improved method for rapid detection of acrosin activity in gels would, therefore, be useful. The present communication describes an improved version of the staining system for detecting acrosin activity using a synthetic arginine derivative of β-naphthylamine. The application of this staining system for the detection of the multiple forms of bovine acrosin is presented. In addition, the stability of the zymograms resulting from three different coupling dyes was investigated using a miniature polyacrylamide gel electrophoretic system.     

Two apparent molecular weight forms of human and monkey phenylalanine hydroxylase are due to phosphorylation

Two-dimensional polyacrylamide gel analyses of purified human and monkey liver phenylalanine hydroxylase reveal that the enzyme consists of two different apparent molecular weight forms of polypeptide, designated H (Mr = 50,000) and L (Mr = 49,000), each containing three isoelectric forms. The two apparent molecular weight forms, H and L, represent the phosphorylated and dephosphorylated forms of phenylalanine hydroxylase, respectively. After incubation of purified human and monkey liver enzyme with purified cAMP-dependent protein kinase and [gamma-32P]ATP, only the H forms contained 32P. Treatment with alkaline phosphatase converted the phenylalanine hydroxylase H forms to the L forms. The L forms but not the H forms could be phosphorylated on nitrocellulose paper after electrophoretic transfer from two-dimensional gels. Phosphorylation and dephosphorylation of human liver phenylalanine hydroxylase is not accompanied by significant changes in tetrahydrobiopterin-dependent enzyme activity. Peptide mapping and acid hydrolysis confirm that the apparent molecular weight heterogeneity (and charge shift to a more acidic pI) in human and monkey liver enzyme results from phosphorylation of a single serine residue. However, phosphorylation by the catalytic subunit of cAMP-dependent protein kinase does not account for the multiple charge heterogeneity of human and monkey liver phenylalanine hydroxylase.     

Distribution of the multiple molecular forms of glucose-6-phosphate dehydrogenase in different physiological states.

The distribution of the multiple molecular forms of rat liver and mammary gland glucose-6-phosphate dehydrogenase was determined by electrophoresis on 5% polyacrylamide gels. In both of these organs, changes in the distribution of enzyme activity among the several forms was slight even when approximately 20- to 40-fold changes in enzyme specific activity were achieved by fasting-refeeding experiments (for liver) or during pregnancy and lactation (for mammary gland). It was concluded that the induction of glucose-6-phosphate dehydrogenase in these two organs occurs without any major redistribution among the multiple molecular forms of this enzyme.     

Multiple forms of alkaline phosphatase in untreated and 5-bromo-2'-deoxyuridine-treated choriocarcinoma cells

The alkaline phosphatases present in choriocarcinoma cells, either untreated or treated with 5-bromo-2′-deoxyuridine (BrdUrd), were purified and characterized. Three forms of phosphatase [I, IIa (or IIIa), and IIb (or IIIb)]were isolated from both the untreated and BrdUrd-treated cells. Although BrdUrd induced the synthesis of all three forms of alkaline phosphatase in these cells, the synthesis of forms IIa and IIb was, however, preferentially stimulated. The forms of phosphatase in choriocarcinoma cells resembled each other in their kinetic properties and thermal lability, but differed in their molecular weights and in their electrophoretic mobilities in nondenaturing polyacrylamide gels. All three phosphatases were inactivated by antiserum to term-placental alkaline phosphatase. The alkaline phosphatases from choriocarcinoma cells differed, however, from the enzyme from term placentas in several physicochemical properties. The phosphatases from choriocarcinoma cells had a lower K_m value for p-nitrophenyl phosphate, were more sensitive to inhibition by l-leucine, levamisole, l-p-bromotetramisole, and EDTA, and were more heat-labile. Phosphatase I comigrated with term-placental alkaline phosphatase on nondenaturing polyacrylamide electrophoretic gels, but phosphatases IIa and IIb migrated more slowly. The apparent molecular weights of phosphatase forms I, IIa, and IIb were estimated by gel filtration and polyacrylamide gel electrophoresis to be 115,000, 240,000, and 510,000, respectively. Although three molecular forms of alkaline phosphatase occurred in choriocarcinoma cells, the subunit molecular weight of these phosphatases appeared to be identical to each other and to the subunit of term-placental alkaline phosphatase (63,000 MW). The alkaline phosphatase in choriocarcinoma cells therefore exists in the dimeric, tetrameric, and octameric forms.     

Direct tissue isoelectric focusing on ultrathin polyacrylamide gels. Applications in enzyme, lectin and immunohistochemistry

Summary Application of cryostal sections directly onto ultrathin polyacrylamide gels and subsequent isoelectric focusing allows elution of proteins, glycoproteins and peptides out of the sections into the gels. The eluted compounds reveal clearly delineated band patterns in the polyacrylamide gels. The advantage of this method is that enzyme histochemical reactions can be directly performed in the gel and in the electroeluted tissue sections. Therefore, this method is suitable for specifying, in more detail, histochemical enzyme reactions and for detecting multiple forms of enzymes even from a single tissue section. Furthermore, the transfer of proteins, glycoproteins and peptides from the gel onto nitrocellulose by a modified Western blot procedure offers the possibility of checking findings obtained by lectin histochemistry and immunohistochemistry.     

A method for horizontal polyacrylamide slab gel electrophoresis

We present a simplified method of preparation of polyacrylamide gels which is totally analogous to the procedure now widely used to pour and run horizontal agarose gels. The acrylamide is poured into an open air gel mold consisting of a glass plate with a masking tape border and a comb. It is subsequently run in a submarine horizontal electrophoresis apparatus. The electrophoretic mobility and resolution of DNA fragments obtained in such gels are identical to results obtained with gels poured and run in the vertical configuration. Numerous advantages of horizontal polyacrylamide gel electrophoresis are discussed.     

Multiple forms of gamma-butyrobetaine hydroxylase (EC 1.14.11.1).

gamma-Butyrobetaine hydroxylase [4-trimethylaminobutyrate, 2-oxoglutarate:oxygen oxidoreductase (3-hydroxylating), EC 1.14.11.1] from human kidney was resolved into three forms by chromatofocusing. After further chromatography on an anion-exchanger, each form appeared as a single band on electrophoresis in polyacrylamide gel containing sodium dodecyl sulphate. The isoelectric points of isoenzymes 1, 2 and 3 were 5.6, 5.7 and 5.8 respectively, as estimated by isoelectric focusing. Their specific activities were 17-29 mu kat/g of protein. The concentrations of the three isoenzymes were about equal, possibly slightly lower for isoenzyme 1. The requirement for Fe2+ and the Km values for gamma-butyrobetaine and 2-oxoglutarate were about the same for the different enzyme forms. L- and D-Carnitine caused decarboxylation of 2-oxoglutarate to the same extent (8 and 29%) with the three forms. The enzyme forms had the same mass, 64 kDa, as determined by gel filtration in nondenaturing media. The same subunit mass, 42 kDa, was obtained for the multiple forms by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Isoenzyme 2 was resolved into two protein bands by isoelectric focusing in polyacrylamide gels containing urea. Isoenzyme 1 contained only one of these bands and isoenzyme 3 the other. The three enzyme forms of gamma-butyrobetaine hydroxylase thus appear to be dimeric combinations of two subunits differing in charge but not in size. gamma-Butyrobetaine hydroxylase from crude extracts of human, rat and calf liver was also separated into multiple forms by a chromatofocusing technique. The isoenzyme pattern was the same in human liver and kidney. The technique used to resolve the mammalian enzymes gave no evidence for the presence of multiple forms of the bacterial enzyme from Pseudomonas sp. AK 1.     

Multiple forms of isocitrate lyase in the matrix of Turbatrix aceti mitochondria

The localization of five separate forms of isocitrate lyase (EC 4.1.3.1) in the free-living nematode Turbatrix aceti was determined by analyzing their distribution among subcellular fractions with continuous (Tris-acetate, pH 7.5) polyacrylamide gel electrophoresis. Enzyme activity on gels was detected either by substrate-dependent Schiff's-aldehyde staining or absorbance of phenylhydrazone at 324 nm. Following rate sedimentation of worm homogenates, the highest specific activity for isocitrate lyase was recovered in the pellet containing intact mitochondria. Glyoxysomes (microbodies) were not observed by electron microscopy in this or any other fraction. Selective removal of mitochondrial outer membranes (and hence components in the intermembrane space) was accomplished by two different procedures: (1) passage of mitochondria in a hypersomotic medium through a French pressure cell at 1500 psi, or (2) treatment with 0.6 mg digitonin mg protein. Electron microscopy revealed essentially complete removal for the former procedure, but only partial removal following digitonin treatment. Equilibrium centrifugation on sucrose gradients was necessary to strip the residual outer membranes from the digitonin-treated mitochondria. Mitoplasts resulting from the two procedures were subfractionated into matrix and inner membrane components by high-pressure disruption (14,000 psi) and subsequent rate sedimentation (144,000g, 60 min). Identical electrophoretic patterns were found using both slab and disc gels, whether stained with Schiff's reagent or scanned at 324 nm, in samples taken from clarified homogenates, intact mitochondria, mitoplasts, or matrix fractions. The results indicate that all five forms of the enzyme occur together in the mitochondrial matrices. Their individual functions are not yet known, but they may be involved in the regulation of isocitrate metabolism common to the tricarboxylic acid and glyoxylate cycles occurring within the same mitochondria.     

Production of multiple forms during purification of Streptomyces aureofaciens DNA polymerase: a study using nondenaturing polyacrylamide gradient gel electrophoresis.

A modified method for the detection of DNA polymerases in cell extracts and purified enzyme preparations after electrophoresis in polyacrylamide gradient cylindrical gels is described. The technique, which is based on direct assay of activity in a reaction mixture during elution of DNA polymerases from gel slices, was applied to the pursuit of enzyme forms of Streptomyces aureofaciens DNA polymerase during purification procedure. In a crude extract of S. aureofaciens mycelium many catalytically active forms of DNA polymerase ranging from 35 to 860 kDa were detected. After purification, that included mycelium homogenization, precipitation of nucleic acids by polyethylene glycol, DEAE-Sephadex, QAE-Sephadex and DNA-Sepharose chromatography, higher molecular mass forms of more than 172 kDa have not been found. The lower molecular mass forms were separated into two groups by DNA-Sepharose chromatography. On the basis of their characterization, it is assumed that the lower molecular mass forms are produced by proteolysis and the major form found after purification of S. aureofaciens DNA polymerase in the presence of suitable proteinase inhibitors should be a protein of 172 kDa.     

Multiple forms of acetylcholinesterase from human erythrocytes

1. Acetylcholinesterase from human erythrocytes was solubilized with Triton X-100 in strong salt solution and partially purified by (NH(4))(2)SO(4) fractionation. This preparation showed three main bands of enzyme activity after electrophoresis on polyacrylamide gel and incubation with either alpha-naphthyl acetate or acetylthiocholine as enzyme substrate. Two of the multiple forms were completely inhibited by 10mum-eserine and one only partially. Treatment with neuraminidase had no effect on the electrophoretic pattern; therefore sialic acid does not appear to determine or affect the ratios of the acetylcholinesterase multiple forms, unlike those of the serum cholinesterase. 2. Chromatography of the preparation on Sephadex G-200 revealed one major peak of enzyme activity and a suggestion of two minor zones of mol.wt. 546000, 184000 and 93000 (i.e. in the proportion 6:2:1). The main peak was almost completely separated from the Triton X-100 and the overall purification was about 600-fold. Further attempts to purify the enzyme by absorption on calcium phosphate gels were unsuccessful. 3. Electrophoresis of the enzyme preparation on a polyacrylamide gradient for 24h revealed three main bands that corresponded to the three values for molecular weights obtained by column chromatography. After 70h of electrophoresis a further three zones of activity developed making six molecular entities, the molecular weights of which were simple multiples of a monomer, thus resembling the cholinesterase found in serum.     

The heterogeneity of sorbitol dehydrogenase from the cytoplasm of mammalian brain cells

The multiple molecular forms of sorbitoldehydrogenase in cytoplasm of brain cells of bull, ground squirrel, guinea-pig, rat, hamster and mouse have been found using the method of electrophoresis in polyacrylamide gel and the subsequent specific dyeing for the fermentative activity. All revealed zones of activity are related to the slowly migrating ones. A set of multiple molecular forms from different sources is various. A form with relative electrophoretic activity 0.385 is found in all analyzed animals. The conditions for obtaining of distinct zones of activity on zymograms are chosen.     

The relationship of molecular weight to electrophoretic mobility of fluorescamine-labeled proteins in polyacrylamide gels

Proteins of known molecular weights were labeled with fluorescamine and then subjected to electrophoresis through polyacrylamide gels. The electrophoretic mobilities of the fluorescamine-labeled proteins were dependent upon their respective molecular weights over a range of 17,000 to 70,000 daltons. The correlation of electrophoretic mobility of fluorescamine-labeled protein to molecular weight was similar to results obtained in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The speed with which data can be obtained with the described procedure is a definite advantage over currently employed procedures. These findings encourage the use of fluorescamine for rapid, sensitive determinations of molecular weights of proteins in polyacrylamide gels.     

Identification of L Forms by Polyacrylamide-Gel Electrophoresis

Crude membranes were obtained from L forms by allowing the cells to lyse in distilled water. The crude membranes were washed several times in distilled water, lyophilized, and extracted with phenol-acetic acid-water. The membrane proteins were separated electrophoretically in polyacrylamide gels run at pH 4.5. Electrophoretic patterns and densitometric tracings of the gels showed distinct, reproducible intergeneric differences among L forms of Proteus, Streptobacillus, Staphylococcus, and Streptococcus. Differences within a genus could not be detected except between the group A streptococcal L forms and the one group D F-24 L form. This electrophoretic method offers possibilities for ready identification of L forms through the use of standard reference strains.     

A highly sensitive technique for staining DNA and RNA in polyacrylamide gels using silver

A technique is described for staining DNA in polyacrylamide gels with silver. It is rapid, requiring about 30 min for whole staining and development procedure, very simple and at least 20 times more sensitive than ethidium bromide for the staining of double-stranded DNA in polyacrylamide gels. This technique can also be applied for the staining of denatured, single-stranded DNA as well as RNA after their electrophoretic separation on polyacrylamide gels, having the same sensitivity as for double-stranded DNA fragments.     

Multiple forms of bacterial hydrogenases

Ackrell, B. A. C. (University of Hawaii, Honolulu), R. N. Asato, and H. F. Mower. Multiple forms of bacterial hydrogenases. J. Bacteriol. 92:828-838. 1966.-Extracts of certain bacterial species have been shown by disc electrophoresis on polyacrylamide gel to contain multiple hydrogenase systems. The hydrogenase enzymes comprising these systems have different electrophoretic mobilities and produce a band pattern that is unique for each bacterial species. Of 20 bacterial species known to possess hydrogenase activity and which were examined by this technique, only the activities of Clostridium tetanomorphum and C. thermosaccharolyticum could be attributed, at pH 8.3, to a single hydrogenase enzyme. This multiplicity of hydrogenase forms was found both in bacteria which contain mostly soluble hydrogenases and in those where the hydrogenase is predominantly associated with particulate material. When solubilization of this particulate material could be effected, at least two solubilized hydrogenases were released, and, of these, one would have the same electrophoretic properties (i.e., R(F)) as one of the soluble hydrogenases already present in small amounts within the cell. Different growth conditions for various types of bacteria, such as the nitrogen source, the degree of aeration, and photosynthetic versus aerobic growth in the dark, as well as the conditions under which the cells were stored, markedly affected the hydrogenase activity of the cells, but not their hydrogenase band pattern. The disc electrophoresis technique proved to be 10 times more sensitive than the manometric technique in detecting hydrogenase activity.