Cetyltrimethylammonium bromide polyacrylamide gel electrophoresis: estimation of protein subunit molecular weights using cationic detergents.

Polyacrylamide gel electrophoresis in the presence of the cationic detergent, cetyltrimethylammonium bromide (CTAB), has been previously used to obtain more accurate estimates of the molecular weight of certain highly charged and membrane protein subunits that exhibit anomalous electrophoretic behavior in the presence of sodium dodecyl sulfate (SDS). The improved method reported herein is comparable to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) method in simplicity, time, and quality of gels, but the CTAB-PAGE method appears to have a wider range of application for diverse types of proteins. The technique may also be used for verification of molecular weight data and thus detection of possible anomalous results obtained using the anionic SDS-PAGE method. The described method eliminates the precipitates formed between ammonium persulfate and cationic detergents during gel polymerization and between cationic detergents and the protein dyes during staining that have complicated previous methods. The reliability of the technique is indicated by the high correlation coefficient (?0.97) between R_f and molecular weight. Data are presented to indicate that the method can be used to estimate the subunit molecular weight of unknown proteins with a 95% level of confidence.     

Lymphocyte receptors for polyclonal T mitogens: II. High-molecular-weight glycoproteins are the best candidate sites for mitogenic action by concanavalin A and galactose oxidase

Murine lymphocytes oxidized by galactose oxidase were radiolabeled by reduction with NaB³H₄. The labeled cells were incubated with Con A and the Con A-Con A receptor complexes formed in situ on the viable cells were isolated by immuno-precipitation with anti-Con A serum and fixed Staphylococcus aureus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography analysis of the precipitates demonstrated four high-molecular-weight glycoproteins which were oxidized by GO and which bound Con A. These same four glycoproteins were also oxidized and labeled by $\text{IO}{}_4\text{NaB}{}^3\text{H}{}_4$. [³H]Tyrosine biosynthetic labeling identified these four plus several other Con A receptors. Because Con A sterically inhibits GO mitogenic stimulation, these four glycoproteins are likely to represent the necessary sites of oxidative mitogenic action and are good candidates for the targets of Con A mitogenesis.     

Purification and Properties of Clostridium botulinum Type F Toxin

Clostridium botulinum type F toxin of proteolytic Langeland strain was purified. Toxin in whole cultures was precipitated with (NH₄)₂SO₄. Extract of the precipitate was successively chromatographed on diethylaminoethyl-cellulose at pH 6.0, O-(carboxymethyl) cellulose at pH 4.9, Sephadex G-200 at pH 8.1, quaternary aminoethyl-Sephadex at pH 4.9, and finally diethylaminoethyl-cellulose at pH 8.1. The procedure recovered 14% of the toxin assayed in the starting culture. The toxin was homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, double gel diffusion serology, and isoelectric focusing. Purified toxin had a molecular weight of 150,000 by gel filtration and 155,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Specific toxicity was 9.6 × 10⁶ mean lethal doses per absorbancy (278 nm) unit. Sub-units of 105,000 and 56,000 molecular weight are found when purified toxin is treated with a disulfide reducing agent and electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. Reciprocal cross neutralizations were demonstrated when purified type F and E toxins were reacted with antitoxins which were obtained with immunizing toxoids prepared with purified toxins.     

Purification and properties of Acid phosphatase-1 from a nematode resistant tomato cultivar

In tomato the acid phosphatase-1 isozyme (Apase-1) is inherited as a single locus linked to the nematode resistance gene (Mi). The Apase-1¹ electrophoretic variant has been purified from a tomato cell suspension culture using ion exchange and concanavalin A sepharose affinity chromatography. A cellulose acetate electrophoresis method was used to distinguish Apase-1¹ rapidly from other Apase isozymes in tomato. The subunit molecular weight of the purified enzyme was estimated to be 31,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native size of the enzyme, which is reported to be a dimer, was determined to be approximately 51,000 by high performance liquid chromatography gel filtration. Apase-1¹ has a lower pH optimum and a distinct substrate specificity as compared to Apases extracted from tomato fruit or from other plant species. The amino acid composition of Apase-1¹ is similar to that of a potato Apase.     

Resolution of the phosphorylated and dephosphorylated cAMP-binding proteins of bovine cardiac muscle by affinity labeling and two-dimensional electrophoresis.

The photoaffinity label 8-azido[32P]adenosine 3':5'-monophosphate (8-azido-cyclic [32P]AMP) was used to analyze both the cAMP-binding component of the purified cAMP-dependent protein kinase, and the cAMP-binding proteins present in crude tissue extracts of bovine cardiac muscle. 8-Azido-cyclic [32P]AMP reacted specifically and in stoichiometric amounts with the cAMP-binding proteins of bovine cardiac muscle. Upon phosphorylation, the purified cAMP-binding protein from bovine cardiac muscle changed its electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels from an apparent molecular weight of 54,000 to an apparent molecular weight of 56,000. In tissue extracts of bovine cardiac muscle, most of the 8-azido-cyclic [32P]AMP was incorporated into a protein band with an apparent molecular weight of 56,000 which shifted to 54,000 upon treatment with a phosphoprotein phosphatase. Thus a substantial amount of the cAMP-binding protein appeared to be in the phosphorylated form. Autoradiograms following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both the pure and impure cAMP-binding proteins labeled with 8-azido-cyclic [32P]AMP revealed another binding component with a molecular weight of 52,000 which incorporated 32P from [gamma-32P]ATP without changing its electrophoretic mobility. Limited proteolysis of the 56,000- and 52,000-dalton proteins labeled with 32P from either [gamma-32P]ATP.Mg2+ or 8-azido-cyclic [32P]AMP showed patterns indicating homology. On the other hand, peptide maps of the major 8-azido-cyclic [32P]AMP-labeled proteins from tissue extracts of bovine cardiac muscle (Mr = 56,000) and rabbit skeletal muscle (Mr = 48,000) displayed completely different patterns as expected for the cAMP-binding components of types II and I protein kinases. Both phospho- and dephospho-cAMP-binding components from the purified bovine cardiac muscle protein kinase were also resolved by isoelectric focusing on polyacrylamide slab gels containing 8 M urea. The phosphorylated forms labeled with 32P from either [gamma-32P]ATP or 8-azido-cyclic [32P]AMP migrated as a doublet with a pI of 5.35. The 8-azido-cyclic [32P]AMP-labeled dephosphorylated form also migrated as a doublet with a pI of 5.40. The phosphorylated and dephosphorylated cAMP-binding proteins migrated with molecular weights of 56,000 and 54,000, respectively, following a second dimension electrophoresis in sodium dodecyl sulfate. The lower molecular weight cAMP-binding component (Mr = 52,000) was also apparent in these gels. Similar experiments with the cAMP-binding proteins present in tissue extracts of bovine cardiac muscle indicate that they are predominantly in the phosphorylated form.     

Studies on the characterization of human erythrocyte acetylcholinesterase and its interaction with antibodies

Human erythrocyte membranes were solubilized in 5% Triton X-100 and the acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was isolated by affinity chromatography utilizing a specific inhibitor, $\text{trimethyl}\text{-p-}\text{aminophenyl}$ ammonium chloride, bound to Sepharose 4B. After a repeated chromatography acetylcholinesterase was found to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunization of rabbits with acetylcholinesterase elicited the formation of an antiserum which gave single precipitin lines with the enzyme on immunodiffusion and rocket, crossed and immuno-electrophoreses. The purified enzyme had a specific activity of 418 units/mg protein. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value of acetylcholinesterase with acetylthiocholine as substrate was $\text{1.5 × 10}{}^{\mathrm{?4}}\text{M}$. Isoelectric focusing of acetylcholinesterase in the presence of Triton X-100 and within the pH ranges of 3–10 and 3–6 exhibited a single peak of enzyme activity with a $\text{P}\text{I}$ of 4.8. The results of amino acid and carbohydrate analyses showed that acetylcholinesterase is a glycoprotein with a carbohydrate/protein weight ratio of 0.16 and glucose, galactose, mannose, glucosamine, galactosamine and sialic acid as the sugar components. The N-terminal amino acid was blocked. Lipid, phosphorus and fatty acid analyses indicated phosphatidylserine and cholesterol as the major lipid components of acetylcholinesterase. The apparent subunit molecular weight estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol was 160 000 and in its presence, 80 000. The kinetic studies showed a competitive inhibition of acetylcholinesterase by its antibodies. Agglutination of human red cells by monospecific antiserum to acetylcholinesterase confirmed that the antigenic site(s) of the enzyme is localized on the outer surface of the erythrocyte membrane.     

Lymphocyte receptors for polyclonal T mitogens: I. Concanavalin A binding to lymphocytes inhibits mitogenesis induced by galactose oxidase

Mitogenic stimulation of mouse lymphocytes by the enzyme galactose oxidase (GO) is inhibited if Con A is present during the enzymatic oxidation. The mechanism of this inhibition appears to involve steric hindrance of GO action at cell surface sites which bind Con A because (a) similar pulse exposure of unoxidized cells to Con A does not affect their subsequent ability to respond to GO stimulation; (b) Con A binding to fetuin interferes with GO oxidation of that glycoprotein substrate; and (c) sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cells labeled by $\text{GO}\text{NaB}{}^3\text{H}{}_4$ in the presence of Con A shows a selective inhibition of labeling of some high-molecular-weight glycoproteins compared to controls labeled in the absence of Con A.     

Detection of iron-sulfur center-containing subunits of mitochondrial NADH dehydrogenase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by high-performance gel permeation chromatography

Soluble NADH dehydrogenase resolved from Complex I of the mitochondrial electron-transfer chain was subjected to gel electrophoresis in the presence of sodium dodecyl sulfate at 4 degrees C, and then the gel was stained for iron with bathophenanthroline disulfonate and thioglycolic acid. The 23,000-dalton subunit was markedly stained, and the 51,000-dalton subunit was also stained, but only slightly. High-performance gel permeation chromatography using an eluant containing 0.1% sodium dodecyl sulfate also demonstrated that these subunits contain an iron-sulfur center: the elution pattern recorded by light absorption at 400 nm gave two peaks corresponding to the positions of the subunits.     

Isolation and biochemical characterization of ten-nanometer filaments from cultured ovarian granulosa cells

Ten-nm filaments have been isolated from control and colchicine-treated primary cultures of rat ovarian granulosa cells. Negative stain electron microscopy indicates an average filament diameter of 10.3 nm in the isolated fiber bundles, which, upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, are found to contain a major polypeptide with a molecular weight of 57,000 and several minor components including actin. One-dimensional peptide mapping and two-dimensional gel electrophoresis demonstrate similarity between the granulosa cell and baby hamster kidney cell 10-nm filament subunit protein, both of which are distinguishable from keratin, desmin, actin, and tubulin. Quantitative gel densitometry experiments demonstrate little difference in the total amount of the 10-nm filament protein in control cells or cells treated with colchicine, accounting for 12 or 15% of the total cellular protein, respectively. The purification procedure, which involves extraction in Triton X-100 and KCl followed by DNase I treatment, yields 709% of the total granulosa cell intermediate filament protein, and 70% of the newly synthesized 57,000 molecular weight component. Two-dimensional gel electrophoresis of cultures labeled with [32P]phosphate show by autoradiography that the 57,000-dalton polypeptide, actin, and a 130,000-dalton protein are the most readily phosphorylated polypeptides in granulosa cell cultures. These studies identify the major intermediate filament subunit protein of granulosa cells as a 57,000-dalton phosphorylatable polypeptide which comprises a substantial portion of the granulosa cell cytoskeleton.     

DNA-binding proteins of Xenopus laevis: synthesis during oogenesis-ovulation and early embryogenesis

DNA-binding proteins of Xenopus laevis synthesized during two periods of early development (oogenesis-ovulation and early embryogenesis) were co-chromatographed on DNA-cellulose. Proteins with an affinity for DNA were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Most of the proteins eluted from DNA-cellulose with 0.6 M NaCl had mol. wts less than 40 000; some of these proteins were synthesized to a greater extent by developing embryos than by oocytes. A DNA-binding protein or group of proteins with a mol. wt of approx. 70 000 was synthesized during oogenesis-ovulation but not during embryogenesis. Differential labeling of developing embryos with $\text{[}{}^3\text{H}\text{]}\text{tryptophan}$ and $\text{[}{}^{14}\text{C}\text{]}\text{lysine}$ indicated that some of the low mol. wt DNA-binding proteins are histones. Some of these proteins also incorporated monosodium $\text{[}{}^{32}\text{P}\text{]}\text{phosphate}$. A greater fraction of the proteins synthesized by oocytes and developing embryos were bound to DNA-histone-cellulose than to DNA-cellulose. A group of low mol. wt proteins made during oogenesis-ovulation were bound more to DNA-histone-cellulose than were proteins with similar mol. wts made by developing embryos.     

Isolation of bovine cytochrome c1 as a single non-denatured subunit using gel filtration or high pressure liquid chromatography in deoxycholate

The non-denatured cytochrome $\text{c}{}_1$ subunit of bovine ubiquinone-cytochrome $\text{c}$ reductase was isolated using either gel filtration or high pressure liquid chromatography in 1% deoxycholate. The preparation was a single band on polyacrylamide gel electrophoresis in dodecyl sulfate, had a heme content of 31 nmol heme/mg protein, had an absorbance ratio $\text{A}{}_{417}\text{A}{}_{278}\text{= 2.65}$, a visible spectrum with maxima at 553, 530, 523.5, 417, 317, and 277 nm for the reduced protein, and an amino acid analysis identical to that previously reported for the isolated denatured protein. The Stokes' radius of this non-denatured deoxycholate solubilized protein was 34Å, indicating that the protein either is a dimer in deoxycholate, is asymmetric, or binds large amounts of detergent.     

Studies on (K+ + H+)-ATPase. V. Chemical composition and molecular weight of the catalytic subunit

(1) A $\text{(K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}\text{)-ATPase}$ preparation from porcine gastric mucosa is solubilized in sodium dodecyl sulfate, and is subjected to gel filtration. (2) A main subunit fraction is obtained, which is a protein carbohydrate lipid complex, containing 88% protein, 7% carbohydrate and 5% phospholipid. The detailed composition of the protein and carbohydrate moieties are reported. (3) Sedimentation analysis of the subunit preparation, after detergent removal, reveals no heterogeneity, but the subunits readily undergo aggregation. (4) Acylation of the subunit preparation with citraconic anhydride causes a clear shift of the band obtained after SDS gel electrophoresis, but the absence of broadening and splitting of the band pleads against subunit heterogeneity. (5) Treatment of the subunit preparation with dansyl chloride indicates that the NH₂ terminus is blocked, which favors the assumption of homogeneity of the protein. (6) Binding studies with concanavalin A indicate that at least 86% of the subunit preparation is composed of glycoprotein. (7) These findings, taken together, strongly suggest that there is a single subunit which is a glycoprotein and which represents the catalytic subunit of the enzyme. From sedimentation equilibrium analysis a molecular mass value of 119 kDa (S.E. 3, $\text{n = 6}$) is calculated for protein + carbohydrate and of 110 kDa (S.E. 3, $\text{n = 6}$) for protein only. (8) In combination with the molecular mass of 444 kDa (S.E. 10, $\text{n = 4}$) obtained for the intact enzyme by radiation inactivation we conclude that the enzyme appears to be composed of a homo-tetramer of catalytic subunits.     

Immunoreactivity of subunits of the (Na+ + K+)-ATPase Cross-reactivity of the α,α+ and β forms in different organs and species

The immunologic cross-reactivity of the α and α+ forms of the large subunit and the β subunit of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from brain and kidney preparations was examined using rabbit antiserum prepared against the purified holo lamb kidney enzyme. As previously reported by Sweadner ((1979) J. Biol. Chem. 254, 6060–6067) phosphorylation of the large subunit of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in the presence of Na⁺, Mg²⁺, and [$\text{γ-}{}^{32}\text{P}\text{]}\text{ATP}$ revealed that dog and, very likely, rat brain contain two forms of the large subunit (designated α and α+) while dog, rat, and lamb kidney contain only one form (α). The cross-reactivity of the α and α+ forms in these preparations was investigated by resolving the subunits by SDS-polyacrylamide gel electrophoresis. The separated polypeptides were transferred to unmodified nitrocellulose paper, and reacted with rabbit anti-lamb kidney serum, followed by detection of the antigen-antibody complex with ¹²⁵I-labeled protein A and autoradiography. By this method, the α and α+ forms of rat and dog brain, as well as the α form found in kidney, were shown to cross-react. In addition, membranes from human cerebral cortex were shown to contain two immunoreactive bands corresponding to the α and α+ forms of dog brain. In contrast, the brain of the insect Manduca sexta contains only one immunoreactive polypeptide with a molecular weight intermediate to the α and α+ forms of dog brain. The β subunit from lamb, dog and rat kidney and from dog and rat brain cross-reacts with anti-lamb kidney ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ serum. The mobility of the β subunit from dog and rat brain on SDS-polyacrylamide electrophoresis gels is greater than the mobility of the β subunit from lamb, rat or dog kidney.     

Comparison of plasma membranes and endoplasmic reticulum fractions obtained from whole white adipose tissue and isolated adipocytes

The influence of the mode of preparation upon some of the characteristics of white adipose tissue plasma membranes and microsomes has been reported. Plasma membrane fractions prepared from mitochondrial pellet were shown to have higher specific activities of $\text{(Mg}{}^{\mathrm{2+}}\text{+ Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ than plasma membranes originating in crude microsomes. Isolation of fat cells by collagenase treatment was found to result in a decrease in specific activity of the plasma membrane enzymes; in plasma membranes prepared from isolated fat cells, the specific activity values obtained for $\text{(Mg}{}^{\mathrm{2+}}\text{+ Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ and 5′-nucleotidase were only 42% and 6.3% respectively of those obtained in plasma membranes prepared from whole adipose tissue. Purification of whole adipose tissue crude microsomes by hypotonic treatment caused extensive solubilization of the endoplasmic reticulum marker enzymes, NADH oxidase and NADPH cytochrome $\text{c}$ reductase. The lability of endoplasmic reticulum marker enzymes, however, was found to be greatly diminished in the preparations from isolated fat cells. The possibility that NADH oxidase and NADHPH cytochrome $\text{c}$ reductase activities found in the plasma membranes are microsomal enzymes adsorbed by the plasma membranes is discussed. The peptide patterns as well as the NADH oxidase and NADPH cytochrome $\text{c}$ reductase activity patterns of plasma membranes and purified microsomes were compared by means of sodium dodecyl sulfate or Triton X-100 polyacrylamide gel electrophoresis.     

The product of the his4 gene cluster in Saccharomyces cerevisiae. A trifunctional polypeptide.

The his4 region of yeast encodes the information for the third (phosphoribosyl-AMP cyclohydrolase), second (phosphoribosyl-ATP pyrophosphohydrolase), and tenth (histidinol dehydrogenase) steps in the histidine biosynthetic pathway. These three activities co-purify with a single protein which has a subunit molecular weight of 95,000 (95,000 protein), as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Extracts of yeast strains which carry nonsense or deletion mutations in various portions of the his4 region, purified in parallel by affinity chromatography on AMP-agarose columns, were examined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis slabs. All such mutant extracts examined were found to lack the 95,000 protein found in a strain carrying a wild type his4 allele. The presence of a protease inhibitor, phenylmethylsulfonyl fluoride, during the purification of the trifunctional enzyme prevented the degradation of the 95,000 protein to polypeptides of lower molecular weight. Monospecific antibody prepared against the 95,000 protein removed all three of the activities specified by his4 from solution; active 95,000 protein was recovered in the resuspended immunoprecipitates. All this evidence shows that the product of the his4 region is a trifunctional, 95,000-dalton protein. Preliminary evidence from two-dimensional gel electrophoresis, NH2-terminal analysis, and gel filtration column chromatography indicates that the native trifunctional enzyme is a dimer of identical 95,000-dalton subunits.     

Alterations in plasma membrane proteins associated with the proteolytic activation of adenylate cyclase

Treatment of intact normal rat kidney fibroblasts, or of purified NRK plasma membranes, with trypsin or papain markedly enhances adenylate cyclase activity [ATP pyrophosphatelyase (cyclizing) EC 4.6.1.1]. Limited proteolysis (25 μg/ml trypsin for 7 min) of confluent cells grown with unheated calf serum significantly increases cyclase activity, whereas similar treatment of sparse cells causes only a marginal increase in cyclic AMP formation. To determine which membrane protein(s) is altered under conditions which result in proteolytic activation of adenylate cyclase, purified plasma membranes and intact normal rat kidney cells were subjected to limited proteolysis and membrane proteins analyzed by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. Membranes prepared from intact confluent normal rat kidney cells exposed to mild trypsinization showed a decrease in proteins of 56,000, 46,000, 37,000, and 32,000 daltons. Trypsin treatment of intact, sparse cells does not activate the cyclase system and does not lead to modification of the 46,000-dalton membrane protein. Treatment of purified normal rat kidney plasma membranes results in the loss of numerous bands in the high molecular mass region (>150,000 daltons) as well as decreases membrane proteins of 56,000, 49,000, 46,000, and 23,000 daltons. Compared with trypsin, the proteolytic action of papain appears to be quite specific, causing a discernible decrease in only the 46,000-dalton protein. The correlation between modification of the 46,000-dalton membrane component and the activation of the cyclase system suggests that perhaps this protein is proteolytically modified to elicit activation of adenylate cyclase.     

Abnormal neutrophil myeloperoxidase from a patient with chronic myelocytic leukemia

Neutrophil myeloperoxidase from a patient with chronic myelocytic leukemia was isolated under conditions designed to minimize proteolysis. Those methods yielded an α₂β₂ form of myeloperoxidase from normal individuals. Purified enzyme from the patient had electronic absorbances ($\text{A}{}_{430}\text{A}{}_{280}\text{= 0.85}$), enzymatic activity, and electrophoretic and Chromatographic behavior indistinguishable from that of normal myeloperoxidase. Edman degradation and physical studies after reduction and denaturation, however, showed that as compared to normal enzyme, one 55,000-dalton α subunit of the patient's myeloperoxidase was replaced by a 39,000-dalton peptide with a different amino-terminal sequence, a mixture of smaller peptides, and an heme derivative. Myeloperoxidase from the leukemic neutrophils appeared to have been partially degraded in vivo by lysosomal proteases.     

The hydrophobic nature of the pig intrinsic factor receptor in the intestine

The pig intestinal intrinsic factor receptor has been isolated and dissociated into its α and β subunits. The β subunit was found to be more hydrophobic than the α subunit. In a detergent solution only the α subunit was accessible to digestion with papain. The whole isolated receptor was introduced into artificial single bilayer liposomes where is apparently was randomly oriented. Liposomes containing the receptor were digested with papain and the polypeptide segments that stayed in the lipid fraction were extracted and analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Four species were found with $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ values of 23 000, 45 000, 70 000 and 86 000.     

A simple logarithmic amplifier for the photographic quantitation of DNA in gel electrophoresis

For the quantitative determination of nonradioactive DNA fragments by gel electrophoresis, it is usually necessary to photograph the gel after staining with ethidium bromide and evaluate the negative by densitometry. It has previously been shown that, because of the logarithmic nature of the photographic process, it is not the optical density (E) of the film which is proportional to the amount of DNA in the gel but instead the value $\text{10}{}^{\mathrm{<mtext>E</mtext><mtext>\gamma </mtext>}}$, γ being a film constant. We describe the design of a simple instrument that converts E into $\text{10}{}^{\mathrm{<mtext>E</mtext><mtext>\gamma </mtext>}}$. The instrument can be built in any electronic workshop at low cost. When it is used together with a standard recording densitometer, densitometric tracings of $\text{10}{}^{\mathrm{<mtext>E</mtext><mtext>\gamma </mtext>}}$ are obtained directly. These tracings can be quantitated by simple peak area measurements, thereby circumventing complicated mathematical transformations. Quantitative analyses of a linear and an exponential densitogram of restriction nuclease digested plasmid DNA are presented to demonstrate the usefulness of the instrument.     

Isolation and characterization of isocitrate lyase of castor endosperm

Isocitrate lyase (threo-D_S-isocitrate glyoxylate-lyase, EC 4.1.3.1) has been purified to homogeneity from castor endosperm. The enzyme is a tetrameric protein (molecular weight about 140,000; gel filtration) made up of apparently identical monomers (subunit molecular weight about 35,000; gel electrophoresis in the presence of sodium dodecyl sulfate). Thermal inactivation of purified enzyme at 40 and 45 °C shows a fast and a slow phase, each accounting for half of the intitial activity, consistent with the equation: , where A₀ and A_t are activities at time zero at t, and k₁ and k₂ are first-order rate constants for the fast and slow phases, respectively. The enzyme shows optimum activity at pH 7.2–7.3. Effect of [S]on enzyme activity at different pH values (6.0–7.5) suggests that the proton behaves formally as an “uncompetitive inhibitor.” A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pK_a equal to 6.9. Successive dialysis against EDTA and phosphate buffer, pH 7.0, at 0 °C gives an enzymatically inactive protein. This protein shows kinetics of thermal inactivation identical to the untreated (native) enzyme. Full activity is restored on adding Mg²⁺ (5.0 mm) to a solution of this protein. Addition of Ba²⁺ or Mn²⁺ brings about partial recovery. Other metal ions are not effective.