Dodecyl Sulfate-polyacrylamide Gel Research Articles

Multiple immunoblot: A sensitive technique to stain proteins and detect multiple antigens on a single two‐dimensional replica

A multiple immunoblotting technique was developed to positively identify up to three different antigens on a single nitrocellulose replica of a two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel. Three highly sensitive immunoblot assays were selected, including: horseradish peroxidase/luminescence, alkaline phosphatase, and silver-enhanced immunogold. As a major advantage, the method permits a simultaneous detection of up to three different antigens without eluting the antibody-dye complex between staining of single polypeptides, thus providing a highly accurate identification of closely migrating components. The staining procedure is summarized in a flow chart. In addition to the multiple immunoblot staining, some suggestions are provided for a sensitive protein staining.

Isolation and Preliminary Characterization of Immuno-inhibitory Factors from Dog Erythrocytes and Liver

Crude dog liver extract (DLE) inhibits proliferation of dog and human lymphocytes stimulated by phytohemagglutinin and alloantigens. While purifying this activity from dog liver, we observed that dog liver inhibitory factor (DLIF) shared properties with hemoglobin. DLIF migrated with hemoglobin during DEAE cellulose chromatography, and DLIF had oligomeric (61,900) and subunit (17,900 and 15,700) apparent molecular weights (AMW) similar to concurrently analyzed Sigma canine hemoglobin (61,100, 16,700 and 14,900). After separate cross linking, the proteins comigrated on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) gels. We therefore isolated dog erythrocyte inhibitory factor (DEIF) from red blood cells to determine if DEIF was a hemoglobin derivative. DEIF, like DLIF, separated at an isoelectric point of 5.6. DEIF also had similar subunit AMW (17,600 and 15,300) by SDS PAGE, but DEIF had much lower lymphocyte inhibitory activity (LIA = 2.27) than DLIF (LIA = 100.00). We conclude that DLIF and DEIF are similar to each other and hemoglobin, but further studies are needed to determine the function and exact structure of DLIF and DEIF.

A new major transmembrane glycoprotein, gp155, in goat erythrocytes. Isolation and characterization of its association to cytoskeleton through binding with band 3-ankyrin complex.

Erythrocyte membranes from goat contain a considerable amount, more than 10% of the total amount, of a glycoprotein with Mr = 155,000 (gp155) on sodium dodecyl sulfate-polyacrylamide electrophoresis gel. This report describes the first isolation and characterization of gp155. This gp155 has major trypsin-sensitive sites at each side of the plasma membrane to generate membrane-bound fragments, indicating that the gp155 spans the lipid bilayer several times. This protein consists of a single polypeptide containing about 1,200 amino acid residues corresponding to Mr = 134,000 and some complex type N-linked oligosaccharide chains. A fraction (15-20%) of the gp155 is recovered in nonionic detergent-extracted ghosts along with 25-30% of band 3 and other cytoskeletal proteins and is completely released into solution by extraction with 1 M KCl. Immunoprecipitation with anti-gp155 and anti-ankyrin antibodies of detergent-solubilized membranes separated on a gel permeation chromatography column showed that a part of the gp155 is tightly linked to band 3 with a molar ratio of 1:2 to 1:3. This gp155-band 3 complex in turn is associated to ankyrin through the binding of band 3 to ankyrin. These data indicate that, in native erythrocyte membranes, as well as in detergent solution, gp155 could play a physiological role in controlling cellular integrity and elasticity by forming the gp155-band 3-ankyrin complex. Partial amino acid sequences of the tryptic peptides are also determined.

Penicillin-binding proteins of protoplast and sporoplast membranes of Streptomyces griseus strains

Membrane-bound penicillin-binding proteins (PBPs) of two Streptomyces griseus strains that sporulate well in liquid and solid medium have been investigated during the course of their life-cycle. The PBP patterns were analyzed by sodium dodecylsulphate polyacrylamide-gel electrophoresis and fluorography. One strain (No. 45 H) has only a single band (mol wt: 27,000) in early log phase, and two additional PBPs of higher mol wt (69,000 and 80,000) in the late log phase. The other strain (No. 2682) possessed two bands with mol wts 27,000 and 38,000 which did not change during its vegetative phase. In strain No. 2682, a new PBP with a mol wt of 58,000 appeared in spore membranes while one of those (mol wt 38,000) present in mycelial membranes disappeared. Our results suggest that appearance of the new PBP in the spore may be associated with the sporulation process. The major PBP band (mol wt: 27,000) present in all stages of the life cycle of these strains, may be characteristic of S. griseus while the other PBPs reflect certain stages of the life cycle. A new method was developed for the production of spore protoplasts by consecutive enzymatic treatments.

Boar sperm surface glycoproteins: isolation, localization, and temporal expression during spermatogenesis.

Boar sperm glycoprotein fractions were isolated by Lens culinaris hemagglutinin affinity chromatography of detergent-solubilized ejaculated spermatozoa, followed by preparative sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. In order to develop methods for further investigations of the sperm proteins, we proceeded with two of the isolated glycoproteins. Antibodies were raised in female rabbits against each of the two sperm glycoproteins. By a combination of immunosorbent chromatography, using the antibodies obtained, and preparative SDS polyacrylamide gel electrophoresis, highly purified sperm proteins were isolated. The sperm proteins were immobilized on Sepharose gel columns and specific immunoglobulin Fab fragments were enriched by affinity chromatography. The specificity of the Fab fragments was ascertained by immunoprecipitation analysis. The Fab fragments were used in indirect immunofluorescence analysis to localize the corresponding antigens on the surface of boar spermatozoa. Both antigens were exclusively confined to the postacrosomal region. Immunohistochemical staining of boar testis sections revealed that both antigens are expressed from the spermatid stage. This technique also revealed that one of the antigens congregated at the Golgi complex-acrosome region during spermatogenesis.

Differential labeling of rat hepatic Golgi and serum very low density lipoprotein apoprotein B variants.

The synthesis of apoB-100 and apoB-48 by rat liver was investigated by studying the apoB complement of very low density lipoproteins (VLDL) from hepatic perfusates and Golgi fractions. The relative amounts of apoB-100 and apoB-48 in perfusate and Golgi VLDL as determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis were similar to those in serum VLDL. To investigate the relative rates of synthesis of the VLDL B proteins, rats were injected intraportally with tritiated amino acid, and hepatic Golgi and serum VLDL were isolated from 7.5 to 120 min later. In hepatic Golgi VLDL, apoB-100 and apoE were maximally labeled at 15 min after the tritiated amino acid pulse. In contrast, VLDL apoB-48 attained maximum radioactivity at 30 min after isotope injection. In serum VLDL, apoB-100 and apoE were maximally labeled at 30 min post-isotope injection, while activity in apoB-48 peaked at 60 min. The data suggest that the synthesis of the B proteins and incorporation into rat liver nascent VLDL are independently regulated. The differential labeling patterns of the VLDL B proteins may be explained by an intracellular pool of apoB-48 that is larger than that of apoB-100. An alternative explanation of the results is that apoB-100 is a precursor to apoB-48.

Cloning and sequencing of complementary DNAs encoding the alpha-subunit of translational initiation factor eIF-2. Characterization of the protein and its messenger RNA.

A clone encoding the alpha-subunit of eukaryotic initiation factor 2 (eIF-2 alpha) was isolated from a lambda gt11 expression library of rat brain cDNAs. The fusion protein expressed by the recombinant phage reacts with eIF-2 alpha antiserum except when the serum is preadsorbed with pure eIF-2. The translation of hybrid-selected HeLa cell mRNA produces two proteins which are indistinguishable from authentic HeLa eIF-2 alpha and its phosphorylated form when analyzed by electrophoresis in two-dimensional isoelectrofocusing/sodium dodecyl sulfate-polyacrylamide gels and by partial protease digestion. HeLa cell eIF-2 alpha mRNA migrates as a single band of about 1600 nucleotides. The rat cDNA insert was sequenced, and the region coding for eIF-2 alpha was identified. A human cDNA clone was obtained by hybridization screening with the rat cDNA, and its sequence was determined also. Both rat and human eIF-2 alpha proteins comprise 315 amino acids (36.1 kDa) and differ by only three amino acids. The eIF-2 alpha mRNA is found exclusively in polysomes containing 10 or more ribosomes in exponentially growing HeLa cells. In serum-depleted cells which synthesize eIF-2 and bulk protein more slowly than exponential cells, the level of eIF-2 alpha mRNA is not changed, the average polysome size is reduced to 7, and little or no eIF-2 alpha mRNA is detected in the ribonucleoprotein fraction. These results are consistent with the view that eIF-2 alpha mRNA translation is very efficient compared to other mRNAs in the cell.

Similar control mechanisms regulate the insulin and type I insulin-like growth factor receptor kinases. Affinity-purified insulin-like growth factor I receptor kinase is activated by tyrosine phosphorylation of its beta subunit.

Insulin-like growth factor I (IGF-I) receptors are partially purified from human placenta by sequential affinity chromatography with wheat germ agglutinin-agarose and agarose derivatized with an IGF-I analog. Adsorption specificity to this affinity matrix demonstrates that low coupling ratios of IGF-I analog to agarose yield preparations that are highly selective in purifying IGF-I receptor with minimal cross-contamination by the insulin receptor present in the same placental extracts. Incubation of the immobilized IGF-I receptor preparation with [gamma-32P]ATP results in a marked phosphorylation of the receptor beta subunits, which appear as a doublet of Mr = 93,000 and 95,000 upon electrophoresis on dodecyl sulfate-polyacrylamide gels. The 32P-labeled receptor beta subunit doublet contains predominantly phosphotyrosine and to a much lesser extent phosphoserine and phosphothreonine residues. The immobilized IGF-I receptor preparation exhibits tyrosine kinase activity toward exogenous histone. The characteristics of the IGF-I receptor-associated tyrosine kinase are remarkably similar to those of the insulin receptor kinase. Thus, prior phosphorylation of the immobilized IGF-I receptor preparation with increasing concentrations of unlabeled ATP followed by washing to remove the unreacted ATP results in a progressive activation of the receptor-associated histone kinase activity. A maximal (10-fold) activation is achieved between 0.25 and 1 mM ATP. The concentration of ATP required for half-maximal (30 microM) activation of the IGF-I receptor kinase is similar to that of the insulin receptor kinase. Like the insulin receptor kinase, the elevated kinase activity of the phosphorylated IGF-I receptor is reversed following dephosphorylation of the receptor beta subunit with alkaline phosphatase. Furthermore, the phosphorylation of the IGF-I receptor beta subunit doublet is enhanced by 7-8-fold when reductant is included in the reaction medium, as is observed for the insulin receptor kinase. Significantly, the dose responses of both receptor types to reductant are identical. Both of the 32P-labeled IGF-I receptor beta subunit bands are resolved into six matching phosphopeptide fractions when the corresponding tryptic hydrolysates are resolved by reverse phase high pressure liquid chromatography. Significantly, four out of the six phosphopeptide fractions derived from the trypsinized IGF-I receptor beta subunits are chromatographically identical to those from the tryptic hydrolysates of 32P-labeled insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation of D-myo-inositol 1:2-cyclic phosphate 2-inositolphosphohydrolase from human placenta.

We have isolated D-myo-inositol 1:2-cyclic phosphate 2-inositolphosphohydrolase (EC 3.1.4.36) from human placenta. This enzyme catalyzes the conversion of inositol 1:2-cyclic phosphate to inositol 1-phosphate. The enzyme was purified 1300-fold to apparent homogeneity from the soluble fraction of human placenta. The enzyme requires Mn2+ or Mg2+ ions for activity, has an apparent Km for inositol 1:2-cyclic phosphate of 0.15 mM and forms 2.2 mumol of inositol 1-phosphate/min/mg protein. The enzyme does not utilize the cyclic esters of inositol polyphosphates as substrates. The molecular weight determined by gel filtration chromatography is approximately 55,000. Upon electrophoresis in polyacrylamide gels in sodium dodecyl sulfate, the molecular weight was found to be 29,000 both in the presence and absence of beta-mercaptoethanol. The enzyme was inhibited by inositol 2-phosphate (IC50 = 4 microM) and to a lesser degree by inositol 1-phosphate (IC50 = 2 mM) and inositol (IC50 = 4 mM). Zn2+ is a potent inhibitor of enzyme activity (IC50 = 10 microM). Neither Li+ nor Ca2+ had any effect on enzyme activity. This enzyme may serve to generate inositol from inositol cyclic phosphate metabolites produced by the phosphoinositide signaling pathway in cells.

Characterization of antigens from extracts of fed ticks using sera from rabbits immunized with extracted tick antigen and by successive tick infestation

Immune resistance to infestation by an ixodid tick, Rhipicephalus appendiculatus, the vector of the African cattle disease, East Coast Fever, was induced in rabbits by either repeated tick feeding or immunization with tick extracts. In addition to resistance to tick infestation, tick extract immunization led to a reduction in the viability of eggs laid by ticks feeding on the immunized host. Resistance to infestation/by ixodid ticks has previously been reported by others to have a humoral immune component. Therefore, antibodies from resistant host animals were used to detect the tick antigens they recognized as an-approach to identification of the target antigen(s) for the observed immune responses on feeding ticks. In crossed immune electrophoresis two antigens were detected using sera from animals made resistant by multiple tick infestations. Sera from extract immunized animals detected these antigens and nine others. The tick antigens detected by both sets of sera in crossed immune electrophoresis were radiolabelled with [35S]amino acids. No labelled antigens were detected by Staphylococcus aureus mediated immune precipitation with sera from hosts made resistant by multiple infestations. Antibodies from extract-immunized animals identified nine protein antigens by S. aureus immunoprecipitation. The molecular weights of these antigens as assessed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis were 180,000; 140,000; 130,000; 98,000; 92,000; 88,000; 85,000 and 82,000. The rate of synthesis of these antigens appeared to vary in relation to the tick feeding cycle.

Electrophoretic analysis of membrane proteases in the luteinized rat ovary.

Membrane fractions from fully luteinized rat ovaries contained proteolytic enzymes that were solubilized and reversibly inhibited by the anionic detergent sodium dodecyl sulfate. Electrophoretic analysis in substrate-containing sodium dodecyl sulfate-polyacrylamide slab gels revealed the presence of numerous protease bands in the mol wt range of 26,000 to more than 200,000. When casein served as protease substrate in the gels, enzyme bands of Mr 26,000, 28,000, 30,000, and 90,000 were produced by crude (2,000 X g pellet) ovarian membranes. Gels containing casein and plasminogen also revealed the presence of two plasminogen activators of Mr 63,000-65,000 and 42,000. Subcellular fractionation of luteinized ovaries by centrifugation in sucrose density gradients indicated that the Mr 90,000 protease and the two plasminogen activators were uniformly distributed among microvillous membranes, basolateral membranes (BLM), and the mitochondrial-lysosomal fraction (MLF). The Mr 26,000, 28,000, and 30,000 proteases were enriched in BLM and MLF. Analysis of crude membranes in slab gels which contained gelatin as the protease substrate revealed the presence of two additional enzymes of Mr 52,000 and more than 200,000. The Mr greater than 200,000 protease was present in microvillous membranes, BLM, and MLF. The Mr 52,000 protease was found exclusively in BLM. This enzyme was not consistently demonstrable in crude membranes but could be generated upon incubation of membranes at 30 C. This finding indicated that Mr 52,000 protease can exist in an inactive and/or zymogen form. The Mr 90,000 protease was inhibited by tosyl-lysine chloromethyl ketone and dansyl-glutamyl-glycylarginine chloromethyl ketone. The gelatinase activity of the Mr 52,000 protease was blocked by tosyl lysine chloromethyl ketone, dansyl-glutamyl-glycylarginine chloromethyl ketone and tosylamide-2-phenyl chloromethyl ketone. The activity of the Mr 26,000, 28,000, and 30,000 proteases was not affected by any of the above mentioned inhibitors. These findings demonstrate that the proteolytic potential of ovarian membranes is not limited to plasminogen activators. Numerous plasminogen-independent proteases are also present, and these may play a role in ovulation, luteolysis, and mediation of hormonal stimulation.

Lateral nasal gland secretion in the anaesthetized dog.

The effects of pharmacological and nervous stimuli on the flow of secretion from the dog lateral nasal gland following catheterization are described. Drugs were injected close-arterially into the arterial supply to the nose, or intravenously. Cholinergic agonists (pilocarpine, methacholine), given intravenously (I.V.) or intra-arterially (I.A.), and stimulation of the vidian nerve produced a copious flow of secretion which was blocked by atropine. The adrenoceptor agonists phenylephrine (alpha) and salbutamol (beta 2), given I.V. or I.A., and stimulation of the vagosympathetic nerve produced a small but consistent flow of secretion. Histamine (50 micrograms), substance P (0.1 micrograms) and prostaglandin E1 (1-5 micrograms), injected I.A., produced small flows of secretion. Bradykinin (25 ng-50 micrograms), 5-hydroxytryptamine (100 ng-50 micrograms) and vasoactive intestinal peptide (VIP) (10 ng-50 micrograms) did not cause secretion. The total protein content, the composition of secretions as revealed by sodium dodecyl sulphate-polyacrylamide agarose gel electrophoresis, and changes in [Na] and [K] in relation to flow of secretion are described. Differences in ion and protein concentrations, and in protein composition, are described for vidian nerve-induced and vagosympathetically induced secretions. Electron microscopy revealed that the gland contains serous cells in the secretory region, and ducts morphologically similar to the intercalated, striated and excretory ducts of salivary glands.

Lipolysis-induced degradation of apolipoproteins B and E of human very low density lipoproteins.

We have found that in vitro lipolysis of human very low density lipoproteins (VLDL) by purified bovine milk lipoprotein lipase (LpL) promotes degradation of the apolipoprotein (apo) B moiety of VLDL. Analysis by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis showed that lipolysis of VLDL by purified LpL for 1 h at 37 degrees C induced the selective degradation of the high Mr apo-B (apo-B-100) from most hypertriglyceridemic VLDL and from a few normolipidemic VLDL into several small fragments with molecular weights ranging from 90,000-490,000. No detectable degradation of apo-B occurred in control VLDL when incubated without LpL. The apo-E moiety of VLDL from certain individuals was also degraded following lipolysis of VLDL, and the extent of degradation of apo-B and -E in VLDL was varied among the individual VLDL. The major degradation products of apo-E, identified from the gel, were 31,000- and/or 28,000-Da species. In contrast to the apo-E moiety of VLDL, purified apo-E was not degraded when incubated with LpL. Incubation of low density lipoproteins (LDL) with LpL showed only a minimal effect on the apoproteins of LDL. When high density lipoprotein (HDL) was included in the lipolysis mixture as an acceptor of lipolytic surface remnants, the apoproteins of HDL remained unaltered, while the apo-B moiety of VLDL remnants in the mixture was degraded. Inclusion of protease inhibitors in the lipolysis mixture prevented the degradation of apo-B, but the hydrolysis of VLDL-triglyceride was minimally affected. A selective degradation of apo-B in VLDL also occurred during lipolysis of VLDL when VLDL was perfused through rat hearts. These results suggest that conformational changes in apo-B and apo-E caused by VLDL lipolysis may increase the susceptibility of apo-B and apo-E to degradation by the proteases co-isolated with VLDL. The consequences of the lipolysis-induced degradation of apo-B and apo-E on changes in metabolic properties of VLDL remnants remain to be determined.

Induction of the primitive streak in the chick blastoderm embryo: patterns of protein synthesis.

Induction of the primitive streak is correlated with specific qualitative and quantitative changes in protein synthesis in the component areas of chick blastoderm. Blastoderm embryos at the initial to intermediate primitive streak stage were labeled with L-[35S] methionine. Radioactively labeled proteins separated by two-dimensional sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis revealed differences in the number and density of spots among the component areas of the epiblast and hypoblast. Protein patterns of the area opaca, marginal zone and central area of the epiblast are very similar qualitatively but show distinct quantitative differences. A comparison between any of the component areas of the epiblast and the hypoblast in chick blastoderm embryos, however, reveals both qualitative and quantitative differences. A protein with a molecular weight of 30,000 unique to the component areas of the epiblast, and proteins with a molecular weight of 22,000 and 37,000 unique to the hypoblast are prominent and seem to be related to the initial appearance of the primitive streak.

Electrophoresis and immunoblot of cerebrospinal fluid proteins in spasmodic torticollis.

Protein patterns of cerebrospinal fluid (CSF) from patients with spasmodic torticollis (ST) were investigated to determine whether abnormalities previously reported could be detected and further identified. CSF was collected from 12 patients with ST and 6 normal controls. The CSF proteins were analyzed using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and silver staining. In 11 of the 12 patients with ST, a CSF protein pattern was observed which differed from that in the controls. The identity of the abnormal proteins was ascertained by blotting and immunostaining with specific antisera to IgG and ceruloplasmin (Cp). CSF from 2 of 12 patients had distinct bands staining for IgG and 7 had abnormal immunostaining for Cp.

Properties of S-adenosylmethionine decarboxylase from rabbit liver: effects of some hydrazone compounds on its activity.

S-Adenosylmethionine decarboxylase (EC 4.1.1.50) has been partially purified from rabbit liver by ammonium sulphate fractionation and gel filtration and anion exchange chromatographies. Sodium dodecylsulphate-polyacrylamide disc gel electrophoresis analysis showed an approximate dimeric subunit mol. wt of 34,000. The enzyme showed a pH optimum at 7.5 (in phosphate buffer) and did not require bivalent cations for catalysis. The enzyme showed sigmoid kinetics to S-adenosylmethionine with a Hill coefficient of 1.7, which became michaelian with Km 70 microM in the presence of 2.5 mM putrescine. Methylglyoxal bis(guanylhydrazone) was an effective inhibitor of the enzyme, but phenylated derivatives of this compound as phenylglyoxal bis(guanylhydrazone) and diphenylglyoxal bis-(guanylhydrazone) inhibited less well.

The expression of a gene for eukaryotic elongation factor Tu in Artemia during development. Translation of poly(A)+ RNA and the use of a synthetic oligonucleotide to detect the presence of eukaryotic elongation factor Tu-specific mRNA.

Total in vivo proteins from Artemia embryos at different developmental stages were examined by two-dimensional gel electrophoresis. A variety of peptides change during development, with one of them, the eukaryotic elongation factor Tu (eEF-Tu), presenting a dramatic increase from dormant embryos to nauplii. When poly(A)+ RNA is translated in vitro, the same relative increase is seen for eEF-Tu during development. Based on the amino acid sequence for Artemia eEF-Tu (Amons, R., Pluijms, W., Roobol, K., and Möller, W. (1983) FEBS Lett. 153, 37-42), a synthetic oligodeoxynucleotide was prepared and used to prime the synthesis of cDNA with poly(A)+ RNA from 12-h developing embryos as template. Direct sequence analysis of the 900-base primary cDNA product shows it to be specific for the 5' end of Artemia eEF-Tu mRNA. Hybridization of a "Northern" blot of denatured (poly(A)+ RNA from different developmental stages with this cDNA reveals a major band migrating at about 1800 bases, which increase in intensity as development proceeds, paralleling the increase in eEF-Tu seen by in vitro translation. When poly(A)+ RNA is separated on a nondenaturing gel, blotted to poly(U) paper, and hybridized with the eEF-Tu cDNA, a single band is observed migrating faster than 18 S. Elution and in vitro translation of this band results in a major product migrating with eEF-Tu in a dodecyl sulfate-polyacrylamide gel and which is precipitable with eEF-Tu-specific antibodies.

Effect of Various Sodium Dodecyl Sulfate Sample Buffers on the Exospore and Spore Polypeptide Profiles of Nosema locustae (Microspora: Nosematidae)

Several studies using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) have produced conflicting results regarding exospore polypeptide profiles. Streett and Briggs (1982, Journal of Invertebrate Pathology 40:159165) did not detect exospore polypeptides in samples of intact spores treated with 1% SDS and 0.01 M dithiothreitol. In contrast, Irby (1983, Master's Thesis, North Carolina State University, 99 p.) observed unique exospore profiles for several species of microsporidia treated with 2% SDS and 5% beta-mercaptoethanol. Irby concluded from his investigation that Streett and Briggs (1982, loc. cit.) did not use a sufficient amount of reducing agent (dithiothreitol). Consequently, for this investigation, Nosema locustae Canning spore homogenates and intact spore suspensions were treated with sample buffers containing various concentrations of SDS and reducing agent. Nosema locustae was propagated in Melanoplus differentialis (Thomas) and isolated by differential centrifugation (Henry and Oma, 1981, Pest control by Nosema locustae, a pathogen of grasshoppers and crickets. In Microbial control of pests and plant diseases, H. D. Burges (ed.). Academic Press, New York, New York, pp. 573586). A spore suspension (1.2 x 109 spores) was homogenized and separated by electrophoresis as reported earlier (Streett and Briggs, 1982, loc. cit.). Both intact and homogenized spore pellets (each with 1.2 x 109 spores) were resuspended in 1.2 ml of 0. 1 M Tris buffer pH 7.2, and divided into 6 aliquots for centrifugation at 500 g for 5 min in microcentrifuge tubes. The supernatant was removed and each aliquot of intact and homogenized spores was treated with 60 ,l of sample buffer. The 6 sample buffers used to treat N. locustae intact and homogenized spore preparations were: 1% SDS with 0.01 M dithiothreitol; 2% SDS with 0.01 M dithiothreitol; 2% SDS with 0.02 M dithiothreitol; 4% SDS with 0.02 M dithiothreitol; 2% SDS with 5% beta-mercaptoethanol; 4% SDS with 10% beta-mercaptoethanol. N. locustae spore profiles were compared and evaluated visually. An exospore polypeptide profile was detected when intact spores were treated with each of the sample buffers except 1% SDS with 0.01 M dithiothreitol (Fig. 1A). When examined with phase contrast microscopy intact spores treated with 1% SDS and 0.01 M dithiothreitol were refractile and appeared normal. However, intact spores treated with the other sample buffers were not refractile, which indicates either spore germination or a lack of an exospore layer. The exospore polypeptide profile for treated intact spore preparations ranged from approximately 11,000 daltons to 86,000 daltons. This generally corroborates the results reported by Irby (1983, loc. cit.) for N. locustae spores treated with 2% SDS and 5% beta-mercaptoethanol. The spore polypeptide profiles of homogenized N. locustae treated with the various sample buffers are shown in Figure IB. N. locustae homogenized spores treated with SDS sample buffers using dithiothreitol as a reducing agent are nearly identical with the exception of 1% SDS with 0.01 M dithiothreitol. The 1% SDS and 0.01 M dithiothreitol sample buffer appears to be inadequate for dissociating either exospore or spore polypeptides. Spore polypeptide profiles for homogenized spores treated with SDS sample buffers containing dithiothreitol ranged from approximately 10,000 daltons to 200,000 daltons. No significant differences are detected in the profiles of N. locustae homogenized spores treated with 2% SDS and either 5% or 10% beta-mer-

Purification and properties of 3-ketovalidoxylamine A C-N lyase from Flavobacterium saccharophilum.

3-Ketovalidoxylamine A C-N lyase was purified about 900-fold from the cell-free extract of Flavobacterium saccharophilum by ammonium sulfate fractionation, column chromatography on CM cellulose and gel filtration on Sephacryl S-200. The purified enzyme was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 36,000 by gel filtration on Sephacryl S-200 and by SDS polyacrylamide gel electrophoresis, indicating that the enzyme is a monomer. The optimum pH was found at 9.0. The enzyme activity was inhibited by EDTA or ethyleneglycol bis(beta-aminoethylether)-N,N'-tetraacetic acid and the inhibition was reversed by Ca2+ ion. The enzyme was able to eliminate p-nitroaniline or p-nitrophenol from p-nitrophenyl-3-ketovalidamine (IV) or p-nitrophenyl-alpha-D-3-ketoglucoside (VI), but not from p-nitrophenyl-1-epi-3-ketovalidamine or p-nitrophenyl-beta-D-3-ketoglucoside. Apparent Km values for IV and VI were 0.24 mM and 0.5 mM, respectively.

Purification of the HhaII restriction endonuclease from an overproducer Escherichia coli clone.

An Escherichia coli K12 strain carrying the HhaII methylase and restriction genes on two separate compatible plasmids, pSK5 and pSK7, is used to overproduce the restriction endonuclease. Plasmid pSK5 expresses the methylase gene constitutively from its chloramphenicol resistance gene promoter, and plasmid pSK7 expresses the restriction endonuclease under control of the lacUV5 promoter. Induction of the two-plasmid clone with 1 mM isopropyl-1-thio-beta-D-galactopyranoside results in a 15-fold increase in HhaII endonuclease activity. The enzyme has been purified to apparent homogeneity. It migrates as a 23-kilodalton polypeptide on denaturing sodium dodecyl sulfate-polyacrylamide electrophoretic gels and as a 52-kilo-dalton native protein dimer on a high pressure liquid chromatography sizing column.