Secreted adenylate cyclase of Bordetella pertussis: calmodulin requirements and partial purification of two forms.

The extracellular adenylate cyclase of Bordetella pertussis was partially purified and found to contain high- and low-molecular-weight species. The high-molecular-weight form had a variable molecular weight with a peak at about 700,000. The smaller species had a molecular weight of 60 to 70,000 as determined by gel filtration. The low-molecular-weight form could be derived from the high-molecular-weight species. The high-molecular-weight complex purified from the cellular supernatant was highly stimulated by calmodulin, while the low-molecular-weight enzyme was much less stimulated. Active enzyme could be recovered from sodium dodecyl sulfate (SDS) gels at positions corresponding to molecular weights of about 50,000 and 65,000. Active low-molecular-weight enzyme recovered from SDS gels migrated with a molecular weight of about 50,000, which coincides with a coomassie blue-stained band. However, when both high- and low-molecular weight preparations were analyzed in 8 M urea isoelectrofocusing gels, the enzyme activity recovered did not comigrate with stained protein bands. The enzyme recovered from denaturing isoelectrofocusing or SDS gels was activated by calmodulin, indicating a direct interaction of calmodulin and enzyme. The high-molecular-weight form of the enzyme showed increasing activity with calmodulin concentrations ranging from 0.1 to 500 nM, while the low-molecular-weight form was fully activated by calmodulin at 20 nM. Adenylate cyclase on the surface of living cells was activated by calmodulin in a manner which resembled that found for the high-molecular-weight form.     

Purification and properties of a low-molecular-weight α-mannosidase from Cellulomonas sp.

Cellulomonas sp. isolated from soil produces a high level of α-mannosidase (α-mannanase) inductively in culture fluid. The enzyme had two different molecular weight forms, and the properties of the high-molecular-weight form were reported previously (Takegawa, K. et al.: Biochim. Biophys. Acta, 991, 431–437, 1989). The low-molecular-weight α-mannosidase was purified to homogeneity by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was over 150,000 by gel filtration. Unlike the high-molecular-weight form, the low-molecular-weight enzyme readily hydrolyzed α-1,2- and α-1,3-linked mannose chains.     

Hepatitis B surface antigen polypeptides: artifactual bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis caused by aggregation.

Hepatitis B surface antigen, subtype ad, was purified and studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Two major bands with molecular weights of 23,500 and 27,500 and several weaker bands with higher molecular weights were observed. When the low-molecular-weight bands and the group of high-molecular-weight bands were excised from the gel, eluted, and reelectrophoresed, neither the low-molecular-weight bands nor the high-molecular-weight bands ever appeared alone, but both high- and low-molecular-weight bands always appeared. It was concluded that the apparently high-molecular-weight bands represented aggregates of the two small polypeptides whose monomers formed the major bands. The preparation thus contained only two polypeptides.     

Methionyl-tRNA synthetase from sheep mammary gland. Purification of a fully active monomeric enzyme derived from high-molecular-weight complexes by controlled proteolysis

Methionyl-tRNA synthetase from sheep lactating mammary gland is found predominantly in the form of high-molecular-weight complexes. Controlled proteolysis of these aggregates generates a low-molecular-weight species of the enzyme with full maintenance of activity as assessed by the rate of aminoacylation of tRNA. The product of proteolysis, which has been purified to homogeneity with a yield of 23%, is a monomeric enzyme of molecular weight 78 000. It has a specific activity of 405 units/mg at 25 degrees C. These findings clearly demonstrate that the aggregated state of methionyl-tRNA synthetase is not a prerequisite for full expression of catalytic activity. Furthermore, the results emphasize the need to provide effective protection against proteolytic damage in studies dealing with the characterization of high-molecular-weight complexes of aminoacyl-tRNA synthetases.     

Purification of rabbit and human serum paraoxonase.

Rabbit serum paraoxonase/arylesterase has been purified to homogeneity by Cibacron Blue-agarose chromatography, gel filtration, DEAE-Trisacryl M chromatography, and preparative SDS gel electrophoresis. Renaturation (Copeland et al., 1982) and activity staining of the enzyme resolved by SDS gel electrophoresis allowed for identification and purification of paraoxonase. Two bands of active enzyme were purified by this procedure (35,000 and 38,000). Enzyme electroeluted from the preparative gels was reanalyzed by analytical SDS gel electrophoresis, and two higher molecular weight bands (43,000 and 48,000) were observed in addition to the original bands. This suggested that repeat electrophoresis resulted in an unfolding or other modification and slower migration of some of the purified protein. The lower mobility bands stained weakly for paraoxonase activity in preparative gels. Bands of each molecular weight species were electroblotted onto PVDF membranes and sequenced. The gas-phase sequence analysis showed that both the active bands and apparent molecular weight bands had identical amino-terminal sequences. Amino acid analysis of the four electrophoretic components from PVDF membranes also indicated compositional similarity. The amino-terminal sequences are typical of the leader sequences of secreted proteins. Human serum paraoxonase was purified by a similar procedure, and ten residues of the amino terminus were sequenced by gas-phase procedures. One amino acid difference between the first ten residues of human and rabbit was observed.     

Pig liver monoamine oxidase: Studies on the subunit structure

The molecular weight of pig liver MAO has previously been shown to be about 115,000 with 1 mole of covalently bound FAD per mole of enzyme. Gel filtration of purified enzyme on Sepharose 4B in 6 m guanidine and 0.1 m mercaptoethanol (MCE) and analytical ultracentrifugation in 0.1% sodium dodecyl sulfate (SDS) and 0.1% MCE yielded molecular weights of 55,000 and 63,000, respectively. By polyacrylamide electrophoresis in 0.1% SDS + MCE one band of 60,000 MW appeared. These results seem to imply that the enzyme is composed of two subunits of which one carries the active site. If MCE was omitted during the gel electrophoresis two equally large bands of about 60,000 MW were formed. By using enzyme inhibited by [¹⁴C]pargyline, a MAO-inhibitor blocking the active site of the enzyme in a 1:1 molar ratio, it was found, however, that both bands contained pargyline. Furthermore, amino acid analyses yielded the same amino acid composition of the two bands. The results are interpreted that the enzyme is composed of two subunits of identical molecular size (about 60,000) of which only one contains the active site and that the enzyme preparation contained two forms of the enzyme presumably differing in the number of disulfide bonds.     

Isolation and purification of a low-molecular-weight skeletal growth factor from human bones

A low-molecular-weight potent bone cell mitogen termed human skeletal growth factor (human SGF) was purified to homogeneity from human bone matrix. Extraction and initial purification steps were done under dissociative conditions to separate human SGF from high-molecular-weight complexes of bone matrix proteins. SGF activity was extracted from human femoral heads by demineralization with 10% EDTA in the presence of 4 M guanidine-HCl and proteinase inhibitors and was purified by hydroxyapatite, HPLC gel-filtration and HPLC reverse-phase chromatography. Human SGF thus purified was homogeneous by HPLC reverse-phase chromatography and SDS-polyacrylamide gel electrophoresis. The relative molecular mass of human SGF purified under dissociative conditions was 11,000. Human SGF stimulated bone cell proliferation ([3H]thymidine incorporation and cell number) at picomolar concentrations, with half maximum activity at 2-3 ng/ml (180-270 pM). Human SGF constitutes 0.00024% of organic bone matrix by weight.     

Purification and characterization of multicatalytic proteinase from eggs of the ascidian Halocynthia roretzi

A multicatalytic (high-molecular-weight) proteinase has been purified from eggs of the ascidian Halocynthia roretzi by a procedure including column chromatographies on DEAE-cellulose and hydroxylapatite and gel filtration on Sepharose 6B. The purified enzyme seemed to be homogeneous, as judged by disc-polyacrylamide gel electrophoresis, isoelectrofocusing, sedimentation velocity, and gel filtration. The molecular weight of the enzyme was estimated to be 610,000 by gel filtration. The isoelectric point and the sedimentation coefficient (S20,w) were 6.2 and 22.8S, respectively. The enzyme showed several protein bands with molecular weight ranging from 25,000 to 33,000 on SDS-polyacrylamide gel electrophoresis and a cylindrical or ring-like structure composed of several subunits under the electron microscope, indicating that the enzyme exists as a large molecule consisting of several protein components. The enzyme exhibited chymotrypsin-like and trypsin-like activities whose pH optima were both 7.0. Chymostatin and its analog, calpain inhibitor I, and elastatinal inhibited both activities, whereas leupeptin and antipain only inhibited the latter. The former activity was stimulated by a low concentration of SDS or fatty acid, whereas the latter was not. Thus, the properties of the enzyme purified from ascidian eggs are similar to those of multicatalytic proteinases from mammalian tissues.     

Purification from Dictyostelium discoideum of a low-molecular-weight myosin that resembles myosin I from Acanthamoeba castellanii

A low-molecular-weight myosin has been purified 1500-fold from extracts of Dictyostelium discoideum, based on the increase in K+,EDTA-ATPase specific activity. The purified enzyme resembles the single-headed, low-molecular-weight myosins IA and IB from Acanthamoeba castellanii, and differs from the conventional two-headed, high-molecular-weight myosin previously isolated from Dictyostelium, in several ways. It has higher K+,EDTA-ATPase activity than Ca2+-ATPase activity; it has a native molecular mass of about 150,000 and a single heavy chain of about 117,000; the 117,000-dalton heavy chain is phosphorylated by Acanthamoeba myosin I heavy chain kinase; phosphorylation of its heavy chain enhances its actin-activated Mg2+-ATPase activity; and the 117,000-dalton heavy chain reacts with antibodies raised against the heavy chain of Acanthamoeba myosin IA. None of these properties is shared by the low-molecular-weight active fragment that can be produced by chymotryptic digestion of conventional Dictyostelium myosin. We conclude that Dictyostelium contains an enzyme of the myosin I type previously isolated only from Acanthamoeba.     

Purification and Properties of beta-N-Acetylhexosaminidase from Mucor fragilis Grown in Bovine Blood

Mucor fragilis grown on bovine blood powder as the sole carbon source abundantly produced beta-N-acetylhexosaminidase. The enzyme activity was several times higher than that of a culture obtained with glucose medium. The enzyme had two different molecular weight forms. The high-molecular-weight form had somewhat higher beta-N-acetylgalactosaminidase activity than the lower-molecular-weight enzyme which had beta-N-acetylgalactosaminidase activity equivalent to about 40% of its beta-N-acetylglucosaminidase activity. Bovine blood seemed to induce both enzymes, but N-acetylamino sugars specifically induced the low-molecular-weight form. N-Acetylgalactosamine had an especially marked effect on activity. The low-molecular-weight form of enzyme was purified from the culture filtrate by fractionation with ammonium sulfate and various column chromatographies. The purified enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The optimum pH was 4.0 to 5.0 for beta-N-acetylglucosaminidase activity and 5.5 to 6.5 for beta-N-acetylgalactosaminidase activity. The enzyme hydrolyzed natural substrates such as di-N-acetylchitobiose, tri-N-acetylchitotriose, and a glycopeptide obtained by modification of fetuin.     

Identity of neutral alpha-glucosidase AB and the glycoprotein processing enzyme glucosidase II. Biochemical and genetic studies

We have previously partially purified, characterized, and chromosomally mapped a human isozyme of alpha-glucosidase which is active at neutral pH. This isozyme appears as a doublet of enzyme activity on native gel electrophoresis and was termed neutral alpha-glucosidase AB. We now report genetic and biochemical evidence that neutral alpha-glucosidase AB is synonymous with the glycoprotein processing enzyme glucosidase II. We have found that a mutant mouse lymphoma line which is deficient in glucosidase II is also deficient in neutral alpha-glucosidase AB, as defined electrophoretically and quantitatively (less than 0.5% of parental). In contrast, both mutant and parental cell lines exhibited several lysosomal hydrolases which are processed by glucosidase II. We have also further purified the human neutral alpha-glucosidase A component of neutral alpha-glucosidase AB 740-fold from placenta in order to compare its biochemical properties with those described for rat liver and pig kidney glucosidase II. Both glucosidase II and neutral alpha-glucosidase AB are high-molecular mass (greater than 200,000 dalton) anionic glycoproteins which bind to concanavalin A, have a broad pH optima (5.5-8.5), and have a similar Km for maltose (4.8 versus 2.1 mM) and the artificial substrate 4-methylumbelliferyl-alpha-D-glucopyranoside (35 versus 19 microM). Similar to human neutral alpha-glucosidase AB, purified rat glucosidase II migrates as a doublet of enzyme activity on native gel electrophoresis. Although rat glucosidase II has been reported to have a subunit size of 67 kDa, pig glucosidase II has been found to have a subunit size of 100 kDa, like the 98-kDa major protein in purified human neutral alpha-glucosidase A. Although we have not demonstrated that neutral alpha-glucosidase AB is microsomal nor that it hydrolyzes the natural substrate of glucosidase II, we believe that the genetic evidence is compelling for and the biochemical data consistent with the hypothesis that neutral alpha-glucosidase AB and glucosidase II are synonymous. These and previous results would localize glucosidase II to the long arm of human chromosome II.     

Studies on the Wolfgram High Molecular Weight CNS Myelin Proteins: Relationship to 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase

Evidence is presented that the major protein components of the high molecular weight CNS myelin proteins designated as the Wolfgram protein doublet (W1 and W2) contain the enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37, CNP). CNP is a basic hydrophobic protein containing about 830 to 840 amino acid residues. When electrophoresed on SDS polyacrylamide gels, CNP appears as a protein doublet, separated by a molecular weight difference of about 2500-3000 in bovine, human, rat, guinea pig, and rabbit. A similar protein doublet has been identified as the Wolfgram proteins W2 and W1 in myelin and in the chloroform-methanol-insoluble pellet obtained from myelin. Moreover, the relative Coomassie blue staining intensity of the CNP2 plus CNP1 protein doublet among the species examined was remarkably similar to that observed for electrophoresed myelin and chloroform-methanol-insoluble pellet derived from myelin. Antisera raised against purified bovine CNP recognized the W1 and W2 proteins isolated from bovine and human brain. The amino acid composition of pure bovine CNP is presented and compared with the compositions of several rat and bovine Wolfgram proteins obtained by other investigators. Our electrophoretic, compositional, and immunological data support the contention that the enzyme CNP is a major component of the Wolfgram protein doublet.     

Role of starch as a substrate for Bacteroides vulgatus growing in the human colon.

Bacteroides vulgatus is the numerically predominant Bacteroides species in the human colonic microflora. Unlike other colonic Bacteroides species, B. vulgatus is not a versatile utilizer of polysaccharides. The only types of polysaccharide that support rapid growth and high growth yields by all strains are the starches amylose and amylopectin. Amylase and alpha-glucosidase activities are among the highest found in a bacterial fraction obtained from human feces. This observation raised the question of whether B. vulgatus was the source of the fecal enzymes. Both alpha-glucosidase and amylase were produced at 20- to 40-fold-higher levels when B. vulgatus was grown on maltose, amylose, or amylopectin than when B. vulgatus was grown on glucose or other monosaccharides. Both enzymes had the same pI (4.6 to 5.0) and undenatured molecular weight (150,000). The pIs and molecular weights of the B. vulgatus amylase and alpha-glucosidase were the same as those of the fecal enzymes. To determine whether the B. vulgatus alpha-glucosidase was identical to the fecal alpha-glucosidase, we partially purified the B. vulgatus enzyme and raised an antiserum against it. Using this antiserum, we showed that all strains of B. vulgatus produced the same enzyme. The antiserum did not detect the B. vulgatus alpha-glucosidase in the bacterial fraction from human feces, even when a partially purified preparation of the fecal enzyme was used. Thus the alpha-glucosidase activity in the bacterial fraction from human feces is not the B. vulgatus enzyme.     

Role of starch as a substrate for Bacteroides vulgatus growing in the human colon

Bacteroides vulgatus is the numerically predominant Bacteroides species in the human colonic microflora. Unlike other colonic Bacteroides species, B. vulgatus is not a versatile utilizer of polysaccharides. The only types of polysaccharide that support rapid growth and high growth yields by all strains are the starches amylose and amylopectin. Amylase and alpha-glucosidase activities are among the highest found in a bacterial fraction obtained from human feces. This observation raised the question of whether B. vulgatus was the source of the fecal enzymes. Both alpha-glucosidase and amylase were produced at 20- to 40-fold-higher levels when B. vulgatus was grown on maltose, amylose, or amylopectin than when B. vulgatus was grown on glucose or other monosaccharides. Both enzymes had the same pI (4.6 to 5.0) and undenatured molecular weight (150,000). The pIs and molecular weights of the B. vulgatus amylase and alpha-glucosidase were the same as those of the fecal enzymes. To determine whether the B. vulgatus alpha-glucosidase was identical to the fecal alpha-glucosidase, we partially purified the B. vulgatus enzyme and raised an antiserum against it. Using this antiserum, we showed that all strains of B. vulgatus produced the same enzyme. The antiserum did not detect the B. vulgatus alpha-glucosidase in the bacterial fraction from human feces, even when a partially purified preparation of the fecal enzyme was used. Thus the alpha-glucosidase activity in the bacterial fraction from human feces is not the B. vulgatus enzyme.     

Protein Components of the Purified Sodium Channel from Rat Skeletal Muscle Sarcolemma

Abstract: Sensitive detection systems have been used to study the protein components of the sodium channel purified from rat skeletal muscle sarcolemma. This functional, purified sodium channel contains at least three subunits on 7–20% gradient sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis: a large glycoprotein which migrates anomalously in the high-molecular-weight range, a 45,000 molecular weight polypeptide, and a third protein often seen as a doublet at 38,000. The large glycoprotein runs as a diffuse band and stains very poorly with Coomassie blue, but is adequately visualized with silver staining or iodination followed by autoradiography. This glycoprotein exhibits anomalous electrophoretic behavior in SDS-polyacrylamide gels. The apparent molecular weight of the center of the band varies from ～230,000 on 13% acrylamide gels to ～130,000 on 5% gels; on 7–20% gradient gels a value of 160,000 is found. Plots of relative migration versus gel concentration suggest an unusually high apparent free solution mobility. Lectin binding to purified channel peptides separated by gel electrophoresis indicates that the large glycoprotein is the only subunit that binds either concanavalin A or wheat germ agglutinin, and this component has high binding capacity for both lectins. The smaller channel components run consistently at 45,000 and 38,000 molecular weight in a variety of gel systems and do not appear to be glycosylated.     

Measurements of the molecular size of the simian virus 40 large T antigen.

A measure of the molecular weight of the large simian virus 40 T antigen was sought by SDS-polyacrylamide gel electrophoresis, random-coil chromatography, and sedimentation-velocity analysis in a density gradient. Large T antigen obtained from a simian virus 40-transformed human cell line either by immunoprecipitation or by standard preparatory methods migrated like a 94,000-molecular-weight (approximately 94K) polypeptide in SDS-gels but was found to have an approximate was observed with T antigen obtained from lytically infected monkey cells. In view of the strong theoretical basis for the guanidine method and the agreement with the sedimentation data, these findings suggest that the molecular weight of this protein is approximately 75 to 80K as opposed to 94 to 100K and, therefore, that considerably less than the entire early region of simian virus 40 is required to encode it. This size estimate is in keeping with earlier results which revealed a normal-size T antigen in cells infected with viable deletion mutants lacking as much as 10% of the early region.     

Purification and characterization of catabolic dehydroquinase, an enzyme in the inducible quinic acid catabolic pathway of Neurospora crassa.

Catabolic dehydroquinase which functions in the inducible quinic acid catabolic pathway in Neurospora crassa has been purified 8000-fold. The enzyme was purified by two methods. One used heat denaturation of contaminating proteins; the other used antibody affinity chromatography. The preparations obtained by these two methods were identical by all criteria. The purified enzyme is extremely resistant to thermal denaturation as well as denaturation 0y urea and guanidine hydrochloride at 25 degrees. It is irreversibly inactivated, although not efficiently dissociated, by sodium dodecyl sulfate and guanidine hydrochloride at 55 degrees. At pH 3.0, the enzyme is reversibly dissociated into inactive subunits. At high concentrations catabolic dehydroquinase aggregates into an inactive, high molecular weight complex. The native enzyme, which has a very high specific activity, has a molecular weight of approximately 220,000 and is composed of identical subunits of 8,000 to 12,000 molecular weight each. The native enzyme and the subunit are both asymmetric.     

Solubilization of membrane-bound acid alpha-glucosidase by proteolysis

The membrane-bound acid alpha-glucosidase was purified partially (400-fold) from human placenta by solubilization with trypsin, concanavalin A-Sepharose chromatography, Ultrogel AcA-34 gel filtration, and Sephadex G-100 affinity chromatography. Two molecular forms of the enzyme were found in the final preparation of the purified enzyme. They were identical in molecular weight with a precursor (110 kDa) and an early intermediate form (105 kDa) of this enzyme. Also direct incubation of the membrane fraction without trypsin resulted in a release mainly of the 105 kDa form, which was inhibited by N-ethylmaleimide, but not by leupeptin, pepstatin or phenylmethylsulfonylfluoride. It was concluded that the precursor of acid alpha-glucosidase is an intrinsic membrane protein, which is transported into lysosomes after solubilization by proteolysis.     

Neutral alpha-glucosidase in granule fractions from guinea pig polymorphonuclear leukocytes: enzymic characterization and comparative studies with monoclonal antibodies

Neutral alpha-glucosidase was partially purified from granular fractions isolated from guinea pig polymorphonuclear leukocytes (PMNL). The native enzyme had a high molecular weight, about 417,000, with a subunit of 43,000. The purified enzyme hydrolysed 4-methylumbelliferyl alpha-glucoside and maltose, but not isomaltose, trehalose, and glycogen. The enzyme was strongly inhibited by bromoconduritol and castanospermine, but only slightly by turanose. Monoclonal antibodies which can bind specifically to the enzyme were prepared by immunizing mice with the partially purified enzyme. Hybridomas producing the monoclonal antibodies were selected by an enzyme-linked immunosorbent assay. The seven monoclonal antibodies were found to react with the enzyme from PMNL, but not with the glycoprotein-processing alpha-glucosidase isolated from liver microsomes nor with the macrophage enzyme. The results indicated that PMNL contain a particulate neutral alpha-glucosidase enzymologically and immunologically distinct from other alpha-glucosidases.     

Electrophoresis of glutamic acid decarboxylase (EC 4.1.1.15) from mouse brain in sodium dodecyl sulphate polyacrylamide gels

Electrophoretically and ultracentrifugally homogeneous glutamic acid decarboxylase purified from mouse brain showed multiple protein bands after electrophoresis in SDS polyacrylamide gel. The positions and intensities of the multiple bands were constant despite different treatments of the enzyme with various concentrations of SDS, β-mercaptoethanol, and urea at different temperatures. The major band had an apparent molecular weight of approximately 60,000 daltons and there were three minor bands of molecular weights, about 120,000, 90,000, and 75,000 daltons, respectively. The molecular weights of almost all bands were approximately integral multiples of 15,000. The possible subunit structure of this enzyme has been discussed in the light of the latter data and data previously reported from ultracentrifugation and gel filtration studies. We suggest that this enzyme may be a hexamer consisting of 15,000-dalton sub-units and that dissociation of these sub-units in SDS is accompanied by reassociation into a variety of aggregates, the probability of whose formation is determined by structural features that are more important than the differences encountered under the environmental conditions employed in these studies.