Fractionation,Purification and Some Properties of Proteolytic Enzymes from Stem Bromelain

The heterogeneity of the proteolytic enzymes in the stem bromelain was investigated by the isoelectric focusing with carrier ampholytes. The isoelectric focusing of the stem bromelain demonstrated the presence of two types of proteolytic enzymes which were distinguishable from each other by their isoelectric points. One of these was a basic protein having an isoelectric point of 9.45. This basic enzyme comprised almost all of basic protein which are found in stem bromelain. The other was an acidic protein having an isoelectric point near pH 4.7. This was a minor compooent. The purification of the two enzymes was carried out by use of chromatographies on CM-Sephadex, DEAE-Sephadex and Sephadex at pH 7.0.     

Studies on the structure of sphingomyelinase. Amino acid composition and heterogeneity on isoelectric focusing.

Sphingomyelinase, purified to apparent homogeneity from human placenta, is an acidic protein, as judged from its amino acid composition and by isoelectric focusing of the carboxymethylated protein. The amino acid composition is characterized by an approximately equal content of hydrophobic and polar amino acid residues. The reduced-alkylated polypeptides were separated into two groups. Most of the polypeptides were heterogeneous with pI values of 4.4-5.0, but an additional more minor component was observed at pI 5.4. Liquid isoelectric focusing resolved the purified enzyme into a single major component (pI 4.7-4.8), a minor component (pI 5.0-5.4) and a plateau region of activity (pI 6-7). On thin-layer isoelectric focusing, the protein profile obtained from each of these regions was the same. In addition, the substrate specificity, Km values and effect of inhibitory substances were identical. We conclude that sphingomyelinase is an acidic, microheterogeneous protein that likely exists as a holopolymer of a single major polypeptide chain. the heterogeneity of the intact protein on isoelectric focusing appears to reflect this microheterogeneity, which is influenced by a tendency to associate with itself and with detergents such as Triton X-100.     

Purification and properties of the gamma-butyrobetaine-binding protein from an Agrobacterium sp.

A binding protein for gamma-butyrobetaine was purified from osmotic shock fluid of an Agrobacterium sp. It was a monomeric protein with an apparent molecular weight of 52,000 or 53,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. The isoelectric point was 4.3, as determined by isoelectric focusing. Amino acid analysis of the protein showed that Asx and Glx were predominant components and that the protein contained no cysteine. The dissociation constant of this protein for gamma-butyrobetaine was found to be 0.7 microM by equilibrium dialysis. Attempts to sequence the amino-terminal end with the Edman method failed, suggesting that this region of the protein is blocked.     

Purification and characterization of protein IIIb, a mammalian brain phosphoprotein

The phosphorylation of a 55,000-dalton protein (Protein IIIb) present in mammalian brain was previously shown to be increased by depolarizing agents in the presence of calcium, by cyclic nucleotides, and by appropriate neurotransmitters. We now report that Protein IIIb has been purified 660-fold to near homogeneity and partially characterized. The hydrodynamic properties of the purified protein indicate that it exists as an elongated monomer. cAMP-dependent protein kinase catalyzes the incorporation of 0.82 mol of phosphate into serine/mol of protein. The protein is heterogeneous in isoelectric focusing, exhibiting multiple forms with isoelectric points ranging in pH from 6.6 to 7.3.     

Some properties of phosphatidylcholine exchange protein purified from beef liver

A phospholipid exchange protein has been purified 2680-fold from beef liver. The assay of the exchange activity of the protein was based on the transfer of [¹⁴C]phosphatidylcholine from microsomes labeled with [¹⁴C]phosphatidylcholine to liposomes. The homogeneity of the protein has been established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoelectrophoresis and isoelectric focusing. The protein has a molecular weight of approximately 22000 and an isoelectric point of 5.8. The amino acid composition has been determined. The protein contains one disulfide bridge and has glutamic acid as the N-terminal amino acid. Phospholipid, tentatively identified as phosphatidylcholine, was found to be present in the protein preparation. The protein stimulated specifically the exchange of phosphatidylcholine between mitochondria and microsomes from rat liver.     

Nucleotide catabolism and nucleoside cycles in human thymocytes. Role of orthophosphate.

Phosphatidylinositol 4-phosphate (PtdIns4P) kinase was purified from cytosolic and particulate material of rat brain. The purification procedure of the enzyme from cytosol consisted of (NH4)2SO4 precipitation. DEAE-cellulose column chromatography and preparative isoelectric focusing. Other methods after DEAE-cellulose column chromatography failed to achieve further purification of the PtdIns4P kinase, probably caused by the tendency of the enzyme to aggregate with contaminating proteins. The final purification was 67-fold, and the recovery was 0.6%. After isoelectric focusing the fraction containing the highest PtdIns4P kinase activity showed only one protein as visualized by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and silver staining. The apparent Mr of this protein was 45 kDa and the isoelectric point about 5.8. The activity of PtdIns4P kinase was dependent on the concentration of divalent cations in the incubation medium. PtdIns4P kinase activity was found to be optimal at 10-30 mM-Mg2+. In an attempt to compare the cytosolic with the membrane-derived kinase activity, a Triton/KCl extract from synaptic membranes was subjected to the same purification procedure as the cytosolic enzyme. A difference in isoelectric focusing was observed, possibly due to a higher tendency to form aggregates. However, we tend to conclude that also in the membranes the PtdIns4P kinase activity is present as a 45 kDa protein, identical with that found in the cytosol.     

Purification and characterization of trichomaglin--a novel ribosome-inactivating protein with abortifacient activity

Trichomaglin, a novel ribosome-inactivating protein, has been isolated from root tuber of a plant Maganlin (Trichosanthes Lepiniate, Cucurbitaceae). The isolation and purification procedure included ammonium sulfate precipitation, Sephadex G-75 chromatography and CM-Sephadex C-50 chromatography. The protein was identified to be homogeneous by SDS-PAGE and FPLC analysis. Its molecular weight is 24,673 dalton and isoelectric point is 5.8, determined by electrospray ionization mass spectroscopy and isoelectric focusing gel electrophoresis respectively. Trichomaglin can inhibit protein synthesis in rabbit reticulocyte lysate with ID50 of 10.1 nM. When rat ribosome was incubated with trichomaglin, a diagnostic RNA fragment appeared on polyacrylamide gel after ribosomal RNAs were treated with acidic aniline. It was concluded that trichomaglin is an RNA N-glycosidase. In addition, it has been verified to be an abortifacient protein.     

Isoelectric-focusing behavior of acid hydrolases in rat kidney lysosomes. Effects of the pH gradient, autolysis and neuraminidase.

Isoelectric focusing was used to study the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase in lysosomes isolated from rat kidney. The isoelectric points of the main protein and hydrolase peaks were 1-1.5 units lower when electrofocusing was done in a pH 3-10 gradient than in a pH 10-3 gradient, apparently because the lysosomal constituents aggregated strongly at their isoelectric points and tended to settle somewhat in the gradient due to gravity. In the extended pH gradient the acidic form of each hydrolase occurred as asingle, relatively discrete peak. However, when pooled acidic fractions were refocused in a restricted pH gradient (pH 6-3 or 3-5) multiple acidic enzyme and protein components were resolved with isoelectric points between 2.7 and 5.1. When autolysis was minimized by extracting lysosomal fractions at alkaline pH (0.2% Triton X-100, 0.1%p-nitrophenyloxamic acid, 0.1 M glycine buffer, pH9) and including 0.1%p-NITROPHENYLOXAMIC ACID, AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND CATHEPSIN D, in the pH gradient, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in two forms, an acidic form with an isoelectric point of about 4.4, and a basic form with an isoelectric point close to 6.2, 6.7 and 8.0, respectively. Acid phosphatase occurred in three forms with isoelectric points of 4.1, 5.6 and 7.4. When some autolytic digestion was permitted by extracting lysosomal fractions in an acidic medium (0.2% Triton X-100, 0.1 M sodium acetate buffer, pH 5.2) AT 0-4DEGREES C and omitting p-nitrophenyloxamic acid from the gradient, the acidic form of beta-glucuronidase and the intermediate form of acid phosphatase were lost, the isoelectric points of the acidic forms of acid phosphatase, arylsulfatase and beta-N-acetylhexosaminidase were increased 0.6-1.2 units, and the isoelectric point of the basic forms of acid phosphatase, arylsulfatase and beta-glucuronidase was increased 0.5 unit. When lysosomal extracts were incubated with bacterial neuraminidase before electrofocusing, the acidic forms of acid phosphatase, arylsulfatase and beta-glucuronidase were largely lost, the isoelectric point of the acidic form of beta-N-acetylhexosaminidase was increased from 4.5 to 6.4, and the isoelectric points of the basic forms of all four hydrolases were increased 0.5-1.5 units. Autoincubation of lysosomal extracts in vitro at pH 5.2 PRODUCED SIMILAR, THOUGH LESS MARKED, effects. cont'd     

Characterization and purification of lupus antigen La, and RNA-binding protein.

HeLa cell La antigen, an RNA-binding protein, was characterized by using two-dimensional gel electrophoresis. Eight isoelectric forms (pI 6 to 7) were observed, many containing phosphate. An in vitro translation product similar in size and antigenicity was identified. The HeLa cell protein purified by using an assay based on ribonucleoprotein reconstitution with adenovirus VA RNAI also comprised several isoelectric forms.     

Purification of a basic phospholipid transfer protein from maize seedlings

A phospholipid transfer protein has been purified 125-fold from maize seedlings. The successive steps of purification comprised gel filtration on Sephadex G-75, DEAE- and CM-chromatography and chromatofocusing. The homogeneity of the protein was determined by polyacrylamide gel electrophoresis with and without SDS and by isoelectric focusing. The protein has an apparent molecular weight of 20000, as estimated from SDS electrophoresis, and an isoelectric point of 8.8 ± 0.2. The amino acid composition of the protein is characterized by a high content of alanine, glycine, cysteine and serine and a small amount of lysine. A molecular weight of 14058 was calculated from this amino acid composition. The protein only loses 25% of its activity after 5 min heating at 95°C. The purified protein is able to transfer phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine between liposomes and mitochondria at the rates of, respectively, 100, 56 and 1.6. After incubation of the purified protein with [³H]phosphatidylcholine, a labelled phosphatidylcholine-protein complex was obtained after chromatofocusing. This suggests that the protein acts by carrying phosphatidylcholine from a membrane toward another one.     

The biochemical and functional heterogeneity of circulating human plasma fibronectin

Human plasma fibronectin purified by affinity chromatography, consisted of homogeneous 215 kD protein subunits when assessed by SDS polyacrylamide gel electrophoresis. On isoelectric focusing however, 5 separate fractions were present, with isoelectric points ranging from 5.6 to 6.1. Isoelectric focusing and immunofixation of native plasma produced similar but not identical appearances. Only 15% of the total plasma fibronectin opsonically stimulated the ingestion of Saccharomyces cerevisiae by human peripheral blood monocytes, and this opsonic fibronectin was confined to the fraction with an isoelectric point of 6.1.     

Low molecular mass phosphoproteins from the frog rod outer segments form a complex with 48 kDa protein.

Upon separation of cAMP-dependent low molecular mass phosphoproteins [Components I and II; Polans et al. (1979) J. gen. Physiol. 74, 595-613] from the frog rod outer segments by gel-chromatography, isoelectric focusing, non-denaturating electrophoresis and ion-exchange chromatography, they behave like subunits of the oligomeric complex. Apparent molecular mass of the complex determined by gel-chromatography is 52-57 kDa and by non-denaturating gradient electrophoresis is 62-66 kDa. The isoelectric point of the complex is 5.5. The elution profile of Components I and II upon gel-chromatography and ion-exchange chromatography coincides with that of major rod outer segment 48 kDa protein. The isoelectric point for them also coincides with the isoelectric point of 48 kDa protein. The amount of low molecular mass phosphoproteins is sealed rods is equal to one molecule per 60 rhodopsin molecules and coincides with that of a 48 kDa protein. It is suggested that in solution Components I and II form an oligomeric complex with 48 kDa protein.     

Purification and identification of a 42-kilodalton abscisic acid-specific-binding protein from epidermis of broad bean leaves

Purification of abscisic acid (ABA)-binding proteins is considered to constitute a major step toward isolating ABA receptors. We report here that an ABA-binding protein was for the first time, to our knowledge, purified from the epidermis of broad bean (Vicia faba) leaves via affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, and isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis two-dimensional electrophoresis of the purified ABA-binding protein all identified a single protein band with a molecular mass of 42 kD and an isoelectric point 4.86. The Scatchard plot for the purified protein showed a linear function with a maximum binding activity of 0.87 mol mol(-1) protein and an equilibrium dissociation constant of 21 nM, indicating that the purified protein may be a monomeric one, possessing one binding site. The ABA-binding protein was enriched more than 300-fold with a yield of 14%. (-)ABA and trans-ABA were substantially incapable of displacing (3)H-(+/-)ABA bound to the ABA-binding protein, and (+/-)ABA was less effective than (+)ABA in the competition. These findings allow establishment of the stereospecificity of the 42-kD protein and suggest its ABA receptor nature. Pretreatment of the guard cell protoplasts of broad bean leaves with the monoclonal antibody raised against the 42-kD protein significantly decreased the ABA specific-induced phospholipase D activity in a dose-dependent manner. This physiological significance provides more clear evidence for the potential ABA-receptor nature of the 42-kD protein.     

Effect of divalent ions on protein precipitation with polyethylene glycol: mechanism of action and applications.

Polyethylene glycol (PEG) is extensively employed for protein purification by fractional precipitation. Efficiency of precipitation is highest when the solution pH is near the isoelectric point of the target protein. At pH values far from the isoelectric point of the target protein, proteins develop a net positive or negative charge and are not more resistant to precipitation. We have found that divalent cations (Ba2+, Sr2+, and Ca2+) or divalent anions (SO4(2-)) significantly change the pattern of PEG precipitation when the ion is chosen so as to counteract the expected net charge on the target protein. At moderate (5-50 mM) concentrations of Ba2+, negatively charged proteins can be precipitated from solution at pH values as high as 10 with efficiency unchanged from precipitation at pH values near their isoelectric point values. The mechanism of PEG precipitation of protein at these high pH values appears to be unchanged from the mechanism operative at the protein isoelectric point. Precipitation is rapid and the capacity for protein precipitation is high. There is no detectable coprecipitation of small molecules (AMP, ATP, and NADH) or soluble proteins (carbonic anhydrase) induced when large quantities of protein are precipitated by this method. The purification of bovine carbonic anhydrase from erythrocyte lysate is more efficient at pH 10 in the presence of Ba2+ than is conventional PEG precipitation carried out at the isoelectric point of carbonic anhydrase. Application of these observations should broaden the utility of protein purification by fractional precipitation with PEG.     

Purification and partial characterization of a cohaemolysin (CAMP-factor) produced by Streptococcus canis

Abstract A cohaemolysin from the culture supernate of a canine pathogenic group G streptococcus ( S. canis ) was purified to electrophoretic homogeneity. The purification procedure involved ammonium sulphate precipitation, ultrafiltration, gel filtration and preparative isoelectric focusing. The cohaemolysin consisted of a single polypeptide chain, 18.6 kDa, with an isoelectric point at pH 5.1. The protein reacted with an homologous anti-serum, appeared to be trypsin-sensitive and relatively heat-stable. The cohaemolysin did not show any non-specific IgG binding activities.     

Purification and characterization of recombinant cell surface protein antigen A of Streptococcus sobrinus B13N.

1. It has been reported that immunization of rhesus monkeys with the surface protein antigen I/II from Streptococcus mutans significantly reduced dental caries. 2. The surface protein antigen A (SpaA) from Streptococcus sobrinus is known to correspond antigenically to I/II. MD51 is an Escherichia coli host containing pMD51, a plasmid encoding the SpaA gene from Streptococcus sobrinus B13N. 3. The recombinant SpaA (rSpaA) was purified from cell extracts of Escherichia coli clone MD51. 4. The purified recombinant SpaA was homogeneous with a molecular weight of 210 kDa according to SDS-PAGE and had an isoelectric point of 4.2 based on isoelectric focusing. 5. Amino acid composition of rSpaA showed a relatively high amount of hydrophobic amino acids (39.7%).     

Antigen-dependent heterogeneity of human migration inhibitory factor

Human migration inhibitory factor (MIF) produced by peripheral blood mononuclear cells stimulated with purified protein derivative, tetanus toxoid, streptokinase-streptodornase, or Candida albicans antigen was analyzed by gel filtration and isoelectrofocusing. In all cases, supernatants harvested after a 24-hr exposure of the mononuclear cells to the antigen yielded only one MIF species with an isoelectric point of 5. In contrast, isoelectrofocusing of supernatants obtained from cells exposed to the antigen for an additional 24 hr demonstrated that different antigens induce the elaboration of different MIF species. Streptokinase-streptodornase and tetanus toxoid induced the production of one MIF species with an isoelectric point of 5 (pH 5-MIF). Stimulation of cells with Candida antigen elaborated a MIF species with an isoelectric point of 3 (pH 3-MIF). In contrast, stimulation of cells with purified protein derivative induced the production of both pH 3-MIF and pH 5-MIF.     

A new inhibitor of plasmin and trypsin from porcine leukocytes.

A new intracellular inhibitor of plasmin and trypsin was isolated from porcine leukocytes by ion exchange chromatography and affinity chromatography. In dodecyl sulphate gel electrophoresis a single protein band with an apparent molecular mass of 15 kDa was found under reducing conditions. On isoelectric focusing three protein bands with isoelectric points between pH 4.0 and 4.5 were found. The association rate constants and the inhibition constants were determined for porcine plasmin and bovine trypsin. The inhibitor shows no immunologic cross-reactivity with any of the tested leukocyte inhibitors. On the basis of its N-terminal amino-acid sequence a great degree of similarity to Kunitz-type inhibitors was observed.     

Properties and Specific Functional Features of Wheat Grain α-Amylase/Subtilisin Inhibitor

A protein bifunctional inhibitor of endogenous α-amylase and subtilisin has been isolated from wheat grain and purified. The inhibitor specifically inactivates α-amylase isozymes with high isoelectric point values (group α-AMY1) and has almost no effect on the α-AMY2 isozymes with low isoelectric point values. This enzyme does not belong to glycoproteins and has a molecular weight of 21 kDa and an isoelectric point of 7.2. The protein displays a relatively high thermostability and pH optimum of 8.0; its inhibitory activity requires the presence of Ca²⁺ cations. The inhibition of excess α-amylase in wheat grain with a low falling number by the purified protein is studied.     

Recombinant-derived interleukin-1 alpha stabilized against specific deamidation

Recombinant-derived human interleukin-1 alpha (IL-1 alpha), purified from Escherichia coli, was resolved by isoelectric focusing on polyacrylamide gels into two species of isoelectric points (pI) 5.45 and 5.20, which constituted approximately 75% and approximately 25% of the total IL-1 alpha protein respectively. The pI 5.45 and pI 5.20 species were separated by chromatofocusing and subjected to N-terminal sequence analysis. The pI 5.45 species contained the expected Asn residue at position 36 of the mature protein sequence whereas the pI 5.20 species contained an Asp residue at the same position. A mutant protein in which Asn-36 was substituted for a Ser residue was isolated from E. coli and shown to be homogeneous on isoelectric focusing analysis with a pI = 5.45. 1H-n.m.r. and circular dichroism analyses of wild-type and the mutant IL-1 alpha indicated a similar conformation which was also indicated by the identical receptor binding affinities of IL-1 alpha with Asn, Asp or Ser in position 36. The mutant protein was stabilized against specific base-catalysed and temperature-induced deamidation, and may be more suitable than the wild-type position for physical and structural studies.