Dalton Monomer Research Articles

Purification of a bacteriolytic enzyme from Bacillus sp. active against Streptococcus mutans

Bacillus sp. strain YU-1006, which was isolated from soil, produced a bacteriolytic enzyme which was active against Streptococcus mutans KCTC 3283. The enzyme was purified 60 fold with a 6% yield by CM-cellulose column chromatography and CM-Sephadex G-50 column chromatography. Lytic activity was stable in the range of pH 6.0~9.0 up to 42°e active enzyme is a 24,000 dalton monomer as estimated by SDS-PAGE.

Preliminary X-ray Crystallographic Studies of Ribonuclease I from Escherichia coli

Single crystals of ribonuclease I from Escherichia coli have been obtained by the vapor diffusion method. The crystals belong to the tetragonal space group P4 12 12 or its enantiomer P 4 32 12 and have cell parameters a = b=119·01 Å and c=34·40 Å. There is one 27,000 dalton monomer in the asymmetric unit. The crystals diffract to beyond 3·0 Å resolution.

Chicken transferrin receptor expression during erythroid differentiation and by retrovirus transformed cells

Antiserum prepared against sucrose gradient purified reticuloendotheliosis virus (REV) recognized the chicken transferrin receptor. Molecules immunoprecipitated from red blood cells (RBC) obtained from embryonic chickens with either the anti-REV reagent or a chicken transferrin immunomatrix were demonstrated to be identical by co-migration in both reducing and nonreducing SDS-polyacrylamide gels and in two-dimensional isoelectric focusing analyses, reciprocal immunodepletion analyses and by peptide mapping. The chicken transferrin receptor was shown to be a 190,000 dalton cell surface membrane molecule consisting of two similar disulfide-bonded subunits of approximately 95,000 daltons. The chicken transferrin receptor was expressed on erythroid cell surface membranes as 95,000 dalton monomers as well as 190,000 dalton dimers. The chicken transferrin receptor was expressed on all differentiation/maturation stages, including mature RBC, of both the primitive and definitive type I erythroid cell series. In adult chickens, the transferrin receptor was expressed by immature erythroid cells in the bone marrow, but not by mature circulating RBC. REV-transformed immature lymphoid cells and avian erythroblastosis virus (AEV)-transformed erythroid cells expressed dimers composed of 95,000 and 110,000 dalton subunits. Comparisons among V8 protease derived peptides from 95,000 dalton transferrin receptors obtained from RBC and REV-transformed lymphoid cells revealed a high degree of homology; however, the 95,000 dalton molecules isolated from REV-transformed lymphoid cells exhibited a 56,000 dalton peptide that was unique. Cloned AEV-transformed erythroleukemia cells induced to differentiate by supplementation of the media with 1 mM butyric acid expressed elevated transferrin receptor levels. Both serological and peptide mapping studies demonstrated the human transferrin receptor on K562 cells and the chicken transferrin receptor to be distinct. However, chicken transferrin was shown to be capable of reacting with the human transferrin receptors on K562 cells.

Hepatic clearance and metabolism in the rat of a human breast cancer associated glycoprotein (GCDFP-15).

Gross Cystic Disease Fluid Protein (GCDFP-15) is a 60,000 dalton glycoprotein isolated from human breast cyst fluid, composed of four 15,000 dalton monomers. Carbohydrate analysis indicates that each monomer has a single carbohydrate chain of the complex type. GCDFP-15 intravenously injected into rats showed a rapid circulatory clearance, the rate of clearance being faster in female animals [t1/2 = 12.8 (+/- 2.0) min. females, and 16.7 (+/- 2.6) min. males]. The major organs of clearance were the liver (70%) and kidneys (15%). Immunoperoxidase staining showed localization in Kupffer cells and the proximal convoluted tubules of the kidney. Removal of sialic acid from GCDFP-15 resulted in a more rapid clearance (t1/2 = 2.2 min) by the liver (85%). This clearance was inhibited by coinjection of asialo alpha 1 acid glycoprotein. About 3% of GCDFP-15 was excreted in bile with a transit time through the liver of 38 min. Examination of the uptake of GCDFP-15 by isolated rat Kupffer cells showed that yeast mannan, fucosylated BSA, and carcinoembryonic antigen (CEA) failed to inhibit uptake, though the binding of GCDFP-15 was clearly saturable. This suggests that a novel receptor system on the rat Kupffer cell may be responsible for GCDFP-15 clearance.

Non-human primate (baboon) anti-gross cystic disease fluid protein-15 antibody infusion in four women with metastatic breast carcinoma.

Four women with metastatic breast carcinoma and elevated plasma levels of human breast gross cystic disease fluid protein of 15,000 dalton monomer size (GCDFP-15) were treated IV with non-human primate (baboon) anti-GCDFP-15 antibody. Three patients were given a single IV infusion of antibody, while the fourth patient received four sequential IV infusions. Antibody dosage patients, after antibody infusion the plasma level of GCDFP-15 decreased to 0 ng/ml and remained there as long as "free" circulating anti-GCDFP-15 antibody was present. The plasma half-life of the antibody ranged between 1 and 40 h and the duration of detectable free antibody ranged from 6 to 240 h. No toxicity was observed for the dosage range of antibody tested. No anti-baboon antibody response was detected. In the patient who received four sequential infusions of antibody partial regression of subcutaneous metastatic nodules occurred. The other three patients showed no clinically detectable changes from the antibody infusion.

Purification and properties of galactosylceramide sulfatase activator from human liver.

Activator protein for galactosylceramide sulfatase (GSase) was purified from human liver. The activator has an approximate molecular weight of 22,000, is glycoprotein in nature, and is most probably a trimer consisting of an 8,000 dalton monomer. Monospecific rabbit antiserum raised against the activator strongly inhibited the activity of the activator. In the presence of a 10-fold or more excess of galactosylceramide sulfate (GS) on a molar basis, GS binding to the GSase activator occurred, and was saturated at an equimolar ratio. Binding studies on the GSase activator were conducted using affinity chromatography on derivatives of GS as ligands, and gel filtration of mixtures containing glycolipids and the activator. A "GS-acid" derivative, which was prepared by oxidative cleavage of sphingosine moiety in GS, and a sulfonamide derivative of GS as ligands still retained affinity for the GSase activator, while a hydrophobic ligands, an aminohexyl group did not bind completely the activator. A ligand of "galactosylceramide-acid" had weak affinity for GSase activator. These results suggest that the sulfate group and one of the two hydrocarbon chains in GS are not essential for the binding of the activator. The affinity of galactosylceramide for the GSase activator was confirmed by the detection of the lipid-protein complex on gel filtration. The activator weakly stimulated porcine GM1-beta-galactosidase activity.

REGULATION OF ENZYMES BY LIGAND INDUCED CHANGE IN POLYMERIZATION: 211

Many enzymes readily change their aggregation state, by dissociation or association of subunits. This constitutes a mechanism for regulation, if the different molecular weight species vary in their specific activity, and if the equilibrium between these species is readily perturbed by physiological effectors. Our studies show that several enzymes involved in the synthesis or degradation of pyrimidines exhibit such regulation: The multifunctional protein UMP synthase (EC 2.4.2.10 + EC 4.1.1.23), and uridine kinase (EC 2.7.1.48) from Ehrlich ascites cells, and N-carbamoyl-β-alanine amidohydrolase (EC 3.5.1.6) from rat liver. Generally, substrates are positive effectors, while immediate or more distant end products are negative effectors.UMP synthase shows only positive regulation: The 50,000 dalton monomer is inactive; effectors result in association to the active dimer. Uridine kinase shows negative regulation: the native enzyme exists as polymers of 4 or more subunits, and CTP or UTP produce dissociation to the 31,000 dalton subunit and loss of activity. NCβA amidohydrolase shows bimodal regulation: the native enzyme has an Mr of 235,000; the substrate produces association to 470,000 daltons; the product, β-alanine, causes dissociation to 120,000 daltons.Ligand induced changes in polymerization may be a general mechanism for regulating enzyme activity.

Glycoprotein Synthesis of Renal Collecting Duct Epithelium Cultured as Globular Bodies

Cortical kidney explants from newborn New Zealand rabbits were cultured in Dulbecco's minimum essential medium (DMEM) containing 10% fetal calf serum. Within 24 h the explants formed 'globular bodies' which were completely covered by a monolayered epithelium. The cells were differentiated and resembled collecting duct epithelium. By culturing the globular bodies in Dulbecco's MEM with D-valine instead of L-valine, a monolayer of collecting duct cells was obtained and used for control experiments. For analysis and identification of synthesized glycoproteins, the globular bodies were incubated with various labelled carbohydrates and amino acids, and then fractionated. Glycoproteins secreted into the culture medium were not detected. Cell-associated glycoproteins were found in crude membrane fractions and then extracted with Triton X-100 for column chromatography, SDS-polyacrylamide electrophoresis in 6 M urea, isoelectrofocusing, and two-dimensional electrophoresis. Two prominent glycoproteins containing galactose and glucosamine were synthesized during the spreading of the epithelium, with an apparent molecular weight of 150,000 and 85,000 (SDS-PAGE). The synthesized glycoproteins differ in their content of radioactive glycoprotein precursor and leucine. The 85,000 dalton monomer glycoprotein has an isoelectric point of 3.5 and was identified by two-dimensional electrophoresis.

PERMEABILITY AND STRUCTURAL CHANGES INDUCED BY PHYTOCHROME IN LIPID VESICLES

Abstract— This paper describes a method for rapidly monitoring early changes in electrolyte permeability induced by phytochrome from salt‐loaded liposomes. The method allows for the continuous monitoring of low‐level ion efflux from liposomes by measuring the conductivity of a liposome suspension medium which has osmotic and chemical potentials that promote a slow. passive efflux of the compartmented electrolytes. The addition of the far‐red absorbing form of phytochrome (Pfr) to this system at 20°C immediately produces efflux rates which are 2–3 times greater than if the red‐absorbing form (Pr) is added. This differential effect is not evident at 4°C and varies with the lipid composition of the liposomes. Under conditions in which Pfr induces a 2‐fold greater change in the electrolyte permeability of liposomes than Pr. only about 18% more 125I‐labeled Pfr than 125I‐labeled Pr binds to the liposomes. At equimolar concentrations. the photochromic small peptide of phytochrome (60 000 dalton monomer) and the more native‘large’phytochrome (120000 dalton subunits) induced equivalent changes in the electrolyte permeability of liposomes. No differential leakage of ATP, glucose, or trvpsin from liposomes was observed after Pr and Pfr reacted with vesicles enclosing these substances. The Pfr form of phytochrome promoted greater turbidity in liposome suspensions and a greater degree of aggregation and/or vesiclc fusion than Pr. The kinetics of these changes suggested that they were not the hasis of the differential permeability effects of Pr and Pfr.

Purification and properties of two forms of human α- l-fucosidase

High (100 000) and low (50 000) molecular weight forms of α- l-fucosidase (α-fucosidase I and II) were purified to apparent homogeneity from human spleen, liver brain and kidney on the basis of differential affinity for ϵ-aminocaproyl fucosamine-agarose bead columns. α-Fucosidase I (the “bound” form) consisted of two 50 000 dalton monomers; however, both forms can aggregate to tetramer and hexamer forms. Most previous studies on α-fucosidase have been carried out on this form of human α-fucosidase although the bound and unbound forms of the enzyme were present in equal amounts in human spleen. The bound (100 000) form is a sialoglycoprotein whereas the unbound (50 000) form, is a neutral mannose-rich glycoprotein. Other differences with respect to amino acid composition, pH optimum, electrophoretic mobility, K m , thermal stability, and natural substrate specificities were observed. All preparations stability, and natural substrate specificities were observed. All preparations hydrolysed 4-methylumbelliferyl-α- l-fucoside, fucosyllactose, and lacto-N- fucopentaose I, but the unbound fraction (α-fucosidase II) preferentially hydrolysed lacto-N- focupentaose II. The unbound (mannose-rich) α-fucosidase II was taken up by human skin fibroblasts with higher affinity (5% per 2 h per 2 · 10 5 cells) than the bound (sialo-) α-fucosidase I (<1% per 2 · 10 5 cells). Uptake was inhibited by other lysosomal hydrolases, fetal calf serum, mannose 6-phosphate and phosphomannans and to a lesser extent by heparins. Our studies suggest that α-fucosidase I is not a simple dimer of α-fucosidase II and represents a less-biologically active form of the enzyme.

Purification and biochemical properties of complex flagella isolated from Rhizobium lupini H13-3

1. 1. The complex flagella of Rhizobium lupini H13-3 differ from plain bacterial flagella in the fine structure of their filaments dominated by conspicuous helical bands, in their fragility and their resistance against heat decomposition. To elucidate the basis of these differences, the composition of complex filaments and their subunits was analysed. 2. 2. Isolated complex flagella containing the filament and hook portions were purified by differential centrifugation. Hooks were separated by ultracentrifugation after acid degradation of filaments at pH 2. The complex filaments consist of 43 000 dalton monomers (cx-flagellin), the hooks are composed of 41 000 dalton subunits. 3. 3. Amino acid analysis of cx-flagellin indicated the presence of approx. 417 amino acid residues. These comprise 47% hydrophobic residues and 21% Asp and Glu (or amides), but no Cys, His, Pro and Trp. No carbohydrate, phosphate or lipid moieties have been detected. Fingerprint analysis after tryptic digestion yields approx. 36 peptides, about half of them clustered in the neutral region. A comparison with the composition of various known flagellins from plain flagella indicates a 7% higher content of hydrophobic amino acid residues in complex filaments; this is largely compensated for by the higher content of Glu and Asp (presumably as Gln and Asn) in plain filaments. 4. 4. Immunodiffusion and immunoelectrophoresis of cx-flagellin yield single precipitin bands indicating homogeneity. In contrast, isoelectric focusing lead to three close-running bands around pH 4.7. When isolated, the two major bands again produced an “isoelectric spectrum” suggesting that it reflects an allomorphism of cx-flagellin. 5. 5. Self-assembly experiments with cx-flagellin lead to coiled fibres including helical regions, but not to intact filaments. The products resemble heat-denatured complex filaments and may represent intermediates between monomers and complete polymers.

Erythrocruorins of Euzonus mucronata treadwell: Evidence for a dimeric annelid extracellular hemoglobin

1. 1. The extracellular hemoglobin of the opheliid polychaete Euzonus mucronata is unusual in that it consists of a 3.2 × 10 6 as well as a 6–7 × 10 6 molecular weight hemoglobin at pH 7.O. 2. 2. Electron microscopy of the 3.2 x 10 6 dalton material shows the typical annelid hemoglobin pattern of a two-tiered hexagonal array of submultiples whereas the 6–7 × 10 6 dalton pigment has a four-tiered hexagonal arrangement, a putative dimer of a 3.2 × 10 6 molecular weight material. A few six-tiered trimeric stacks are also present. 3. 3. The dimeric material, which represents 10–15% of the total pigment, is present in unpurified vascular fluid as well as in purified hemoglobin. The constant stoichiometry between the 3.2 × 10 6 dalton monomer and the dimeric pigment under different methods of purification as well as in hemoglobin examined directly from the worm suggests that the dimer is not a preparative artifact. The dimer is not in a state of rapid dissociation-association equilibrium with the monomer nor does the dimer seem to be the result of a ligand-linked association of the 3.2 × 10 6 dalton monomer. 4. 4. The subunit molecular weights of both the monomer and dimer are the same with polypeptide chains of 36,700 ± 800, 31,400 ± 800. 14,400 ± 300. and 12,500 ± 300 as determined by sodium dodecyl sulfate gel electrophoresis. The same molecular weight heterogeneity is present for globin which has been performic acid oxidized, carboxymethylated in 7–8 M guanidine HC1 and 0.1 M in 2-mercaptoethanol, or treated with 8 M urea, 0.1 M in 2-mercaptoethanol. 5. 5. The hemoglobin contains one heme per 22,400 g protein. The amino acid composition of this protein is presented.

Partial characterization of haemoglobin of the bug, Buenoa confusa

The molecular weight of haemoglobin (Hb) from 5th instar larvae of Buenoa confusa was estimated from Sephadex gel filtration and SDS-acrylamide gel electrophoresis. Two Hb fractions eluted from G-100 or G-200 Sephadex columns ranged from 12000 to 14000 and 52000 to 61300 daltons. Another fraction elutes with the void volume in each case. SDS-gel electrophoresis of 3H δ-ALA (δ-aminolevulinic acid)-labelled Hbs treated with SDS and mercaptoethanol shows that most of the high molecular weight Hbs are aggregates of 14000 dalton monomers (corresponding to the low molecular weight Hb eluted from Sephadex filtration).

Multiple peptide composition of the large and small sub-units of Nicotiana tabacum fraction I protein ascertained by fingerprinting and electrofocusing

14C-labeled Fraction I protein was synthesized by Nicotiana tabacum leaves during 20 h of photosynthesis in the presence of 14CO 2. The crystalline protein was carboxymethylated, dissociated by sodium dodecylsulfate and subjected to Sephadex G-100 chromatography which resolved the large and small subunits in a 3:1 ratio according to 14C content of the peptides. The large subunit migrated as 56 000 dalton monomers during sodium dodecylsulfate-polyacrylamide-gel electrophoresis; the small subunits as 12 500 dalton monomers. Fingerprinting resolved 54–56 tryptic peptides from the large subunit whereas 53–55 were expected from the number of arginine-lysine residues. Electrofocusing of carboxymethylated Fraction I protein showed the large subunit to be composed of three peptide bands which resolved in the pH 7 region and were nearly equal in density of staining. The number of tryptic peptides of the small subunit was twice the number of arginine-lysines; electrofocusing produced two approximately equal peptide bands in the pH 6 region. The same number of peptides as well as proportions of each was achieved by either staining of the bands or by autoradiography of 14C-labeled Fraction I protein. The three bands of different isoelectric points composing the large subunit have proved to be exceptionally useful as phenotypic markers for chloroplast DNA genes.

Triphosphopyridine Nucleotide-linked Aldehyde Reductase

A broad substrate specificity, low molecular weight, TPNH-linked aldehyde reductase has been purified from the soluble cytoplasm of pig kidney cortex. The enzyme reduces aromatic aldehydes, such as 3-pyridinecarboxaldehyde, 4-nitrobenzaldehyde, 4-carboxybenzaldehyde, and 4-cyanobenzaldehyde; aldoses, such as d -glyceraldehyde, d -lactaldehyde, pyruvaldehyde, and d -glucuronate; and α-diketones, such as (+)-camphorquinone and diacetyl. It also catalyzes a slow TPN-linked oxidation of the corresponding alcohols, as well as Tris. The enzyme, a 30,200 dalton monomer, is homogeneous by gel filtration, ultracentrifugation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis; but on standard polyacrylamide disc gel electrophoresis it gives multiple bands which have the same relative substrate specificity. A diagnostic property of this reductase is its sensitivity to barbiturate inhibition. Due to its kinetic constants and pH optima, this enzyme probably functions physiologically as an aldehyde reductase rather than an alcohol dehydrogenase. The broad substrate specificity of this reductase overlaps several previously described, apparently unrelated, but perhaps identical, TPN-specific mammalian alcohol dehydrogenases.