Oligomeric hepatoproliferin disaggregates into an active monomer that was purified and forcefully dissociated into two different ionic species

Hepatoproliferin (HPF), a liver regeneration factor, was isolated initially as an aggregated molecule (big-HPF) and was purified into two homogeneous, bioactive species of 14 kDa and 18.5 kDa. These two big-HPFs were disaggregated to completion into two monomeric forms (small-HPFs) when incubated for 10 days in 0.15 M ammonium bicarbonate at 25 degrees C. Both monomeric forms were purified to homogeneity as active entities, one with a molecular mass of 944 Da and one with a molecular mass of 1066 Da. Each of the two (35)S-labelled small-HPFs was found, by enzymic analysis, to contain a charged sulfonated saccharide, which was neutralized by a specific amine. Monomeric HPF is therefore a stable ionic complex formed between these two ionic species. So strong was the electrostatic association that small-HPF remained intact in solution and no amine was displaced by the ammonium ions of the buffer. Small-HPF remained unimpaired during purification, since all activity was retained despite alternating acidic and basic conditions. However, when small-HPF was brought into contact with either a cationic or an anionic resin, it was dissociated to completion when mixed continuously with the resin for 4 days. The ionic entity that was released had no bio-activity and was either a pure radioactively labeled saccharide or a non-labeled amine, depending on the kind of resin used. When incubated together, the separated counterions combine to regain full activity after 2 days of reassociation. However, with incubation for longer, this reassociated small-HPF formed different oligomeric HPFs by aggregation. Small-HPF is therefore a new kind of growth enhancer, consisting of an acidic sulfonated saccharide and a basic amine assembled into a stable active ionic complex that has a tendency to aggregate.     

On the structure of heparitin sulfates. Analyses of the products formed from heparitin sulfates by two heparitinases and a heparinase from Flavobacterium heparinum.

The analyses of the products formed from heparitin sulfates by the action of two heparitinases and a heparinase from Flavorbacterium heparinum is reported. Heparitin sulfates A and B are degraded by heparitinase I yielding two disaccharides, one of them composed of N-acetylucosamine and an unsaturated uronic, joined by alpha(1 lead to 4) linkage, and the other, with the same composition but with an O-sulfate at the hexosamine moiety. A third disaccharide is also formed from heparitin sulfate B, by the action of the same enzyme, composed of glucosamine N-sulfate and an unsaturated uronic acid joined probably by alpha(1 lead to 4) linkage. Besides these three disaccharides, heparitin sulfate B yields, by the action of heparitinase I, an oligosaccharide (with an average molecular weight of 6000) which is completely degraded by the heparitinase II yielding a disaccharide composed of glucosamine 2,6-disulfate and unsaturated uronic acid. All the disaccharides are further degraded by alpha-glycuronidase from Flavobacterium heparinum yielding the respective monosaccharides. Based on these and other analyses the possible structures of the heparitin sulfates are proposed.     

Characterization of soluble vs membrane-bound human placental 5'-nucleotidase

Three forms of 5'-nucleotidase purified from human placenta (two membrane-bound forms, one sensitive and one resistant to cleavage by phosphatidylinositol-specific phospholipase C, as well as a soluble form) had the same molecular weight before (73,000 Da) and after (56,000 Da) digestion with N-glycosidase F and showed similar amino acid compositions, N-terminal amino acid sequences, and KMs for IMP (9.6 to 11.9 microM). Thus, these three forms of 5'-nucleotidase appear to have very similar structures. The form sensitive to phosphatidylinositol-specific phospholipase C contained nearly 1 mol myo-inositol/mol of protein as determined by mass spectrometry, indicating a glycosyl phosphatidylinositol membrane anchor. Soluble 5'-nucleotidase contained a similar quantity of myo-inositol, suggesting that it was previously membrane-anchored via glycosyl phosphatidylinositol. The form resistant to phosphatidylinositol-specific phospholipase C contained less myo-inositol, leaving open the possibility of a third form of 5'-nucleotidase with a conventional transmembrane anchor.     

On the structure of heparitin sulfates Analyses of the products formed from heparitin sulfates by two heparitinases and a heparinase from Flavobacterium heparinum

The analyses of the products formed from heparitin sulfates by the action of two heparitinases and a heparinase from Flavobacterium heparinum is reported. Heparitin sulfates A and B are degraded by heparitinase I yielding two disaccharides, one of them composed of N-acetylglucosamine and an unsaturated uronic, joined by α(1 → 4) linkage, and the other, with the same composition but with an O-sulfate at the hexosamine moiety. A third disaccharide is also formed from heparitin sulfate B, by the action of the same enzyme, composed of glucosamine N-sulfate and an unsaturated uronic acid joined probably by α(1 → 4) linkage. Besides these three disaccharides, heparitin sulfate B yields, by the action of heparitinase I, an oligosaccharide (with an average molecular weight of 6000) which is completely degraded by the heparitinase II yielding a disaccharide composed of glucosamine 2,6-disulfate and unsaturated uronic acid. All the disaccharides are further degraded by α-glycuronidase from Flavobacterium heparinum yielding the respective monosaccharides. Based on these and other analyses the possible structures of the heparitin sulfates are proposed.     

Structural elucidation of the EPS of slime producing Brevundimonas vesicularis sp. isolated from a paper machine

The slime forming bacteria Brevundimonas vesicularis sp. was isolated from a paper mill and its EPS was produced on laboratory scale. After production, the exopolysaccharide (EPS) was purified and analysed for its purity and homogeneity, HPSEC revealed one distinct population with a molecular mass of more than 2,000 kDa. The protein content was around 9 w/w%. The sample was analysed to determine its chemical structure. The EPS was found to consist of rhamnose, glucose, galacturonic acid and glucuronic acid. Due to the presence of uronic acids the molar ratio between the four sugars found varies from 3:5:2:4 by sugar composition analyses after methanolysis to 1:1:1:1 found by NMR. A repeating unit with a molecular mass of 678 Da was confirmed by MALDI-TOF mass spectrometry after mild acid treatment. 13C and 1H hetero- and homonuclear 2D NMR spectroscopy of the native and partial hydrolysed EPS revealed a repeating unit, no non-sugar substituents were present.     

Grucuronic acid-containing glycopeptide from squid cartilage

A glycopeptide fraction containing glucuronic acid as a component sugar was extracted and purified from squid cartilage to give a single band migrating much slower than hyaluronic acid in cellulose acetate electrophoresis. The molecular weight of the glycopeptide was fairly large since its K_av value in Sephadex G-200 chromatography was 0.18; however, it was soluble in 66% ethanol. This glycopeptide contained glucuronic acid, glucosamine, galactosamine, galactose, and fucose. The total amino acid content was 1.87 μmol of amino acid per mg of the glycopeptide. Threonine, serine and proline represented 80% of the amino acids. Digestion with chondroitinase ABC or reaction with nitrous acid did not result in degradation of the glycopeptide; however, it was completely degraded by reaction with 0.5 M KOH at 37°C. Two hexasaccharides were separated from the alkaline degradation products, and they both contained glucuronic acid, fucose, galactosamine, and reducing terminal glucosamine in the molar ratio, 2:1:2:1. These results indicated that the glycopeptide contains glucuronic acid-containing sugar chains that are distinct from any known glycosaminoglycan.     

Substrate specificity of a heparan sulfate-degrading endoglucuronidase from human placenta.

A heparan sulfate-degrading endoglucuronidase was isolated from human placenta and partially purified by affinity chromatography on heparan sulfate-Sepharose 4B. The endoglucuronidase has a molecular weight of approximately 100 000 estimated by gel chromatography and a broad pH optimum between pH4 and pH6. Carboxyl reduced heparan sulfate is not split by partially purified endoglucuronidase, but inhibits the action of that enzyme towards non-modified heparan sulfate. Low molecular weight heparan sulfate (Mr approximately 3 000) is not attacked by the endoglucuronidase. N-Desulfated heparan sulfate and heparin are only weak substrates. The amino sugar adjacent to the glucuronic acid residue appearing at the reducing terminal of heparan sulfate fragments liberated by the endoglucuronidase appears to be exclusively N-acetylated glucosamine.     

Purification,molecular cloning,and characterization of pyridoxine 4-oxidase from Microbacterium luteolum

Pyridoxine 4-oxidase (EC 1.1.3.12, PN 4-oxidase), which catalyzes the oxidation of PN by oxygen or other hydrogen acceptors to form pyridoxal and hydrogen peroxide or reduced forms of the acceptors, respectively, was purified for the first time to homogeneity from Microbacterium luteolum YK-1 (=Aureobacterium luteolum YK-1). The purified enzyme required FAD for its catalytic activity and stability. The enzyme was a monomeric protein with the subunit molecular mass of 53,000 +/- 1,000 Da. PN was the only substrate as the hydrogen donor. Oxygen, 2,6-dichloroindophenol, and vitamin K3 were good substrates as the hydrogen acceptor. The gene (pno) encoding PN 4-oxidase was cloned. The gene encodes a protein of 507 amino acid residues corresponding to the molecular mass of the subunit. PN 4-oxidase was expressed in Escherichia coli and found to have the same properties as the native enzyme from M. luteolum YK-1. Comparisons of primary and secondary structures with other proteins showed that the enzyme belongs to the GMC oxidoreductase family. M. luteolum YK-1 has four plasmids. The pno gene was found on a chromosomal DNA. Search for genes similar in sequence in other organisms suggested that a nitrogen-fixing symbiotic bacterium, Mesorhizobium loti, which harbors two plasmids, has a PN degradation pathway I in chromosomal DNA.     

Mechanism of inhibition of the class A beta -lactamases PC1 and TEM-1 by tazobactam. Observation of reaction products by electrospray ionization mass spectrometry

The reactions of class A beta-lactamases PC1 and TEM-1 with tazobactam (TZB), a potent penicillanic sulfone inhibitor for class A beta-lactamases, were studied using electrospray ionization mass spectrometry (ESI/MS). Following inactivation of the beta-lactamases by TZB, new abundant high mass components were observed including three with molecular masses of 52, 70, and 88 Da greater than PC1 and TEM-1, respectively, and a component with a molecular mass of 300 Da greater than PC1. In addition, three TZB reaction products with molecular masses of 248, 264, and 280 Da were observed. High performance liquid chromatography (HPLC)/ESI/MS analysis of the TZB-PC1 adduct digested with Glu-C revealed three new components with masses 52, 70, and 88 Da greater than that of the peptide composed of amino acid residues 58-82 and one new component with a mass 70 Da greater than that of the peptide composed of amino acid residues 125-141. HPLC/ESI/MS/MS analysis of the two digested peptides whose masses increased by 70 Da indicated that Ser-70 and Ser-130 were the most likely TZB-modified amino acid residues. Based on these data, a mechanism for the inactivation of the class A beta-lactamases by TZB is proposed. In this scheme, initial acylation of Ser-70 by TZB and opening of the lactam ring are followed by one of several different events: (1) the rapid decomposition of TZB with loss of the enamine moiety to form the propiolylated enzyme, (2) an intramolecular nucleophilic displacement of the imine or enamine moiety by Ser-130 to form a cross-linked vinyl ether, and (3) hydrolysis of the imine or enamines to form a Ser-70-linked aldehyde.     

Cell wall and sheath constituents of the cyanobacterium Gloeobacter violaceus

Sheaths isolated from Gloeobacter violaceus were found to be composed of a major polysaccharide moiety (glucose, galactose, rhamnose, mannose, arabinose), a protein moiety, and negatively charged components (glucuronic acids, phosphate, sulfate). Outer membrane polypeptide patterns were dominated by two major peptidoglycan-associated proteins (M_r 62,000 and 53,000). Lipopolysaccharide constituents were glucosamine, 3-hydroxy fatty acids (3-OH-14:0, anteiso-3-OH-15:0, 3-OH-16:0, 3-OH-18:0), carbohydrates, and phosphate. A1-type peptidoglycan and non-peptidoglycan components (mannosamine, glucose, mannose, and glucosamine) indicated the presence of a peptidoglycan-polysaccharide complex in the cell walls of Gloeobacter violaceus.Abbreviations A₂pm diaminopimelic acid - ATCC American Type Culture Collection - CE cell envelope - CM cytoplasmic membrane - CW cell wall - dOcla 3-deoxy-d-manno-2-octulosonic acid - GalN galactosamine - GlcN glucosamine - GlcUA glucuronic acid - HF hydrofluoric acid - LPS lipopolysaccharide - ManN mannosamine - M relative molecular mass - MurN muramic acid - MurN-6-P muramic acid-6-phosphate - OMe O-methyl - PAGE polyacrylamide gel electrophoresis - PCC Pasteur Culture Collection - SDS sodium dodecyl sulfate - SH sheath     

Characterization of rat adipose tissue lipoprotein lipase using a monospecific antibody

An antibody to a highly pure enzyme preparation was developed to facilitate detailed studies of rat adipose tissue lipoprotein lipase regulation. Lipoprotein lipase was purified by heparin-Sepharose affinity chromatography followed by preparative isoelectric focusing. The enzyme migrated as a single broad band on SDS disc gel and two-dimensional gel electrophoresis with an apparent molecular mass of 67 000 and 62 000 Da, respectively. The amino acid composition of the purified rat enzyme was virtually identical to that of bovine milk. A major protein component with no lipase activity co-eluted with the enzyme from the affinity column, but was separated by the isoelectric focusing step. The molecular mass was slightly lower (58 000 Da) but the amino acid composition of this protein was similar to that of the enzyme. An antibody raised against the purified rat enzyme was highly potent and was effective in inhibiting rat heart lipoprotein lipase, but not the salt-resistant hepatic lipase. Analysis of crude acetone-ether adipose tissue preparation on SDS slab polyacrylamide gel coupled to Western blotting revealed five protein bands = (62 000, 56 000, 41 700, 22 500, 20 000 Da). Similarly, following affinity purification by immunoadsorption, the purified antibody reacted with five equivalent protein bands. Fluorescent concanavalin A binding data indicated that the 56 kDa band is a glycosylated form of lipoprotein lipase. Pretreatment of adipose tissue with proteinase inhibitors revealed that the lower molecular mass proteins (41 700 and 20 000 Da) were degradation products of lipoprotein lipase, and the 22 500 Da band could be accounted for by non-specific binding.     

Seed lectin from pisum arvense: isolation, biochemical characterization and amino acid sequence

A glucose/mannose lectin was purified by affinity chromatography from Pisum arvense seeds (PAL) and the 50 kDa molecular mass in solution determined by size exclusion chromatography. SDS-PAGE and electrospray ionization mass spectrometry showed two distinct polypeptide chains: alpha (Mr. 5591 Da) and beta (19986 Da). The lectin was extensively characterized in terms of its biochemical and biological aspects. The amino acid sequence was established by Edman degradation of overlapping peptides. PAL in solution behaves as a dimer and has its monomeric structure formed by two distinct polypeptide chains named alpha (Mr. 5591 Da) and beta (19986 Da) by Electrospray ionization (ESI) mass spectrometry. PAL possesses identical amino acid sequences to that of pea seed lectin but undoubtedly does not exhibit sequence heterogeneity. It is discussed that P. arvense should be considered as a synonym of P. sativum. Furthermore, like pea lectin, PAL discriminates biantennary fucosylated glycan, determined by surface plasmon resonance.     

Biochemical and EPR characterization of a high potential iron-sulfur protein in Thiobacillus ferrooxidans

Abstract A soluble acid-stable high potential iron-sulfur protein (HiPIP) was purified from Thiobacillus ferrooxidans using the periplasmic extraction method. It was isolated in the form of a tetramer consisting of four subunits with a molecular mass of 5582 Da, and its biochemical and biophysical properties were characterized. The N-terminal amino acid sequence (15 residues) was compared with the nucleotide sequence of the iro gene isolated from another strain and the two sequences were found to be identical. The iron content measurement together with optical and EPR spectroscopic studies of the purified protein were consistent with the presence of one [4Fe-4S] cluster per subunit. The EPR spectrum recorded in the oxidized state was attributed to a [4Fe-4S]³⁺ cluster and the redox potential has been determined to be +380 mV.     

Purification and characterization of 2,6-dichloro-p-hydroquinone chlorohydrolase from Flavobacterium sp. strain ATCC 39723.

The biochemistry of pentachlorophenol (PCP) degradation by Flavobacterium sp. strain ATCC 39723 has been studied, and two enzymes responsible for the conversion of PCP to 2,6-dichloro-p-hydroquinone (2,6-DiCH) have previously been purified and characterized. In this study, enzymatic activities consuming 2,6-DiCH were identified from the cell extracts of strain ATCC 39723. The enzyme was purified to apparent homogeneity by a purification scheme consisting of seven steps. Gel filtration chromatography showed a native molecular weight of about 40,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein of 42,500 Da. The purified enzyme converted 2,6-DiCH to 6-chlorohydroxyquinol (6-chloro-1,2,4-trihydroxybenzene), which was easily oxidized by molecular oxygen and hard to detect. The end product, 6-chlorohydroxyquinol, was detected only in the presence of a reductase and NADH in the reaction mixture. The enzyme dechlorinated 2,6-DiCH but not 2,5-DiCH. The enzyme required Fe2+ for activity and was severely inhibited by metal chelating agents. The optimal conditions for activity were pH 7.0 and 40 degrees C. The Kcat for 2,6-DiCH was 35 microM, and the kcat was 0.011 s-1.     

Enzymatic determination of free glucuronic acid with glucuronolactone reductase. I. Isolation and purification of glucuronolactone reductase from rat kidney

Glucuronolactone reductase [EC 1.1.1.20] from rat kidney was purified over 300-fold by ammonium sulfate fractionation, chromatography on DEAE-cellulose and hydroxylapatite columns, and preparative isoelectric focusing. The substrate specificity of the enzyme in the reduction reaction was broad, and hexuronic acid was one of the best substrates among monosaccharides. Km values for D-glucuronic acid, D-glucuronolactone, D-galacturonic acid, and L-iduronic acid were 6, 9, 4, and 6 mM, respectively. An investigation of the activity for aldose led to the finding that triose and tetrose served as good substrates for this enzyme. However, the activity for aldopentose or aldohexose was less than 1% of that for D-glucuronic acid at the same concentration. The enzyme was inactive towards most hexosamines (galactosamine, mannosamine, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine, but not glucosamine), meso-inositol, D-fructose, and tetrasaccharides from hyaluronic acid and chondroitin 4-sulfate. Trisaccharides from hyaluronic acid and chondroitin 6-sulfate which possess glucuronic acid at the reducing end were poor substrates for the enzyme and the activity towards these 4-substituted glucuronic acids was less than 3% of that towards non-substituted glucuronic acid.     

Enhanced degradation of gamma-irradiated lignocelluloses by a new xylanolytic Flavobacterium sp. isolated from soil

An extracellular chitosanase produced by Rhodotorula gracilis CFR-1 that catalyses a limited degradation of chitosan with no detectable generation of glucosamine or reducing groups was identified. Ultracentrifugation, polyacrylamide gel electrophoresis and gel permeation studies suggest that chitosan of average molecular mass 36000 Da was reduced by the enzymic catalysis to nearly one-fourth this size without further hydrolysis of the products. The enzyme, produced constitutively by this yeast, was partially purified and some of its properties were studied.     

Modulation of drug-stimulated ATPase activity of human MDR1/P-glycoprotein by cholesterol

MDR1 (multidrug resistance 1)/P-glycoprotein is an ATP-driven transporter which excretes a wide variety of structurally unrelated hydrophobic compounds from cells. It is suggested that drugs bind to MDR1 directly from the lipid bilayer and that cholesterol in the bilayer also interacts with MDR1. However, the effects of cholesterol on drug-MDR1 interactions are still unclear. To examine these effects, human MDR1 was expressed in insect cells and purified. The purified MDR1 protein was reconstituted in proteoliposomes containing various concentrations of cholesterol and enzymatic parameters of drug-stimulated ATPase were compared. Cholesterol directly binds to purified MDR1 in a detergent soluble form and the effects of cholesterol on drug-stimulated ATPase activity differ from one drug to another. The effects of cholesterol on K(m) values of drug-stimulated ATPase activity were strongly correlated with the molecular mass of that drug. Cholesterol increases the binding affinity of small drugs (molecular mass <500 Da), but does not affect that of drugs with a molecular mass of between 800 and 900 Da, and suppresses that of valinomycin (molecular mass >1000 Da). V(max) values for rhodamine B and paclitaxel are also increased by cholesterol, suggesting that cholesterol affects turnover as well as drug binding. Paclitaxel-stimulated ATPase activity of MDR1 is enhanced in the presence of stigmasterol, sitosterol and campesterol, as well as cholesterol, but not ergosterol. These results suggest that the drug-binding site of MDR1 may best fit drugs with a molecular mass of between 800 and 900 Da, and that cholesterol may support the recognition of smaller drugs by adjusting the drug-binding site and play an important role in the function of MDR1.     

Granulin-like peptide in the mid-gut gland of the bivalve mollusk, Patinopecten yessoensis

A cysteine-rich polypeptide, termed CRP1, with a molecular mass of 5829 Da was found to occur in the mid-gut gland of the scallop Patinopecten yessoensis. CRP1 was purified by reverse phase and cation-exchange chromatographies. The amino acid sequence of CRP1 was deduced from its N-terminal amino acid sequence, amino acid composition and the sequence of a partial cDNA, indicating that CRP1 is a 57-amino-acid polypeptide containing 12 cysteine residues with a calculated molecular mass of 5841 Da (5829 Da when oxidized to form six disulfide bridges). A homology search of databases revealed that the deduced amino acid sequence of CRP1 displays significant similarity to those of granulin/epithelins, a family of growth-modulating factors; all cysteine residues in CRP1 are located at the same positions as those conserved characteristically in other known granulin/epithelins. Purified CRP1 inhibited the proliferation of mouse embryo cells. The results suggest that CRP1 functions as a growth-modulating factor in the scallop, and that granulin/epithelin family polypeptides and their precursors play physiologically important roles in invertebrates.     

Two novel muramidases from skin mucosa of rainbow trout (Oncorhynchus mykiss)

Two novel antibacterial muramidases were purified to homogeneity from skin exudates of rainbow trout (Oncorhynchus mykiss). Unusually, one has an acidic isoelectric point and it is the first anionic muramidase to be reported for fish. Its molecular mass is 14,268 Da, as determined by mass spectrometry. The other muramidase is cationic with a mass of 14,252 Da. Partial N-terminal amino acid sequencing and peptide mapping strongly point to it being a c-type lysozyme, the first to be purified and characterised from skin of a salmonid. Its optimum pH ranges from 4.5 to 5.5 and its optimum temperature, at pH 5.0, is 33-49 degrees C, although it still exhibits activity at 5 degrees C. It is strongly bactericidal to the Gram-(+) bacterium Planococcus citreus, with a minimum bactericidal concentration of 100 U ml(-1), but is neither chitinolytic nor haemolytic. These two muramidases probably contribute to epithelial defence of the fish against microbes, either alone or in synergism with antibacterial peptides.