Gel filtration studies on oxyhemerythrin. II. Effect of temperature and ionic strength on the association-dissociation equilibria.

The effects of temperature and ionic strength on the association of oxyhemerythrin have been studied. deltaH degrees and deltaS degrees for association at pH 7.0 are -2.6 kcal and +16.5 eu per mol of monomer. These values suggest that solvent adjacent to the surface of the protein undergoes rearrangement on association. Increasing ionic strength is observed to promote dissociation while decreasing the rate of attainment of equilibrium between monomers and octamers. Qualitatively similar results are observed on lowering the pH from 7.0 to 4.8, thereby linking the effects of increasing ionic strength to those of protonation of specific amino acid residues at the subunit contacts of hemerythrin. The apparent enthalpy of ionization of the amino acid residue controlling dissociation at acidic pH was found to be -1.9 to +2.1 kcal/mol. These values are consistent with a carboxyl group.     

The hydrolysis of nicotinamide adenine nucleotide by brush border membranes of rat intestine.

The hydrolysis of NAD by rat intestine was studied to determine the subcellular site of this hydrolysis and to identify the niacin-containing products that are formed. Using [nicotinamide-14C]NAD as substrate, and high pressure liquid chromatography for identification and quantification of products, the present study demonstrates two independent reactions for the hydrolysis of NAD; one that forms nicotinamide through hydrolysis of the ribosyl-pyridinium bond and one that forms nicotinamide mononucleotide through the hydrolysis of the pyrophosphate bond. The nicotinamide mononucleotide is subsequently dephosphorylated to nicotinamide riboside. Enzymes which release nicotinamide mononucleotide and nicotinamide riboside are associated with the brush border membrane as determined by analysis of fractionated intestinal homogenates. The enzyme activity which releases nicotinamide from NAD is associated with the brush border membrane fraction and also with a second cellular particulate fraction. Between pH5 and pH6 NAD is hydrolysed principally to nicotinamide. At pH 7.0 rates of nicotinamide and nicotinamide mononucleotide formation are the same. Above pH 7.0 the formation of nicotinamide mononucleotide is preferred.     

Murine epidermal growth factor: heterogeneity on high resolution ion-exchange chromatography

We have shown that epidermal growth factor (EGF) purified either by the classical method of Savage and Cohen, or solely by h.p.l.c. techniques can be resolved into two species, EGF alpha and EGF beta. However, despite the apparent purity of such materials, as determined both chromatographically and by amino acid analysis, they failed to give homogeneous products on radioiodination. Analysis by isoelectric focusing on agarose gels followed by transfer to nitrocellulose and silver staining showed that EGF alpha could be further resolved into three sub-species which focused at pH 4.6, 4.3 and 4.1. EGF beta (which also focused at pH 4.6) contained very small amounts of the species with isoelectric points of 4.1 and 4.3, probably due to slight contamination of this preparation by EGF alpha. Preparative separation of the sub-species of EGF alpha was achieved by high performance anion-exchange chromatography at pH 6.5 on a Pharmacia Mono Q column. Radioiodination of these purified sub-species did not produce significant charge heterogeneity. However, two slightly different forms of [125I]EGF alpha 1 (pH 4.6 species) were separable by anion-exchange chromatography on the Mono Q column. All of the EGF species competed for binding to EGF receptors on A431 cells and were active mitogens for BALB/c 3T3 fibroblasts.     

pH Transitions in ion-exchange systems: role in the development of a cation-exchange process for a recombinant protein

Unexpected transient changes in effluent pH can occur during ion-exchange chromatography. Such changes can occur even if a column that is equilibrated with a buffer receives another solution in the same buffer and of the same pH but of a different salt concentration. An attempt is made to understand the basis for this phenomenon and apply it to the process purification of a recombinant protein on a strong cation-exchange resin. Incomplete column equilibration was eliminated as a possible cause of these effects. Various buffering species and various salt ions were studied at different solution concentrations to investigate pH transitions on strong cation-exchange resins. A further comparison was made between cation-exchange resins with different backbone chemistries. On the basis of these studies, a mechanism is proposed for these phenomena based on competitive equilibria between ions from the buffer salts and H(+)/OH(-) ions. In addition to the equilibria between these ions and the functional groups on the resins, charged groups on the resin backbone were also found to contribute to transient pH changes. The results from this study were applied to the cation-exchange step for a recombinant protein that was sensitive to pH excursions to help maintain activity of the protein during the purification process.     

Photolysis of desmosine and isodesmosine by ultraviolet light.

1. Desmosine and isodesmosine were separated by ion-exchange and paper chromatography, after acid hydrolysis of purified elastin from beef ligamentum nuchae. The fractions obtained by ion-exchange chromatography were clearly mixtures of related compounds. The desmosine fraction could be resolved into seven compounds and the isodesmosine into four by paper chromatography. 2. Desmosine was maximally degraded by irradiation at 274 nm and isodesmosine at 285 nm. These wavelengths did not correspond to the absorption maxima of the cross links, but to shoulders of the main absorption peaks. 3. When irradiated at their optimum wavelengths, but at various pH, both desmosine and isodesmosine seemed quite stable at pH greater than 8.5. Between pH 8 and 5, the photolytic rate was maximum and decreased slightly at more acidic pH. Below pH 4.0, one of the products of photolysis was free lysine. 4. In analogy to the mechanism of the photolytic degradation of N-methyl pyridinium chloride, it appears that the (iso)desmosines were degraded via the formation of an open amino aldehyde, which was hydrolysed at acid pH to give free lysine and a substituted glutaconic aldehyde.     

Purification and properties of phosphofructokinase from fruits of Lycopersicon esculentum

A procedure is described using affinity chromatography on Blue Sepharose and on an immobilized ATP column by which phosphofructokinase has been purified by 260-fold from tomato fruits. The properties of the enzyme are affected by the pH at which the preparation is made and maintained. At the pH optimum, pH 8.0, the enzyme is very heterogeneous with up to three forms present differing in MW. At pH 7.5 a single major form of MW 180 000 is present, and evidence that raising the pH to 8.0 promotes dissociation of the enzyme is discussed.     

Peripheral enkephalin hydrolysis in different animal species: A comparative study

Using column and thin layer chromatography, plasma hydrolysis of leu-enkephalin has been studied in man and several laboratory animals. The hydrolysis kinetics determined in the various species examined are considerably different. In addition, also the enzyme forms evidentiated, their molecular weight distribution and relative ratios have been found to vary greatly in the animals under test. Our data suggest that the widely different hydrolysis kinetics reported by various authors are attributable to the differences between species, rather than to differences in the analytical techniques employed.     

Kinetic light scattering studies on the dissociation of hemoglobin from Lumbricus terrestris.

The kinetics of the pH-induced dissociation of the 3 X 10(6) mol wt hemoglobin from Lumbricus terrestris (the earthworm) have been studied in a light-scattering stopped-flow apparatus. The ligand dependent dissociation data were fit well by a simple sequential model. The data for CO and oxyhemoglobin are consistent with Hb12 leads to 2Hb6 leads to 12Hb. Methemoglobin at pH 7 appears to be hexameric and the dissociation is consistent with the model: Hb6 leads to 6Hb. In a sequential decay scheme for which light-scattering changes are monitored, the relative amounts of rapid and slow phase are determined by the rate constants as well as the molecular weights of intermediate species. Assignment of the hexameric intermediate is supported by an investigation of the sensitivity of the theoretical kinetic curves to the molecular weights of the intermediates. This assignment is further supported by the following: (1) the same model will fit the data for oxy- and CO-hemoglobin at all three temperatures (a 24-29-fold variation in rate constants), (2) evidence from electron microscopy shows hexameric forms, and (3) methemoglobin is apparently stable as a hexamer at pH 7. When CO replaces O2 as the ligand, the dissociation rate increases by a factor of four. The met is about 20 times faster than the initial oxyhemoglobin dissociation rate, but perhaps more relevant for comparing dissociation of the hexamer, the met rate was respectively 100 times and 500 times faster than that for the assumed hexameric forms of CO- and oxy-hemoglobin. The activation energies for the dodecamer to hexamer dissociation and for the dissociation of the hexamer to smaller forms were about 30 kcal/mol for oxy-, CO-, and methemoglobin.     

Purification and characterization of histidine decarboxylase from mouse kidney.

Histidine decarboxylase was purified 800-fold from the kidneys of thyroxine-treated mice. The purification procedure included precipitation of protein from a crude supernatant after heating it to 55 degrees C at pH 5.5, fractionation with (NH4)2SO4, phosphocellulose column chromatography, chromatofocusing, DEAE-Sepharose column chromatography, gel filtration on Sephacryl S-300 and preparative polyacrylamide-gel electrophoresis. The native enzyme had an estimated Mr of 113 000. The protein was analysed in SDS/10%-polyacrylamide gels and formed a single band corresponding to a subunit Mr of 55 000, indicating that it is a dimer. Three forms of the enzyme were resolved on isoelectrofocusing gels, with pI 5.3, 5.5 and 5.7.     

Kinetic study of isomerization of ferricytochrome c at alkaline pH

Kinetic studies of the isomerization reaction of horse heart ferricytochrome c between pH 8.5 and pH 12.1 have been carried out by using stopped-flow and rapid scanning stopped-flow techniques. Below pH 10, our results were in good agreement with the scheme proposed earlier (Davis, L. A., Schejter, A. and Hess, G. P. (1974) J. Biol. Chem. 249, 2624–2632). Above pH 10, another faster first-order process was observed, which suggested the existence of a transient species in the isomerization reaction between the species with and without a 695 nm band. The probable scheme of the isomerization reaction is considered to be

where H denotes a proton, the colored forms are the species predominant at neutral pH with a 695 nm band and the noncolored forms are the species without a 695 nm band. The transient species has a small 695 nm absorbance which suggests that the sixth ligand is still Met-80, although the protein conformation might be different from that at neutral pH.     

Bovine liver formaldehyde dehydrogenase. Kinetic and molecular properties

The dimeric formaldehyde dehydrogenase from bovine liver has been resolved into three nearly homogeneous enzyme forms by the successive use of ion-exchange, affinity, and ampholine (chromatofocusing) chromatography. The different enzyme species were isolated in the approximate proportions 3:2:1, having pI values of 6.5, 6.2, and 6.0, respectively. The subunit molecular weights of the three forms are all similar (Mr congruent to 41,000), on the basis of sodium dodecyl sulfategel electrophoresis. The enzyme species appear to arise from covalent differences unrelated either to partial proteolysis during isolation or to differential sialization of homodimeric protein. Human liver contains a single major form and two minor forms of formaldehyde dehydrogenase having pI values very similar to those found for the bovine liver enzyme. The macroscopic kinetic constants (V, V/K) for the three forms of the dehydrogenase from bovine liver are all similar in magnitude, using NADH and S-hydroxymethylglutathione as substrates. The isotope-sensitive hydride transfer step is not significantly rate-limiting during catalysis by any of the forms, as evidenced by the near-unity primary deuterium isotope effects on both V and V/KS (for S-hydroxymethylglutathione); catalysis may be limited by the rate of dissociation of at least one (and possibly both) of the product molecules. In support of rate-limiting dissociation of NAD+ in the normal reaction, V increases by approximately 22-fold and isotope effects of approximately 1.4 are observed on both V and V/KS, using the coenzyme analog 3-acetylpyridine adenine dinucleotide. Product dissociation from the active site appears to be accelerated by the presence of dilute denaturing agents, perhaps indicative of a rate-limiting conformational transition associated with product release.     

Structure of the lysosomal neuraminidase-beta-galactosidase-carboxypeptidase multienzymic complex.

Lysosomal neuraminidase (sialidase; EC 3.2.1.18) and beta-galactosidase (EC 3.2.1.23), together with a carboxypeptidase, the so-called 'protective protein', were co-purified from the human placenta by affinity chromatography on a concanavalin A-Sepharose column followed by a thiogalactoside-agarose affinity column for beta-galactosidase. Analysis of the purified material by gel-filtration h.p.l.c. revealed three distinct molecular forms, all with high beta-galactosidase specific activity, but only the largest one expressed neuraminidase activity. Rechromatography of each individual species separately indicated that all three are in fact part of an equilibrium system (the neuraminidase-beta-galactosidase-carboxypeptidase complex or NGC-complex) and that these species undergo slow conversion into one another through dissociation and association of protomeric components. Each species was sufficiently stable for the determination of their hydrodynamic properties by gel-filtration h.p.l.c. and sedimentation velocity. The largest species had an apparent sedimentation coefficient S20.w, of 18.8 S and a Stokes' radius of 8.5 nm, giving a molecular mass of 679 kDa and a fractional ratio, f/f min, of 1.47. The latter value indicates that the macromolecule is asymmetric or highly hydrated. This large species is composed of four types of polypeptide chains of molecular mass 66 kDa (neuraminidase), 63 kDa (beta-galactosidase), 32 kDa and 20 kDa (carboxypeptidase heterodimer). The 32 kDa and 20 kDa protomers are linked together by a disulphide bridge. Glycopeptidase F digestion of the NGC-complex transformed the diffuse 66-63 kDa band on the SDS gel into two close but sharp bands at 58 and 56 kDa. The two smaller species which were separated on the h.p.l.c. column correspond to tetrameric and dimeric forms of the 66-63 kDa protomers and express exclusively beta-galactosidase activity. Treatment of the NGC-complex with increasing concentrations of guanidinium hydrochloride up to 1.5 M also resulted in dissociation of the complex into the same smaller species mentioned above plus two protomers of molecular mass around 60 and 50 kDa. A model of the largest molecular species as a hexamer of the 66-63 kDa protomers associated to five carboxypeptidase heterodimers (32 kDa and 20 kDa) is proposed     

Human cationic trypsinogen. Purification, characterization, and characteristics of autoactivation

Human pancreatic cationic trypsinogen has been purified to homogenity from an acetone powder of pancreatic tissue. After an initial ion exchange chromatography step on sulfopropyl (SP)-Sephadex at pH 2.6, cationic trypsinogen was separated from the majority of trypsin activity by passage through an affinity column of lima bean trypsin inhibitor-agarose at high ionic strength. The zymogen was then further purified by affinity chromatography on the same material at low ionic strength. Highly purified trypsinogen was resolved from containing chymotrypsinogen by ion exchange chromatography on SP-Sephadex at pH 6.0. The purified zymogen was shown to be homogeneous by polyacrylamide gel electrophoresis at pH 2.1 and at pH 4.3 as well as by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The autoactivation of human trypsinogen was investigated at pH 5.6 and at pH 8.0. The rate of autoactivation of the human zymogen is rapid at pH 5.6 and is maximal in approximately 1 mM Ca2+. These results are in marked contrast to those previously reported for autoactivation of bovine trypsinogen, which is extremely slow at pH 5.6 and which shows a dependence on at least 50 mM Ca2+ for maximum rate of activation (MacDonald, M. R., AND Kunitz, M. (1941) J. Gen. Physiol. 25, 53-73).     

Studies on brain cytosol neuraminadase. I. Isolation and partial characterization of two forms of the enzyme from pig brain.

1. Two forms of cytosol neuraminidase (EC 3.2.1.18) (neuraminidase A and neuraminidase B) were isolated and purified from pig brain homogenate, by proceeding through the following steps: centrifugation of brain homogenate at 105 000 X g (1h); ammonium sulphate fractionation (35-55% saturated fraction); column chromatography on Biogel A 5 m; column chromatography on hydroxy apatite/cellulose gel; affinity chromatography on Affinose-tyrosyl-p-nitrophenyloxamic acid. The separation of the two forms of neuraminidase was provided by chromatography on hydroxylapatite/cellulose gel. Neuraminidase A was purified about 500-fold; neuraminidase B about 400-fold. 2. The pH optima and the maximum activities in various buffers were different for neuraminidase A and B (for instance the pH optimum was in sodium acetate/acetic acid buffer, 4.7 for neuraminidase A and 4.9 for neuraminidase B). Ions affected in a different way the two enzymes: K+ activated neuraminidase A but not neuraminidase B; Na+ and Li+ inhibited neuraminidase A at a higher degree than neuraminidase B. Neuraminidase B seemed to be moderately activated by some bivalent cations (Ca2+; Mg2+; Zn2+); neuraminidase A did not. The Km values for sialyllactose were different: 2.2-10(-3) M for neuramindase A; 0.46-10(-3) M for neuraminidase B.     

The unusual dodecameric ferritin from Listeria innocua dissociates below pH 2.0.

The stability of the dodecameric Listeria innocua ferritin at low pH values has been investigated by spectroscopic methods and size-exclusion chromatography. The dodecamer is extremely stable in comparison to the classic ferritin tetracosamer and preserves its quaternary assembly at pH 2.0, despite an altered tertiary structure. Below pH 2.0, dissociation into dimers occurs and is paralleled by the complete loss of tertiary structure and a significant decrease in secondary structure elements. Dissociation of dimers into monomers occurs only at pH 1.0. Addition of NaCl to the protein at pH 2.0 induces structural changes similar to those observed upon increasing the proton concentration, although dissociation proceeds only to the dimer stage. Addition of sulfate at pH values >/= 1.5 prevents the dissociation of the dodecamer. The role played by hydrophilic and hydrophobic interactions in determining the resistance to dissociation of L. innocua ferritin at low pH is discussed in the light of its three-dimensional structure.     

Purification and characterization of alkaline phosphatase in cultured rat liver cells.

Alkaline phosphatase has been purified from cultured rat liver cells by butanol extraction, column chromatography on DEAE-cellulose and on Sephadex G-200, and preparative polyacrylamide gel electrophoresis. By electrophoresis on polyacrylamide, the purified enzyme was resolved into two active forms. Both forms have similar molecular weights of around 200,000. The subunit size was found to be 50,000 by SDS-polyacrylamide gel electrophoresis. These results suggest that alkaline phosphatase purified from cultured rat liver cells has a tetrameric structure. The optimum pH was found to be approximately 10.4, using p-nitrophenylphosphate as a substrate in a carbonate buffer system. The apparent Km was estimated to be 2.4 mM, using p-nitrophenylphosphate in carbonate buffer, pH 10.4.     

The intramolecular cyclization 2-Iodo-3-ureidopropionic acid.

2-Iodo-3-ureidopropionic acid resulting from the hydrolysis of 5-iodo-5,6-dihydrouracil catalyzed by either dihydrouracil amidohydrolase or hydroxide ion cyclizizes to yield 2-amino-2-oxazoline-3-carboxylic acid. This cyclization involves intramolecular attack of the ureido oxygen atom on carbon two of the ureidoacid to yield iodide ion and the oxazoline as products. The kinetics of this cyclization indicate that from pH 2 to 9 the reaction rate is pH independent. Below pH 2 the rate is diminished due to protonation of the ureido group. Above pH 12 the rate increases dramatically probably due to proton abstraction which would dramatically increase the nucleophilic character of the ureido function. In the pH independent region the reaction is not subject to catalysis by external buffers.     

Analytical techniques for cell fractions. XIX. The cyclum: an automatic system for cyclic chromatography.

An automatic system, termed a Cyclum, is described which allows column chromatographic separations to be repeated precisely a large number of times. Provision is made for the adjustment during operation of parameters such as equilibration, wash, elution, and sample flow times and duration of fraction collection. The system is applicable to both analytical and preparative use in various types of column chromatography (e.g., affinity, gel filtration, ion-exchange), but has been especially developed for separations based on immunosorption.     

Kinetics of the hemerythrin-oxygen interaction.

The kinetics of the reaction of Golfingia gouldii hemerythrin with O2 have been studied by stopped flow spectrophotometry. For the second order oxygenation process, k1 = 7.4 X 10(6) M-1 s-1, deltaH1++ = 8.2 kcal-mol-1 and deltaS1++ = +1 e.u. at 25 degrees, pH 8.2, and I = 0.015 M. The rate constant is unchanged when protein concentration is varied from 3 to 25 muM, the ionic strength is increased to 0.07 M, and the pH moved to 6.8. The deoxygenation of oxyhemerythrin is studied with stopped flow by scavenging liberated O2 with S2O4(2-). For the first order dissociation, k-1 = 51 s-1, deltaH-1++ = 20.6 kcal-mol-1 and deltaS-1++ = +19 e.u. at 25 degrees, pH 8.2, and I = 0.015 M. The value of k-1 is independent of [protein] = 50 to 200 muM, [S2O4(2-)] = 5 to 100 mM I = 0.015 to 0.30 M and pH 6.8 to 9.0. Using myoglobin instead of S2O4(2-) as scavenger gives similar results. Combination of activation parameters for the oxygenation and deoxygenation processes gives K1 = 1.5 X 10(5) M-1, deltaH = -12.4 kcal-mol-1, and deltaS = -18 e.u., values in good agreement with independent thermodynamic data. Perchlorate ion (0.05 M) enhances k-1 about 3-fold and hardly effects k1. There is no sign of other than a single reaction in either direction, and octameric hemerythrin apparently behaves kinetically as eight single units.     

Relationship between succinate transport and production of extracellular poly(3-hydroxybutyrate) depolymerase in Pseudomonas lemoignei

The relationship between extracellular poly(3-hydroxybutyrate) (PHB) depolymerase synthesis and the unusual properties of a succinate uptake system was investigated in Pseudomonas lemoignei. Growth on and uptake of succinate were highly pH dependent, with optima at pH 5.6. Above pH 7, growth on and uptake of succinate were strongly reduced with concomitant derepression of PHB depolymerase synthesis. The specific succinate uptake rates were saturable by high concentrations of succinate, and maximal transport rates of 110 nmol/mg of cell protein per min were determined between pH 5.6 and 6. 8. The apparent KS0.5 values increased with increasing pH from 0.2 mM succinate at pH 5.6 to more than 10 mM succinate at pH 7.6. The uptake of [14C]succinate was strongly inhibited by several monocarboxylates. Dicarboxylates also inhibited the uptake of succinate but only at pH values near the dissociation constant of the second carboxylate function (pKa2). We conclude that the succinate carrier is specific for the monocarboxylate forms of various carboxylic acids and is not able to utilize the dicarboxylic forms. The inability to take up succinate2- accounts for the carbon starvation of P. lemoignei observed during growth on succinate at pH values above 7. As a consequence the bacteria produce high levels of extracellular PHB depolymerase activity in an effort to escape carbon starvation by utilization of PHB hydrolysis products.