Purification and properties of soybean nodule xanthine dehydrogenase

Xanthine dehydrogenase (EC 1.2.1.37), an essential enzyme for ureide metabolism was purified from the cytosol fraction of soybean nodules. The purified xanthine dehydrogenase was shown to be homogeneous by electrophoresis and a pI of 4.7 was determined by isoelectric focusing. The enzyme had a molecular weight of 285,000 and two subunits of molecular weight 141,000 each. The holoenzyme contained 1.7 (±0.7) mol Mo and 8.1 (±2.0) mol Fe/mol enzyme and the enzyme also contained FMN and is thus a molybdoironflavoprotein. Soybean xanthine dehydrogenase is the second enzyme in plants demonstrated to contain Mo and the first xanthine-oxidizing enzyme reported to contain FMN, rather than FAD as the flavin cofactor.     

Saccharification of Complex Cellulosic Substrates by the Cellulase System from Clostridium thermocellum

True cellulase activity has been demonstrated in cell-free preparations from the thermophilic anaerobe Clostridium thermocellum. Such activity depends upon the presence of Ca²⁺ and a thiol-reducing agent of which dithiothreitol is the most promising. Under these conditions, native (cotton) and derived forms of cellulose (Avicel and filter paper) were all extensively solubilized at rates comparable with cellulase from Trichoderma reesei. Maximum activity of the Clostridium cellulase was displayed at 70°C and at pH 5.7 and 6.1 on Avicel and carboxymethylcellulose, respectively. In the absence of substrate at temperatures up to 70°C, carboxymethylcellulase was much more unstable than the Avicel-hydrolyzing activity.     

Sulfate Reduction and Methanogenesis in the Sediment of a Saltmarsh on the East Coast of the United Kingdom

The rates of sulfate reduction, methanogenesis, and methane loss were measured in saltmarsh sediment at monthly intervals. In addition, dissolved methane and sulfate concentrations together with pS²⁻ and pH were determined. Methane formation from carbon dioxide, but not from acetate, was detected within the same horizon of sediment where sulfate reduction was most active. Sulfate reduction was about three orders of magnitude greater than annual methanogenesis. The two processes were not separated either spatially or temporally, but occurred within the same layer of sediment at the same time of the year. Their coexistence did not seem to be the result of sulfate-depleted microenvironments within which methanogenesis could occur, but the methanogenic bacteria persisted at very low rates of activity within the same environment as the sulfate reducers.     

Photoreactions of cytochrome b6 in systems using resolved chloroplast electron-transfer complexes

Photoreactions of cytochrome b₆ have been studied using resolved chloroplast electron-transfer complexes. In the presence of Photosystem (PS) II and the cytochrome b₆-f complex, photoreduction of the cytochrome can be observed. No soluble components are required for this reaction. Cytochrome b₆ photoreduction was found to be inhibited by quinone analogs, which inhibit at the Rieske iron-sulfur center of the cytochrome complex, by the addition of ascorbate and by depletion of the Rieske center and bound plastoquinone from the cytochrome complex. Photoreduction of cytochrome b₆ can also be demonstrated in the presence of the cytochrome complex and PS I. This photoreduction requires plastocyanin and a low-potential electron donor, such as durohydroquinone. Cytochrome b₆ photoreduction in the presence of PS I is inhibited by quinone analogs which interact with the Rieske iron-sulfur center. These results are discussed in terms of a Q-cycle mechanism in which plastosemiquinone serves as the reductant for cytochrome b₆ via an oxidant-induced reductive pathway.     

Co-purification of soluble human galactosyltransferase and immunoglobulins

Preparations of human malignant effusion galactosyltransferase activity purified according to previously published techniques using enzyme-specific affinity chromatography consistently produced antibodies directed toward immunoglobulins with no detectable antigalactosyltransferase. Double immunodiffusion analysis of the antigen showed the presence of both IgG and IgA. Affinity chromatography with anti-human IgG-Sepharose and anti-human serum-Sepharose resulted in a 48,000-fold purification of galactosyltransferase activity with no detectable IgG by radioimmunoassay. Immunization of rabbits with this preparation produced antibodies directed against galactosyltransferase activity and minimal anti-Ig. The persistence of immunoglobulins during the purification of soluble galactosyltransferase activity through two enzyme-specific affinity chromatographic steps suggests an association of immunoglobulins with galactosyltransferase activity.     

Location in the yeast hexokinase structure of residues related to the enzyme activity

Seven residues implicated as acting directly in substrate binding in yeast hexokinase B have been identified in the crystallographic structure by chemical sequencing. The cysteine which is regarded as a residue critically maintaining the active conformation of yeast hexokinase has been selectively labelled and likewise located in the structure. In some parts of the amino acid sequence predicted from the high-resolution electron density map it is found that alignments of chemically sequenced peptides can be made unambiguously; however, the extent of matching to the predicted sequence varies considerably along the chain.     

Maximal activities of hexokinase, 6-phosphofructokinase,oxoglutarate dehydrogenase,and carnitine palmitoyltransferase in rat and avian muscles

The maximum activities of 6-phosphofructokinase and oxoglutarate dehydrogenase in muscle provide quantitative indices of the maximum capacities of anaerobic glycolysis and the Krebs cycle (i.e. the aerobic capacity) respectively. These activities were measured in red, white, and cardiac muscle of birds and the rat. The activities in the white pectoral muscle of the domestic fowl suggest that the Krebs cycle plus electron transfer could provide only about 1% of the rate of ATP production provided by anaerobic glycolysis whereas in pigeon pectoral muscle the predicted maximal rates from the two processes are similar. In contrast to domestic-fowl pectoral muscle, the white rat muscle, epitrochlearis, contains a significant activity of oxoglutarate dehydrogenase, which indicates that the Krebs cycle could provide about 12% of the maximum rate of ATP formation. This may be explained by a higher proportion of type-I and -IIA fibres in the rat muscle compared to the avian muscle. In the aerobic muscles of the rat the maximum activities of carnitine palmitoyl transferase indicate that fatty-acid oxidation could provide a high rate of ATP formation.     

Protection of cryopreserved Onchocerca microfilariae (nematoda) from dilution shock by the use of serum

This paper describes the use of newborn calf serum during the cooling and warming/dilution phases of the cryopreservation of Onchocerca gutturosa microfilariae using an interrupted slow cool to ?196 °C in the presence of 5% (v/v) methanol. Serum proved detrimental at concentrations above 20% (v/v) in the cooling medium unless it was also present in high concentrations, 60% (v/v) in the warming/ dilution medium.Damage to the organisms occurred predominantly during the thawing/dilution phase of cryopreservation and not the cooling phase and could be reduced greatly by the presence of high serum concentrations when thawing. This indicates that the major protective action of serum is that of reducing dilution shock—shock produced by a rapid influx of water and/or the effects of high solute concentrations established during cooling.     

Vasopressin alters the mechanism of apical Cl− entry from Na+:Cl− to Na+:K+:2Cl− cotransport in mouse medullary thick ascending limb

Summary Experiments were performed usingin vitro perfused medullary thick ascending limbs of Henle (MTAL) and in suspensions of MTAL tubules isolated from mouse kidney to evaluate the effects of arginine vasopressin (AVP) on the K⁺ dependence of the apical, furosemide-sensitive Na⁺:Cl^– cotransporter and on transport-related oxygen consumption (QO₂). In isolated perfused MTAL segments, the rate of cell swelling induced by removing K⁺ from, and adding onemm ouabain to, the basolateral solution [ouabain(zero-K⁺)] provided an index to apical cotransporter activity and was used to evaluated the ionic requirements of the apical cotransporter in the presence and absence of AVP. In the absence of AVP cotransporter activity required Na⁺ and Cl^–, but not K⁺, while in the presence of AVP the apical cotransporter required all three ions.⁸⁶Rb⁺ uptake into MTAL tubules in suspension was significant only after exposure of tubules to AVP. Moreover,²²Na⁺ uptake was unaffected by extracellular K⁺ in the absence of AVP while after AVP exposure²²Na⁺ uptake was strictly K⁺-dependent. The AVP-induced coupling of K⁺ to the Na⁺:Cl^– cotransporter resulted in a doubling in the rate of NaCl absorption without a parallel increase in the rate of cellular²²Na⁺ uptake or transport-related oxygen consumption. These results indicate that arginine vasopressin alters the mode of a loop diuretic-sensitive transporter from Na⁺:Cl^– cotransport to Na⁺:K⁺:2Cl^– cotransport in the mouse MTAL with the latter providing a distinct metabolic advantage for sodium transport. A model for AVP action on NaCl absorption by the MTAL is presented and the physiological significance of the coupling of K⁺ to the apical Na⁺:Cl^– cotransporter in the MTAL and of the enhanced metabolic efficiency are discussed.     

Morphine and Cocaine Exert Common Chronic Actions on Tyrosine Hydroxylase in Dopaminergic Brain Reward Regions

We studied levels of tyrosine hydroxylase immunoreactivity and phosphorylation state in the ventral tegmental area (VTA) and nucleus accumbens (NAc) in an effort to understand better the mechanisms by which these brain reward regions are influenced by opiates and cocaine. In the VTA, chronic, but not acute, administration of either morphine or cocaine increased levels of tyrosine hydroxylase immunoreactivity by 30-40%, with no change observed in the relative phosphorylation state of the enzyme. In the NAc, chronic, but not acute, morphine and cocaine treatments decreased the phosphorylation state of tyrosine hydroxylase, without a change in its total amount. In contrast, morphine and cocaine did not regulate tyrosine hydroxylase in the substantia nigra or caudate/putamen, brain regions generally not implicated in drug reward. Morphine and cocaine regulation of tyrosine hydroxylase could represent part of a common biochemical basis of morphine and cocaine addiction and craving.