Purification of monomeric agmatine iminohydrolase from soybean

Agmatine iminohydrolase (EC 3.5.3.12) was purified to homogeneity from the cytosol of soybean (Glycine max) axes by chromatographic separations on Sephadex G-25, Bio-rex 70, and agmatine-affinity columns. The enzyme was homogeneous by the criteria of analytical gel electrophoresis. Molecular weights estimated by Sephadex G-100 gel and sodium dodecyl sulfate polyacrylamide gel electrophoresis were 70,000, indicating that the soybean axes enzyme is a monomer, in contrast to the dimeric enzymes from corn and rice. The isoelectric point determined by gel electrofocusing was 7.5, higher than that of the corn enzyme (4.7). The optimal pH and temperature for activity were 6.5 and 50 degrees C, respectively. The enzyme has high specificity for agmatine, and the Km for agmatine was 2.5 x 10(-3) molar. The enzyme was sensitive to Cu2+ and also was inhibited by p-hydroxymercuribenzoate.     

Purification of S-adenosylmethionine decarboxylase from soybean.

S-Adenosylmethionine decarboxylase (EC 4.1.1.19) was purified to homogeneity from the cytosol of soybean (Glycine max) axes by ammonium sulfate fractionation, DEAE-Sepharose and methylglyoxalbis(guanylhydrazone)-Sepharose 6B chromatographies. The enzyme was free from diamine oxidase activity. The molecular weight of the enzyme estimated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis was 66,000. The Km value for S-adenosylmethionine was 0.26 mM. The optimum pH and temperature were 7.5 and 40 degrees C. Neither putrescine nor Mg2+ affected the enzyme activity, but the enzyme was inhibited by spermidine, spermine, methylglyoxalbis(guanylhydrazone), sodium borohydride and phenylhydrazine. Agmatine was a novel inhibitor which inhibited S-adenosylmethionine decarboxylase and arginine decarboxylase, preventing the accumulation of decarboxylated S-adenosylmethionine and putrescine, respectively.     

The alpha subunit of type II Ca2+/calmodulin-dependent protein kinase is highly conserved in Drosophila

A monoclonal antibody against rat brain type II Ca2+/calmodulin-dependent protein kinase (CaM kinase) precipitates three proteins from Drosophila heads with apparent molecular weights similar to those of the subunits of the rat brain kinase. Fly heads also contain a CaM kinase activity that becomes partially independent of Ca2+ after autophosphorylation, as does the rat brain kinase. We have isolated a Drosophila cDNA encoding an amino acid sequence that is 77% identical to the sequence of the rat alpha subunit. All known autophosphorylation sites are conserved, including the site that controls Ca(2+)-independent activity. The gene encoding the cDNA is located between 102E and F on the fourth chromosome. The protein product of this gene is expressed at much higher levels in the fly head than in the body. Thus, both the amino acid sequence and the tissue specificity of the mammalian kinase are highly conserved in Drosophila.     

A linkage map of distal mouse chromosome 12

To refine the linkage map of distal mouse Chromosome 12, we have identified DNA restriction fragment variants associated with a creatine kinase gene (Ck-3), the Akt proto-oncogene, an Abelson proviral integration site (D12N1), and the immunoglobulin heavy chain V_H3609 variable region family (Igh-V36). The patterns of inheritance of these markers in backcross progeny and recombinant inbred mouse strains allowed their localization with respect to previously mapped genes to yield the linkage map: Aat-15.8 cM-Ck-3-0.9 cM-(Crip, Akt, Igh-C)-0.3 cM-(D12N1, Igh-V). This map confirms genetically the localization of the Igh-V gene complex distal to Igh-C on the chromosome. It differs from previous maps in placing D12N1 distal to Igh-C, and in suggesting that the Igh-V gene complex spans less than one centiMorgan (cM).Other DNA sequence variants detected with the creatine kinase probe allowed definition of four additional genetic loci: Ck-1 near Lmyc-1 on Chromosome 4; Ck-2 between Upg-1 and Hprt-ps1 (D17Rp10) on distal Chromosome 17; Ck-4 near Mpmv-17 and Mls-3 on Chromosome 16; and Ck-5 near Hba on Chromosome 11.     

Monomer diffusion into polymer domains in sickle hemoglobin.

The gelation of sickle hemoglobin includes the formation of spherulitic arrays of polymers, known as polymer domains, which are an intrinsic result of the polymer formation mechanism. We have observed the diffusion of monomers into domains as they form, which substantially increases the total concentration of hemoglobin within the domain. The maximum total concentration attained is comparable with the pellet concentration of 0.5-0.55 g/cm3 obtained in sedimentation experiments. The half time for this process is approximately 50 s for domains of 25 microns radius, and is approximately independent of temperature. The shape of the diffusion progress curves as well as the deduced diffusion constants, and their weak temperature dependence are consistent with a simple model of hemoglobin monomer diffusion into the domain.     

The effect of unilateral nephrectomy and sham operation on tyrosine content and activity of tyrosine aminotransferase in the rat

During the first four days after unilateral nephrectomy the free tyrosine content in plasma, liver and hypertrophic kidney was decreased by more than 50% as compared with the values observed in intact rat. After sham operation, the content of tyrosine was decreased to the same extent. The activity of tyrosine aminotransferase in liver was doubled two days after sham operation: no such increase was observed after unilateral nephrectomy. At the same time a decline of the enzyme activity in kidney was demonstrated after both types of surgery. Hydrocortisone in a single i.p. dose stimulated enzyme activity in the liver of intact rats three-fold, and more than four-fold after nephrectomy and sham operation. In kidney of intact rat, as a result of hydrocortisone treatment, the enzyme activity was doubled; it was, however, insensitive to this treatment after unilateral nephrectomy, and increased only by 20% after sham operation. It is suggested that the changes in tyrosine content and tyrosine aminotransferase activity observed after unilateral nephrectomy were not due to stress alone, but underwent regulation aimed at assuring a sufficient level of this amino acid for metabolism.     

Immobilization of enzymes on activated carbon: selection and preparation of the carbon support.

Based upon its superior catalytic activity for H2O2 decomposition, a bituminous coal-based activated carbon was selected for investigations of pretreatment and enzyme immobilization methods. Pretreatments considered include acid washing, exposure to strong oxidizing agents, contact with concentrated peroxide solutions, nitration and amination, isothiocyanate derivatization, silanization, and stearic acid coating. Effects of these pretreatments on morphology and trace-metal content of the carbon pellets have been studied using scanning electron microscopy and dispersive analysis of x rays. Immobilization of glucoamylase by adsorption, glutaraldehyde crosslinking, and covalent attachment to carbon activated by water-soluble diimide or diazotization have been examined. These different enzyme-carbon catalysts have been characterized by their enzyme loading, enzyme activity, catalytic activity for H2O2 decomposition, or combinations of these measures of performance.     

Mixed cell agglutination reaction of ABO substances in the saliva and determination of human-type ABO blood groups in the cynomolgus monkey.

For detection of ABO substances in the saliva of the cynomolgus monkey, the mixed cell agglutination reaction (MCAR) gave specific and clear results with a very small amount of saliva. Anti-A and anti-B antibodies in the sera of the same species showed the clearest hemagglutination by the saline agglutination method. The combined use of both methods was demonstrated to be easily and accurately applicable to the determination of human-type ABO blood groups of the cynomolgus monkey.     

Possible involvement of cerebroside sulfate in opiate receptor binding.

Cerebroside sulfate (CS) appears to fulfill most of the structural requirements of a hypothetical opiate receptor. It possesses many of the properties that are thought to be necessary for the identification of an "opiate receptor," exhibiting high affinity and stereoselective binding to a number of narcotic drugs. Although these properties are insufficient to establish identity of the receptor, it is highly significant that the affinity of this binding can be correlated with the analgetic potency of these drugs in both man and rodents. CS is an endogenous component of brain tissue, and a partially purified opiate receptor from mouse brain has been found to be CS. Other experiments indicate that reduced availability of brain CS decreases the analgetic effects of morphine and this is accompanied by a reduction in number of binding sites, suggesting that the interaction of opiates with CS observed in vitro may also have importance in vivo. CS was also found to be a component of the opiate receptor after marking with 125I-labeled diazosulfanilic acid. The possibility that CS or the SO4-2 group of this lipid may be the "anionic site" of the opiate receptor should be considered.