Multiple molecular forms of prostaglandin 15-hydroxydehydrogenase and 9-ketoreductase in chicken kidney

Prostaglandin-15-hydroxydehydrogenase and prostaglandin-9-ketoreductase were purified from chicken kidney. Both enzymes exist in multiple forms as determined by isoelectric focusing. The dehydrogenases catalyze the transformation of the functional group at C-15 but not the functional group at C-9. The preferred cofactors in these reactions are NAD⁺ or NADH. The 9-ketoreductases catalyze the reversible transformation of the functional group at C-9 and also the oxidation or reduction of the C-15 functional group. The preferred cofactors are NADP⁺ or NADPH. Bradykinin does not affect the activities of any of the three prostaglandin 9-ketoreductases. Flavin mononucleotide and the flavonoid, quercetin, as well as indomethacin, ethacrynic acid, and furosemide, inhibit all three 9-ketoreductases. An inhibitor of 9-ketoreductase isolated from chicken breast muscle also inhibits the three separable reductases, but the pattern of inhibition of the reductase that focuses at pH 5.7 differs from that of the reductases focusing at pH 7.8 and 8.2.     

Multiple molecular forms of prostaglandin 15-hydroxydehydrogenase and 9-ketoreductase in chicken kidney.

Prostaglandin-15-hydroxydehydrogenase and prostaglandin-9-keto-reductase were purified from chicken kidney. Both enzymes exist in multiple forms as determined by isoelectric focusing. The dehydrogenases catalyze the transformation of the functional group at C-15 but not the functional group at C-9. The preferred cofactors in these reactions are NAD+ or NADH. The 9-ketoreductases catalyze the reversible transformation of the functional group at C-9 and also the oxidation or reduction of the C-15 functional group. The preferred cofactors are NADP+ or NADPH. Bradykinin does not affect the activities of any of the three prostaglandin 9-ketoreductases. Flavin mononucleotide and the flavonoid, quercetin, as well as indomethacin, ethacrynic acid, and furosemide, inhibit all three 9-ketoreductases. An inhibitor of 9-ketoreductase isolated from chicken breast muscle also inhibits the three separable reductases, but the pattern of inhibition of the reductase that focuses at pH 5.7 differs from that of the reductases focusing at pH 7.8 and 8.2.     

Multiple forms of invertase in developing oat internodes

Three different invertases are found in the developing internodes of oat (Avena sativa cv. Victory). Two soluble invertases (I and II) are separable on diethylaminoethylcellulose and Sephadex columns. They are further distinguished by their kinetic constants, heat stability, and differences in stability and apparent activity optima in response to pH treatments. Relative activities of the two soluble isozymes change considerably during the developmental stages examined. Invertase I activity rises early and begins to fall after maximal activity is reached at 6 hours of incubation. This early increase in activity accompanies the period of most rapid growth rate of the internode. Invertase II activity does not increase significantly during the first 6 hours of internode extension, but rapidly rises to a maximum activity at 16 hours, then declines. The third form of invertase, bound invertase (III), is present in both immature and mature stem tissue. Its activity increases (by 6 hours) during immature growth stages, decreases considerably with maturation, and remains relatively constant in mature tissue.     

Multiple molecular forms of murine thymocyte-stimulating factor

Murine thymocyte stimulating factor (TSF) was found to sediment in sucrose density gradients on a broad band with peaks at about 2.60 S and 2.0 S. Two main peaks of TSF activity (with buoyant densities of 1.34 and 1.28 g/ml) were found in CsCl density gradients. Gel chromatography on Sephadex G-100 columns of the material sedimented in sucrose or CsCl density gradients originated multiple peaks of TSF activity with various molecular weights. Heterogeneity of molecular forms of TSF was also found upon dilution of the factor. The lowest molecular weights found were 4000 and 4700 daltons. When Sephadex fractions containing the low molecular weight material were pooled and rerun on Sephadex columns, molecular species with a wide range of molecular weights were found. Temperature also affects the appearance of the low molecular weight forms of TSF. Most of the experiments presented in this work were carried out with Sephadex-purified TSF. Multiple molecular forms, however, and, in particular, the forms with molecular weights of 4000 and 4700 daltons were found also with TSF-Fraction IIIa, a highly purified preparation of this factor.     

Multiple molecular forms of mouse liver arginase

Hepatic arginase (L-arginine amidinohydrolase, EC 3.5.3.1) is an oligomer composed of three or four subunits. The present studies indicate heterogeneity in the size and charge of arginase subunits in mouse liver. Two types of arginase subunits with molecular weights of approximately 35,000 and 38,000 have been found. These two subunits are detected in liver cytosol or in purified preparations of arginase after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Two dimensional SDS-PAGE revealed multiple ionic forms of arginase for both the 35,000 and 38,000 subunits; the subunits contain basic proteins (pI range 7.8-9.1) and acidic proteins (pI range 5.8-6.4). Limited proteolysis by trypsin eliminated the molecular weight differences between the subunits without substantially affecting either their isoelectric points or activity. Comparative peptide maps and amino acid analyses of the 35,000- and 38,000-Da subunits showed that they were very similar. The data indicate that a neutral peptide (approx 3000 Da) is responsible for the differences in subunit molecular weight and that the multiple sized and charged forms are variants of the same protein.     

Multiple molecular forms of rat brain enkephalinase

Rat brain enkephalinase has been partially purified by ion exchange chromatography, chromatofocusing, and affinity chromatography on immobilized lectins. Ion exchange chromatography resolved two principle forms of enkephalinase designated A1 and A2. Both enkephalinase A1 and A2 are bound to immobilized lentil lectin while chromatography on immobilized wheat germ lectin resolved each of the principle forms into two subforms, A1, 1, A1, 2, A2, 1, and A2, 2. All four enkephalinase forms have similar, if not identical kinetic properties. The possible implications of multiple molecular forms of enkephalinases are discussed.     

Multiple molecular forms of Acanthamoeba lactic dehydrogenase

1. Acanthamoeba lactic dehydrogenase (D-lactate specific, EC 1.1.1.28) has been studied through the use of polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (PAGIEF). 2. Data from PAGE showed the presence of only a single macromolecular form of the enzyme in trophozoites, but PAGIEF data demonstrated that at least three LDH species occur. 3. Molecular weight determinations indicated a tetrameric assembly state for the enzyme. 4. Multiple molecular forms of the enzyme (isoenzymes) are encoded either by genes at two loci or by two alleles at a single LDH locus.     

Multiple molecular forms of lysosomal enzymes in mucolipidosis II

Summary The multiple molecular forms of selected lysosomal enzymes, as determined by analytical isoelectric focusing electrophoresis, from mucolipidosis II fibroblasts have a highly simplified pattern demonstrating a failure to undergo normal oligosaccharide processing. On the other hand, the multiple molecular forms of these same enzymes in mucolipidosis II sera and culture media are indistinguishable from controls.     

Multiple molecular forms of glutamine synthetase in pea seeds

Summary Multiple molecular forms of glutamine synthetase (GS, EC 6.3.1.2) have been studied in pea seeds of different varieties. The number of GS molecular forms in the seeds proved to be related to their colour. Two GS forms in the green seeds have been found and only one of them in the yellow seeds. Green seeds had chlorophyll content amounted to 0.4% of the total pigment content in the leaves. Chloroplasts, somewhat smaller than those in pea leaves of the same variety, have been isolated from green seeds. The presence of the second GS form in the pea green seeds we relate to the chloroplasts. By electrophoretic mobility both forms of GS from the green seeds are not identical to the chloroplast GS and the cytosol GS of leaves. Thus, we believe pea plant to contain, at least, four GS forms.     

Multiple molecular forms of glia maturation factor.

Glia maturation factor from the pig brain can be detected in two molecular forms: the high molecular weight form which is 200 000 dalton in size and the low molecular weight form which is 40 000 dalton in size, as determined by Sephadex gel filtration. The former accounts for 85% of the total biological activity extracted at physiologic pH. The proportion of the low molecular weight form increases following freeze-thawing and ion-exchange chromatography. In addition to the morphological effects, both forms possess mitogenic activity but no esteropeptidase activity. Both forms show similar enzyme susceptibility, being inactivated by papain, ficin and pronase but resistant to subtilisin, thermolysin and trypsin. The high molecular weight form is more resistant to denaturation by low pH, heating and urea than the low molecular weight form. The high molecular weight factor has an isoelectric point of 4.27 whereas the low molecular weight factor has one of 5.04.     

Kinetic mechanism of the molecular forms of chicken liver mitochondrial malate dehydrogenase

• 1.1. The reaction kinetic mechanism (pH 7.4) of the molecular forms of chicken liver m-MDH is of the ordered bi-bi ternary complex type with the existence of the E-oxaloacetate, E-L-malate, E-NAD⁺ oxaloacetate, E-NADH-l-malate, E-NAD⁺-NADH, E-NAD⁺-NAD⁺, E-NADH-NAD⁺ and E-NADH-NADH abortive complexes.
• 2.2. The saturating concentration values of the substrates are notably modified, in certain cases, in the presence of the reaction products.

     

Multiple molecular forms of gonadotropin-releasing hormone in teleost fish brain

Gonadotropin-releasing hormone (GnRH) immunoreactive peptides in extracts of hake (Merluccius capensis) and tilapia (Tilapia sparrmanii) brain were investigated by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera. In hake brain, content and concentration of GnRH was higher in the pituitary gland than in the hypothalamic lobes or extrahypothalamic brain. Hake pituitary gland GnRH was purified by six consecutive HPLC systems. The major GnRH molecular form co-eluted with salmon brain GnRH (Trp7, Leu8-GnRH) in four different HPLC systems which were specifically designed to separate the four natural vertebrate GnRHs (mammalian, salmon, chicken I and II). The immunoreactive peak in the final purification step had a retention time identical to that of Trp7, Leu8-GnRH and an UV absorbance (280 nm) peak appropriate for two tryptophan residues in the peptide, as in Trp7, Leu8-GnRH. Six additional less hydrophobic forms of GnRH were detected. Tilapia brain extract contained two major GnRH molecular forms which had identical retention times to chicken GnRH I (Gln8-GnRH) and Trp7, Leu8-GnRH in an HPLC system which separates the natural vertebrate GnRHs. The immunological properties of these two immunoreactive peaks, determined by relative interaction with four region-specific GnRH antisera raised against vertebrate GnRHs, were identical to those of Gln8-GnRH and Trp7, Leu8-GnRH. Additional GnRH molecular forms were also detected. In summary, these findings indicate that a major GnRH molecule in hake pituitary gland is Trp7, Leu8-GnRH, while tilapia brain contains both Trp7, Leu8-GnRH and Gln8-GnRH. Additional GnRH molecular forms were detected in both species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple molecular forms of glutamine synthetase in carp muscles]

Cytoplasmic and mitochondrial molecular forms of glutamine synthetase (CE 6.3.1.2) have been isolated from the carp muscle with purification degree of 100 and 165 times and output 9.0%. It is established that the temperature optimum of the cytoplasmic form activity is 30 degrees C and that of mitochondrial one--20 degrees C; the pH optimum for the both molecular forms is 6.0 and 8.2. The optimal ratio [Me2+] : [ATP] for the isolated form is 2:1; Km (seeming) of the cytoplasmic form in the presence of Mg2+ is 6.0 mM for glutamate, 0.035 for ammonium, for ATP 0.5 and 0.7 for magnesium ions; these values for the mitochondrial form are: 14.3, 0.048, 1.0 and 0.8 mM, respectively. Activity of the both glutamine synthetases with Mg2+ ions is almost by 50% higher than that of glutamine synthetases with Mn2+ ions. Seasonal regularities of the synthesis of molecular glutamine synthetase forms have been established in vivo. Cytoplasmic form is present in the muscles all year round, while mitochondrial one only in winter at low temperature of the environment and fish starvation. Differences in properties and seasonal character of synthesis of molecular glutamine synthetase forms in carp muscles are a result of diversity of their functional role.