The role of His-134, -147, and -150 residues in subunit assembly, cofactor binding, and catalysis of sheep liver cytosolic serine hydroxymethyltransferase

In an attempt to unravel the role of conserved histidine residues in the structure-function of sheep liver cytosolic serine hydroxymethyltransferase (SHMT), three site-specific mutants (H134N, H147N, and H150N) were constructed and expressed, H134N and H147N SHMTs had K-m values for L-serine, L-allo-threonine and beta-phenylserine similar to that of wild type enzyme, although the k(cat) values were markedly decreased, H134N SHMT was obtained in a dimeric form with only 6% of bound pyridoxal 5'-phosphate (PLP) compared with the wild type enzyme, Increasing concentrations of PLP (up to 500 mu M) enhanced the enzyme activity without changing its oligomeric structure, indicating that His-134 may be involved in dimer-dimer interactions, H147N SHMT was obtained in a tetrameric form but with very little PLP (3%) bound to it, suggesting that this residue was probably involved in cofactor binding, Unlike the wild type enzyme, the cofactor could be easily removed by dialysis from H147N SHMT, and the apoenzyme thus formed was present predominantly in the dimeric form, indicating that PLP binding is at the dimer-dimer interface, H150N SHMT was obtained in a tetrameric form with bound PLP, However, the mutant had very little enzyme activity (<2%). The k(cat)/K-m values for L-serine, L-allo-threonine and beta-phenylserine were 80-, 56-, and SS-fold less compared with wild type enzyme, Unlike the wild type enzyme, it failed to form the characteristic quinonoid intermediate and was unable to carry out the exchange of 2-S proton from glycine in the presence of H-4-folate. However, it could form an external aldimine with serine and glycine, The wild type and the mutant enzyme had similar K-d values for serine and glycine, These results suggest that His-150 may be the base that abstracts the alpha-proton of the substrate, leading to formation of the quinonoid intermediate in the reaction catalyzed by SHMT.

Chemical modification of sheep plasma glycoprotein

Glycoprotein isolated from sheep plasma was chemically modified, and the effect of chemical modification on biological activities and immunological cross reactions has been studied. The removal of sialic acid resulted in a change in the “overall conformation” of the glycoprotein as evidenced by a decrease in viscosity of the glycoprotein solution and an increased susceptibility of the glycoprotein to proteolytic enzymes. Sialic acid-free glycoprotein no longer inhibited the tryptic activity or prolonged the clotting time of plasma. However, it could react with the antiserum to sheep plasma glycoprotein. The periodate oxidation of sheep plasma glycoprotein resulted in a complete loss of inhibition of trypsin activity, prolongation of plasma clotting time, and the ability to cross-react with the rabbit antiserum. The significance of periodate oxidation in relation to the possible sequence of sugars in the carbohydrate prosthetic group in the glycoprotein is discussed. Iodination and heating in buffers of acid and alkaline pH values of sheep plasma glycoprotein resulted in complete loss of trypsin activity and ability to prolong plasma clotting time. Iodination of the glycoprotein did not affect the immunological cross-reactivity.

Isolation and partial characterization of sheep plasma α1-mucoprotein

1. 1. Sheep plasma α1-mucoprotein was isolated in an electrophoretically homogeneous state by a combination of ammonium sulphate saturation, isoelectric precipitation and preparative agar electrophoresis in a yield of approx. 150 mg/l of plasma. 2. 2. The mucoprotein was water-soluble, non-coagulable on heating at 100°, not precipitable by 1.8 M perchloric acid, 10% trichloroacetic acid but precipitable by saturated ammonium sulphate solution, 0.6 M sulfosalicylic acid and 5% phosphotungstic acid in 2 N HCl. It had E1 cm1 % value of 9.57 at 278 mμ in water, refractive-index index increment 1.9·10-4 (g/l) in water, isoelectric point at pH 4.45 (sodium acetate-acetic acid buffer) and was homogeneous in pH range 4.0-11.5 but at pH values 2.6 and 3.5 showed some dissociation. 3. 3. The mucoprotein had the following chemical composition: Nitrogen, 12.4%; polypeptide, 77.4%; total hexose (only mannose and galactose), 7.1%; fucose, 1.0%; glucosamine, 4.9% and sialic acid, 4.8%. It had no N-terminal amino acid.

Fatty acid component of vitamin A ester in sheep liver

Vitamin A, when extracted along with other lipids from sheep liver, had an E1cm.1% value of 14.4, which was raised to 45.57 on removal of the phospholipids by cold acetone. Selective hydrolysis of triglycerides by an extract of acetone-dried sheep pancreas in the presence of HgCl2 as inhibitor of vitamin A esterase, followed by chromatography through alumina gave a product with E1cm.1% value of 276. This on chromatography through magnesium oxide raised the E1cm.1, value to 601.5, representing 64% pure vitamin A ester calculated as palmitate, and the total recovery was 23% of the starting oil. The purified ester preparation, when subjected to reverse-phase chromatography on silicone-impregnated paper, gave a single ultraviolet fluorescent band. The fluorescent band on hydrolysis gave only one fatty acid. This was conclusively identified to be palmitic acid.

Studies on nucleotide pyrophosphatase I. Partial purification and properties of a sheep liver enzyme that catalyzes the hydrolysis of dinucleotides

A partially purified sheep liver enzyme that hydrolyzed dinucleotides at the pyrophosphate bond was obtained by solubilizing the 18,000g sediment with n-butanol and fractionating the solubilized enzyme with acetone. The enzyme activity when measured using FAD as substrate, (FAD → FMN + AMP), was optimal at pH 9.7 and temperatures between 30 °–36 ° and at 60 °. The rate of release of FMN with time occurred with an initial lag of 30 sec, a linear increase for 1 min, and a subsequent irregular rate. In the presence of orthophosphate (Pi; 10 μImage ), FMN was released at an uniformly continuous and enhanced rate. 32Pi was not incorporated into the substrate or products. Sodium arsenate counteracted the effects of Pi. The apparent Km and Vmax were 0.133 mImage and 100 units; and 0.133 mImage and 200 units, in the absence and presence of Pi, respectively. The temperature optimum was 42 ° in the presence of Pi.Negative cooperative interactions observed at low concentrations of FAD were abolished by the addition of Pi. The inhibition by AMP was sigmoid and Pi abolished this sigmoidal response. The enzyme hydrolyzed in addition to FAD, NAD+ and NADP+. Nucleoside triphosphates were potent inhibitors of the enzyme activity. The partial inhibition of the enzyme by o-phenanthroline and by p-hydroxymercuribenzoate could be reversed by Fe2+ ions and by reduced glutathione, respectively.

Elucidation of the mechanism of interaction of sheep spleen galectin-1 with splenocytes and its role in cell-matrix adhesion

The binding of a 14 kDa beta-galactoside animal lectin to splenocytes has been studied in detail. The binding data show that there are two classes of binding sites on the cells for the lectin: a high-affinity site with a K-a ranging from 1.1 x 10(6) to 5.1 x 10(5) M-1 and a low affinity binding site with a K-a ranging from 7.7 x 10(4) to 3.4 x 10(4) M-1 The number of receptors per cell for the high- and low-affinity sites is 9 +/- 3 x 10(6) and 2.5 +/- 0.5 x 10(6) respectively. The temperature dependence of the K value yielded the thermodynamic parameters. The energetics of this interaction shows that, although this interaction is essentially enthalpically driven (Delta H - 21 kJ lambda mol(-1)) for the high-affinity sites, there is a very favorable entropy contribution to the free energy of this interaction (-T Delta S - 17.5 Jmol(-1)), suggesting that hydrophobic interaction may also be playing a role in this interaction. Lactose brought about a 20% inhibition of this interaction, whereas the glycoprotein asialofetuin brought about a 75 % inhibition, suggesting that complex carbohydrate structures are involved in the binding of galectin-1 to splenocytes, Galectin-1 also mediated the binding and adhesion of splenocytes to the extracellular matrix glycoprotein laminin, suggesting a role for it in cell-matrix interactions. Copyright (C) 2000 John Wiley & Sons, Ltd.

A Ca2+-sensitive cyclic-3',5'-nucleotide phosphodiesterase from sheep lung modulated by a tightly bound endogenous calmodulin.

Highly purified sheep lung cyclic-3',5'-nucleotide phosphodiesterase was sensitive to Ca2+/EGTA but insensitive to exogenous calmodulin. The Ca2+-sensitivity was inhibited by trifluoperazine. Heat-treated enzyme could activate a calmodulin-deficient phosphodiesterase, suggesting the presence of endogenous calmodulin in sheep lung cyclic-3',5'-nucleotide phosphodiesterase, possibly associated with the enzyme in a Ca2+-independent manner.

Temperature and thiol-induced desensitization of a Ca2+-sensitive cyclic-3',5'-nucleotide phosphodiesterase from sheep lung

Ca2+-sensitivity of sheep lung cyclic-3',5'-nucleotide phosphodiesterase is provided by endogenous tightly bound calmodulin. The calcium sensitivity of a highly purified enzyme was desensitized by increasing the assay temperature. It could also be desensitized to Ca2+-activation by thiols such as dithiothreitol. The thiol-induced desensitization could be partially reversed by dialysis and almost completely reversed by dilution. The results presented in this paper indicate that thiols are possibly involved in the interaction of calmodulin with cyclic-3',5'-nucleotide phosphodiesterase. This is the first report on temperature and thiol-induced desensitization of Ca2+-sensitivity of a cyclic-3',5'-nucleotide phosphodiesterase.

Mechanism of interaction of O-amino-D-serine with sheep liver serine hydroxymethyltransferase

The mechanism of interaction of 0-amino-D-serine (OADS) with sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) (SHMT) was established by measuring changes in the enzyme activity,absorption spectra, circular dichroism (CD) spectra, and stopped-flow spectrophotometry. OADS was a reversible noncompetitive inhibitor (Ki = 1.8 pM) when serine was the varied substrate. The first step in the interaction of OADS with the enzyme was the disruption of enzyme-Schiff base, characterized by the rapid disappearance of absorbance at 425 nm (6.5 X lo3 M-' s-') and CD intensity at 430 nm. Concomitantly,there was a rapid increase in absorbance and CD intensity at 390 nm. The spectral properties of this intermediate enabled its identification as pyridoxal 5'-phosphate (PLP). These changes were followed by a slow unimolecular step (2 X s-') leading to the formation of PLP-OADS oxime, which was confirmed by its absorbance and fluorescence spectra and retention time on high-performance liquid chromatography. The PLP-OADS oxime was displaced from the enzyme by the addition of PLP as evidenced by the restoration of complete enzyme activity as well as by the spectral properties. The unique feature of the mechanism proposed for the interaction of OADS with sheep liver SHMT was the formation of PLP as an intermediate.

Interactions of methoxyamine with pyridoxal-5'-phosphate-Schiff's base at the active site of sheep liver serine hydroxymethyltransferase

The mechanism of interaction of methoxyamine with sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) (SHMT) was established by measuring changes in enzyme activity, visible absorption spectra, circular dichroism and fluorescence, and by evaluating the rate constant by stopped-flow spectrophotometry. Methoxyamine can be considered as the smallest substituted aminooxy derivative of hydroxylamine. It was a reversible noncompetitive inhibitor (Ki = 25 microM) of SHMT similar to O-amino-D-serine. Like in the interaction of O-amino-D-serine and aminooxyacetic acid, the first step in the reaction was very fast. This was evident by the rapid disappearance of the enzyme-Schiff base absorbance at 425 nm with a rate constant of 1.3 x 10(3) M-1 sec-1 and CD intensity at 430 nm. Concomitantly, there was an increase in absorbance at 388 nm (intermediate I). The next step in the reaction was the unimolecular conversion (1.1 x 10(-3) sec-1) of this intermediate to the final oxime absorbing at 325 nm. The identity of the oxime was established by its characteristic fluorescence emission at 460 nm when excited at 360 nm and by high performance liquid chromatography. These results highlight the specificity in interactions of aminooxy compounds with sheep liver serine hydroxymethyltransferase and that the carboxyl group of the inhibitors enhances the rate of the initial interaction with the enzyme.