Purification and partial characterization of the soluble low Km 5′-nucleotidase from human seminal plasma

doi:10.1016/0167-4838(91)90010-W

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology

Volume 1080, Issue 3, 15 November 1991, Pages 252-258

Biochimica et Biophy...

https://doi.org/10.1016/0167-4838(91)90010-W Get rights and content

Abstract

Soluble low K_m 5′-nucleotidase from human seminal plasma has been purified to homogeneity by one affinity and two gel-filtration chromatographic steps. The pure enzyme had a specific activity of 2000 nmol min⁻¹ mg⁻¹. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified low K_m 5′-nucleotidase revealed a single polypeptide band of 40 ± 7 kDa and a tetrameric structure of 160 ± 10 kDa has been proposed for the native enzyme. The kinetic properties of low K_m 5′-nucleotidase have been determined and rather unique characteristics have been found for this soluble low K_m 5′-nucleotidase: the substrate efficiency was slightly higher for IMP with an optimum pH at 7.5; the enzyme showed an absolute dependence on Mg²⁺ ions. Ca²⁺ could replace Mg²⁺ ions for activity while other divalent cations could not substitute for Mg²⁺; the enzymes were equally activated by ATP and ADP up to 0.1 mM concentrations. At higher concentrations up to 1 mM, ADP was still an activator while ATP caused a gradual decrease of activation to the native activity. This effect could not be related to the Mg-ATP complexes since the enzymic preparation Mg²⁺-free still showed the same biphasic pattern of activation.

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Cited by (12)

Activity of IMP- and AMP-preferring isoforms of 5'-nucleotidase from human seminal plasma with AMP analogues
1999, Molecular Genetics and Metabolism
AMP analogues modified at various positions of the molecule were checked as substrates for the two soluble isoforms of 5′-nucleotidase from human seminal plasma. These isoforms were isolated to near homogeneity by affinity chromatographies. AMP derivatives were differently dephosphorylated by both the isoforms depending on the site of modification in the natural compound. Changes in the phosphate moiety reduced significantly hydrolysis by the IMP-preferring form, whereas the AMP-preferring form was less affected. The AMP-preferring form was characterized by a relatively broad specificity toward substrate analogues indicating that the binding domains for the phosphate moiety of these isoforms are not identical. Substitutions at the C-8 adenine base reduced the hydrolysis rate of both the enzymes and variations of thesyn-anticonformational equilibrium resulted in different effects on catalysis by both forms. Therefore, the orientation of the heterocyclic base around the glycosidic bond may not be the crucial factor affecting binding and catalytic activity. Hydrogen bonding potential of base N-7 was essential for the binding and catalysis of the IMP- but not of the AMP-preferring form. This was the most striking difference between the studied isoforms. Modifications and substitutions of 6-amino function, better accepted by the IMP-preferring form than by the AMP-preferring form, indicated that no essential hydrogen bonding is required for catalytic activity. The binding was however significantly slowed in 6-SH-PuMP. Hydrogen bonding potential of N-1 was significant for the hydrolysis rate of the IMP- but not of the AMP-preferring form. We suggest that these human seminal plasma isoforms of soluble 5′-nucleotidase, characterized by unique features, may represent the tissue-specific expression of the polymorphic gene.
Human seminal plasma soluble 5'-nucleotidase: Regulatory aspects of the dephosphorylation of nucleoside 5'-monophosphates
1997, Biochemical and Molecular Medicine
Human seminal plasma contains two enzyme activities both capable of dephosphorylating all nucleoside 5-monophosphates with different efficiency and specificity. Broad-spectrum soluble 5′-nucleotidase is the object of this paper which deals with the definition of the response of this enzyme to effectors, some physiological and others not naturally occurring. The enzyme did not show any product regulation as all the nucleosides tested caused a moderate effect on the hydrolysis of the substrates. Theophylline and other xanthine derivatives had no effect on enzyme activity, whereas glycerate 2,3bisphosphate, like other soluble 5′-nucleotidases, caused a stimulation of the enzyme, especially toward CMP and UMP. 5-Deoxy-5-isobutylthiadenosine resulted in no inhibition of the hydrolysis of AMP and IMP. The enzyme was affected neither by monovanadate nor by decavanadate, whereas it was strongly inhibited by Ap₅A. Variations in adenylate energy charge did not cause any alteration of the enzyme activity toward AMP and only a slight decrease of the hydrolysis of IMP. These regulatory properties, distinct from those of other soluble 5′-nucleotidases, show that this form, newly isolated from human seminal plasma, is subject to an almost unique, tissue-specific regulation.
The isolation from human seminal plasma of a new form of soluble 5'- nucleotidase
1996, Biochemical and Molecular Medicine
Soluble broad spectrum 5′-nucleotidase from human seminal plasma was purified to homogeneity by a combination of (NH₄)₂SO₄precipitation, affinity chromatography, and gel filtration. The pure enzyme had a specific activity of 4800 nmol min⁻¹mg⁻¹. SDS–PAGE of purified enzyme preparation revealed a single polypeptide band of 53 kDa and a tetrameric structure of 203 kDa was proposed for the native enzyme. This form had modest preference for AMP as substrate; Mg²⁺and Mn²⁺were activators of the enzyme although its activity was not absolutely dependent on the presence of these exogenous bivalent cations. The enzyme, recovered in the nonsedimentable fraction of human seminal plasma, had a pH optimum of 7.5; ATP and ADP were inhibitors of mixed type, P_iwas a potent inhibitor at nonphysiological concentrations, and Con A and adenosine 5-[α,β-methylene]diphosphate had no effect on the enzyme activity. The enzyme described here therefore has some unique properties between truly cytoplasmic and membrane-bound derived forms.
Soluble 5′-nucleotidase from thyroid gland partial purification and properties
1996, Biochemical and Biophysical Research Communications
A soluble 5′-nucleotidase from pig thyroid was purified over 110-fold by chromatography on phosphocellulose, (NH₄)₂SO₄precipitation and gel filtration on Sephadex G-150. The purified 5-nucleotidase was free of non-specific phosphatases. The enzyme had optimum pH at 6.5 and hydrolysed preferentially IMP and GMP. The K_mvalues were 0.66 and 1.0 mM for IMP and GMP, respectively. The enzyme also hydrolysed other nucleotides and showed the following relative V_max: IMP > GMP > CMP > AMP > UMP.Mg²⁺was necessary for the enzyme activity.
The dephosphorylation of AMP and IMP by a soluble low K<inf>m</inf> 5′nucleotidase from human seminal plasma: Some regulatory aspects
1995, International Journal of Biochemistry and Cell Biology
In this study, a soluble low K_m 5′nucleotidase, dephosphorylating IMP with a V_max/K_m ratio 10-times higher than that of AMP, has been purified from human seminal plasma. The effect of inorganic phosphate (P_i) and adenylate energy charge variations on the activity of this enzyme has also been investigated. In the physiological range, with IMP as substrate, the activity of the enzyme does not change whereas the hydrolysis of AMP increases with decreasing energy charge values. In the presence of both the substrates, phosphate exerts an inhibitory effect on the enzyme activity with a similar concentration dependence pattern. The results show that AMP-hydrolysing activity responds to variations of energy charge by increasing the AMP degradation thus protecting the value of energy charge at the expense of a decrease in the total adenylate pool.
In contrast, the dephosphorylation of IMP is not regulated by changes in energy charge. This data suggests that the degradation of IMP and AMP, although carried out by the same enzyme, is controlled by different regulatory mechanisms.
Effects of adenine nucleotides on low K<inf>m</inf> 5′ nucleotidase from human seminal plasma
1993, International Journal of Biochemistry
- 1.
  1. Low K_m 5' nucleotidase purified from human seminal plasma has been used in this study to investigate the response of the enzyme to adenine nucleoside di- and triphosphates in the presence of AMP and IMP as substrates.
- 2.
  2. In the presence of AMP, the addition of 0.5 mM ATP to the enzyme Mg-free results into the highest V_max/K_m ratio value and other experimental combinations of effectors tested cause variation of the kinetic parameters of the enzyme, indicating a control of AMP dephosphorylation by adenine nucleotides.
- 3.
  3. In the presence of IMP, ATP and ADP activate the enzyme but the response to various experimental combinations of effectors shows no significant difference in the kinetic properties of the enzyme, indicating a different control of the dephosphorylation of IMP.

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Purification and partial characterization of the soluble low Km 5′-nucleotidase from human seminal plasma

Abstract

Trends Biochem. Sci.

Metabolism

Biochim. Biophys. Acta

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Methods Enzymol.

J. Biol. Chem.

Biochim. Biophys. Acta

Int. J. Biochem.

Comp. Biochim. Physiol.

J. Biol. Chem.

Biochim. Biophys. Acta

Essays in Biochem.

Annu. Rev. Biochem.

Am. J. Physiol.

J. Immunol.

Purification and partial characterization of the soluble low K_m 5′-nucleotidase from human seminal plasma