The influence of metal ions on the orthophosphatase and inorganic pyrophosphatase activities of human alkaline phosphatase

1. The differential effects of adding Zn(2+) and Mg(2+) on the orthophosphatase and inorganic pyrophosphatase activities of human intestinal alkaline phosphatase were studied. 2. In the presence of excess of Zn(2+), inorganic pyrophosphatase activity is inhibited. At higher concentrations of pyrophosphate, hydrolysis of this substrate takes place, but is inhibited competitively by the Zn(2+)-pyrophosphate complex. This complex also acts as a competitive inhibitor of orthophosphate hydrolysis. 3. Excess of Mg(2+) also inhibits pyrophosphatase action by removal of substrate; at low concentrations, this ion activates pyrophosphatase, as is the case with orthophosphatase. 4. It is concluded that, when interactions between metal ions and pyrophosphate are taken into account, the effects of these ions are consistent with the view that alkaline phosphatases possess both orthophosphatase and inorganic pyrophosphatase activities.     

Inhibition of cellulase, xylanase and beta-glucosidase activities by softwood lignin preparations

The conversion of lignocellulosic biomass to fuel ethanol typically involves a disruptive pretreatment process followed by enzyme-catalyzed hydrolysis of the cellulose and hemicellulose components to fermentable sugars. Attempts to improve process economics include protein engineering of cellulases, xylanases and related hydrolases to improve their specific activity or stability. However, it is recognized that enzyme performance is reduced during lignocellulose hydrolysis by interaction with lignin or lignin-carbohydrate complex (LCC), so the selection or engineering of enzymes with reduced lignin interaction offers an alternative means of enzyme improvement. This study examines the inhibition of seven cellulase preparations, three xylanase preparations and a beta-glucosidase preparation by two purified, particulate lignin preparations derived from softwood using an organosolv pretreatment process followed by enzymatic hydrolysis. The two lignin preparations had similar particle sizes and surface areas but differed significantly in other physical properties and in their chemical compositions determined by a 2D correlation HSQC NMR technique and quantitative 13C NMR spectroscopy. The various cellulases differed by up to 3.5-fold in their inhibition by lignin, while the xylanases showed less variability (< or = 1.7-fold). Of all the enzymes tested, beta-glucosidase was least affected by lignin.     

Properties of alkaline-phosphatase fractions in extracts of human small intestine

1. The chromatographic and electrophoretic heterogeneity of human-intestinal alkaline-phosphatase activity is described. 2. The phosphatase activity has been divided into two main components, of which certain properties have been compared. The components resemble each other in their Michaelis constants for the hydrolysis of phenyl phosphate and in their behaviour towards certain inhibitors, but differ in their stability to heat. 3. The addition of Mg²⁺ or Ca²⁺ ions to the electrophoresis buffers sharpens the electrophoretic zones. 4. The results presented do not support the existence of more than one alkaline phosphatase in small intestine.     

Inhibition by avidin of the ATP-Pi enchange activities associated with preparations of energy transfer factors A and A-D

Two water-soluble protein fractions were isolated from sonic extracts of beef heart mitochondria, which corresponded to the energy transfer factors A and A·D (ATP synthetase) described by Sanadi and coworkers. Both fractions augmented the activities of “urea” particles for ATP-Pi exchange and ATP-dependent DPN reduction by succinate. These activities were strongly inhibited by rutamycin. In the absence of added particles, both soluble fractions exhibited ATP-Pi exchange activities, which were not affected by rutamycin, but were strongly inhibited by avidin. The inhibitory effect of avidin was abolished when it was pretreated with biotin. The soluble fractions also exhibited avidin-sensitive propionyl-coenzyme A carboxylase activities, which were compatible with their ATP-Pi exchange activities.     

Inhibition of inorganic pyrophosphatase of animal mitochondria by calcium

Calcium ion is an uncompetitive inhibitor of the inorganic pyrophosphatases of bovine heart and rat liver mitochondria with respect to substrate MgPPi at pH 8.5 and a non-competitive inhibitor of the former enzyme at pH 7.2. The concentration of Ca2+ required to decrease the maximal velocities for both enzymes at pH 8.5, 0.4 mM Mg2+ was about 10 microM. The inhibition results from the binding of two Ca2+ ions to both free enzymes and their complexes with the substrate. The results suggest that Ca2+ regulates pyrophosphatase activity and hence PPi level in mammalian mitochondria.     

Inhibition of inorganic pyrophosphatase from Escherichia coli with inorganic phosphate

The interaction of inorganic pyrophosphatase from E. coli with inorganic phosphate (Pi) was studied in a wide concentration range of phosphate. The apoenzyme gives two inactive compounds with Pi, a product of phosphorylation of the carboxylic group of the active site and a stable complex, which can be detected in the presence of the substrate. The phosphorylation occurs when Pi is added on a millimole concentration scale, and micromole concentrations are sufficient for the formation of the complex. The formation of the phosphorylated enzyme was confirmed by its sensitivity to hydroxylamine and a change in the properties of the inactive enzyme upon its incubation in alkaline medium. The phosphorylation of pyrophosphatase and the formation of the inactive complex occur upon interaction of inorganic phosphate with different subsites of the enzyme active sites, which are connected by cooperative interactions.     

Location of nucleotide pyrophosphatase and alkaline phosphodiesterase activities on the lymphocyte surface membrane.

1. Isolated mouse spleen lymphocytes hydrolysed UDP-galactose added to the medium. Nucleotide pyrophosphatase activity that accounted for this hydrolysis was enriched to a similar extent as alkaline phosphodiesterase and 5'-nucleotidase in a lymphocyte plasma-membrane fraction. 2. The cell surfaces of mouse spleen and thymus lymphocytes were iodinated with 125I by using the lactoperoxidase-catalysis method. Detergent extracts of the cells were mixed with a purified anti-(mouse liver plasma-membrane nucleotide pyrophosphatase) antiserum and the immunoprecipitates analysed by polyacrylamide-gel electrophoresis. Only one major radioactive component, similar in size (apparent mol.wt 110000-130000) to the liver enzyme, was observed. 3. Electrophoresis of an iodinated spleen plasma-membrane fraction indicated peaks of radioactivity, including one of apparent mol.wt 110000-130000. 4. When detergent extracts of spleen lymphocytes were passed through a Sepharose-bead column containing covalently attached anti-(nucleotide pyrophosphatase) antiserum, the nucleotide pyrophosphatase activity was retained by the beads, whereas protein and leucine naphthylamidase activity were eluted. 5. The results indicate that nucleotide pyrophosphatase and alkaline phosphodiesterase activities are due to the location of the same or similar enzymes at the outer aspect of the lymphocyte plasma membrane. Some possible functions of enzymes at this location are discussed.     

Inhibition of mouse spleen inorganic pyrophosphatase by methylene diphosphonic acid]

Some properties of mouse spleen cytosol inorgainc pyrophosphatase (PPi-ase) (E. C. 3.6.1.1) as well as the effect of methylene diphosphonic acid (PCP) on the PPi-ase activity were studied. Specific staining for the enzyme PAAG disc-electrophoresis was developed; it was shown that the PPi-ase formed only one band in 7.5% PAAG. The enzyme pH optimum being 8.0, the optimal [Mg++]/[PPi] ratio was about 2; Km =7.7x10(-4) M, Vmax=0.77 mkM. min-1. mg protein-1. PCP was shown to competitively inhibit the pyrophosphatase reaction, Ki=2.5x10(-4) M +/- 0.2x10(-4) M.     

Inhibition of intrinsic proteolytic activities moderates preanalytical variability and instability of human plasma

Human plasma and serum proteins are subject to intrinsic proteolytic degradation both during and after blood collection. By monitoring peptides, we investigated the stability of plasma and serum samples and the effects of anticoagulants and protease inhibitors on the plasma samples. Serum and plasma were subjected to time-course incubation, and the peptides (750-3200 Da) were extracted and analyzed with MALDI-TOF MS. Peptides of interest were further identified by MALDI-TOF/TOF MS and ESI-MS/MS analyses. Our observations indicate that plasma peptides are significantly different from serum peptides. Intrinsic proteases cause these differences between plasma and serum samples, as well as the differences among three plasma samples using either EDTA, sodium citrate, or heparin as the anticoagulant, which accounts for partial inhibitory effects on plasma proteolytic activities. Proteases and peptidases, including both aminopeptidases and carboxypeptidases, also cause time-dependent, sequential generation and digestion of the peptides in serum and all three plasmas, specifically during early sample collection and processing. Protease inhibitors within an EDTA-plasma-collection device inhibit both intrinsic plasma peptidases and proteases and moderate the time-dependent changes of the plasma peptides, including bradykinin, and complement C4- and C3- derived peptides. Our results suggest that mixing protease inhibitors immediately with blood during blood collection provides enhanced stabilization of the plasma proteome.     

Cloning and expression profile of human inorganic pyrophosphatase.

We have cloned a 1.23 kb cDNA from a human heart library which encodes a 32 kDa protein that is 94% identical to bovine inorganic pyrophosphatase. The protein contains an aspartate-rich signature sequence that was previously identified in yeast and prokaryotic pyrophosphatases. Our clone detects a single band on Northern blots and is expressed at modest levels in all tissues examined. The cDNA shows linkage to markers on the long arm of chromosome 10.