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A method of efficient enzymatic hydrolysis of polyprenyl pyrophosphates was established. Polyprenyl pyrophosphates such as all-trans-octaprenyl and all-trans-nonaprenyl (solanesyl) pyrophosphate can be hydrolyzed completely to the corresponding primary alcohols by the action of potato acid phosphatase in the presence of 60% (v/v) methanol.
Dimethylallyl pyrophosphate: L-tryptophan dimethylallyltransferase (dimethylallyl tryptophan synthetase) has been purified from Claviceps strain SD58 to a homogeneous crystalline form. The enzyme is pure as judged by polyacrylamide gel electrophoresis and contains two similar subunits of 34,000 molecular weight as shown by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Since the enzyme was determined to have a molecular weight of 70,000 by gel exclusion chromatography, it is dimeric. The substrates dimethylallyl pyrophosphate and tryptophan each have mixed (negative to positive) cooperativity with a minimum Hill coefficient of 0.37 and 0.73, respectively. Calcium ion is a positive allosteric effector and, at 20 mM concentration, deregulates the enzyme. The deregulated enzyme has Km values of 7.2 microM for dimethylallyl pyrophosphate and 8.8 microM tryptophan. The Vmax under deregulated conditions is 953 nmol/min/mg, giving a turnover number of 14. Ca2+ has a S[0.5] of 4.5 mM. The kinetic mechanism is random sequential.
Mevalonate pathway enzyme activities in rat liver mitochondria were investigated, and it was found that isopentenyl pyrophosphate can be utilized for the synthesis of all-trans-polyprenyl pyrophosphates in vitro. In this reaction sequence intermediate formation of farnesyl pyrophosphate (FPP) predominates, and the FPP synthase activity was studied in more detail. The mitochondrial activity constitutes 13% of the total hepatic capacity for FPP synthesis, exceeding the corresponding microsomal, nuclear, and peroxisomal activities by 10-fold. Mitochondrial FPP synthase exhibits trypsin sensitivity only after sonication of intact mitochondria and upon subfractionation the activity is found localized in the matrix. FPP synthase activities at different locations responded distinctly when rats were treated with a diet enriched in cholesterol or containing mevinolin or cholestyramine. With the high cholesterol diet, mitochondrial FPP synthase activity increased 2-fold, while the cytosolic activity was slightly decreased. Both mevinolin and cholestyramine treatment resulted in 3-fold increases in cytosolic FPP synthase activities, without altering the mitochondrial activity. FPP was utilized as substrate for trans-prenyltransferase activity in the inner mitochondrial membrane. The products formed in this reaction were identified as nona- and decaprenyl-PP, and the reaction was influenced by changes in both substrate and Mg2+ concentration, giving more decaprenyl-PP when the concentrations of these substances were increased. These results demonstrate that mitochondria utilize endogenously produced FPP for isoprenoid biosynthesis and that the biosynthetic steps in mitochondria are regulated independently from those occurring in other subcellular compartments.
There is little information concerning the intracellular function of inositol 1,3,4,5,6-pentakis- and hexakisphosphate, despite their being the most abundant inositol polyphosphates. Current opinions that they play passive roles as antioxidants (Graf, E., Mahoney, J. R., Bryant, R. G., and Eaton, J. W. (1987) J. Biol. Chem. 259, 3620-3624) or "housekeeping" molecules (Berridge, M. J., and Irvine, R. F. (1989) Nature 341, 197-205) arises from belief in their metabolic lethargy. However, we have discovered that cell homogenates, incubated with 5 mM fluoride and 5 mM ATP, converted both inositol hexakisphosphate (Km = 2 +/- 0.5 microM, Vmax = 9 +/- 2 pmol/mg of protein/min) and inositol 1,3,4,5,6-pentakisphosphate (Km = 13 +/- 4 microM, Vmax = 11 +/- 5 pmol/mg of protein/min) to more polar products. These reactions were also observed in intact cells treated with 0.5-20 mM fluoride, and the precursor/product relationships were confirmed by comparing the effects of fluoride on cells differentially labeled with [3H]inositol in either short-term or pulse-chase protocols. The novel products were determined to be inositol pyrophosphates because of their relatively specific hydrolysis by tobacco pyrophosphatase and alkaline phosphatase. The pyrophosphates were metabolized rapidly by cell homogenates back to their pentakisphosphate and hexakisphosphate precursors. This endogenous pyrophosphatase activity was inhibited by up to 99% by 5 mM fluoride in vitro. In intact cells incubated with 10 mM fluoride, about 20% of the inositol 1,3,4,5,6-pentakisphosphate pool, and 50% of the inositol hexakisphosphate pool were each converted to pyrophosphate derivatives within 1 h.
The primary structure of pyrophosphate-dependent phosphofructokinase (PFK) from Propionibacterium freudenreichii exhibits a low but significant level of sequence identity with Escherichia coli ATP-dependent PFK, permitting the tentative assignment of residues that may be involved in catalysis. Based on these assignments, the roles in catalysis of 2 aspartyl residues (Asp151 and Asp153) and 2 lysyl residues (Lys80 and Lys85) were examined. Mutagenesis of the Asp151 to alanine and serine reduced kcat by a factors of 2 x 10(4) and 4 x 10(5), respectively, while showing no change in Km for either substrate in the forward reaction or for metal ion in the back reaction. The kcat for Asp153 was decreased by a factor of 700 with no change in Km for pyrophosphate and an increase of about 20-fold in Km for fructose 6-P and close to 4-fold for magnesium ion. That these changes in the mutants were not the result of global conformational changes was indicated by their identical behavior during substrate-specific elution chromatography, ion-exchange chromatography, and limited proteolysis by trypsin and subtilisin. Mutations of Lys80 and Lys85 showed no significant changes in kinetic parameters, suggesting no involvement in mechanism or substrate binding. These and other results permit preliminary modeling of the active site of pyrophosphate-dependent PFK.
A randomized, two-compartment calculus clinical study of twelve-weeks duration was conducted among a group of calculus-forming subjects in the St. Louis area. The purpose of this parallel and double-blind clinical study was to compare the effect of a dentifrice containing 5.0% potassium nitrate, 1.3% soluble pyrophosphate, 1.5% PVM/MA copolymer and 0.243% sodium fluoride in a silica base, to that of a placebo dentifrice, with regard to supragingival calculus formation. The study examiner, using the Volpe-Manhold Calculus Index, selected a panel of calculus-prone men and women who had completed a one-month placebo regimen. The Volpe-Manhold Calculus Index scores and the number of completely calculus-free sites were recorded. One-hundred and fifteen subjects were entered into the study. After an oral soft and hard tissue examination, the subjects were given a complete oral prophylaxis and randomly assigned to use either the placebo or test dentifrice for a 12-week home-use period. They were prohibited from using any other means of oral hygiene during the study. After completing 12 weeks of twice-daily brushing at home using their assigned toothpaste and a standard soft-bristled toothbrush, subjects were again assessed for supragingival calculus deposits and calculus-free sites. The dentifrice containing 5.0% potassium nitrate, 1.3% soluble pyrophosphate, 1.5% PVM/MA copolymer and 0.243% sodium fluoride in a silica base, inhibited supragingival calculus formation by 54.4%, as compared to a 0.243% sodium fluoride silica-based placebo dentifrice. The mean Volpe-Manhold Calculus Index scores were compared statistically and the difference indicated statistical significance at probability of 0.01 by means of an analysis of covariance (ANCOVA). The number of calculus-free sites, a second parameter of efficacy, also was compared and demonstrated an absolute difference of 32.6% in favor of the group using the dentifrice containing potassium nitrate, soluble pyrophosphate and PVM/MA copolymer compared to a placebo dentifrice (37.8% vs 5.2%, respectively). An analysis of covariance indicated that this improvement showed statistical significance at a probability of 0.01.
Whereas radiographic lesions of the sacroiliac joints are common in patients with calcium pyrophosphate deposition crystal disease, they are rarely accompanied with clinical symptoms. We report two cases of acute sacroiliitis probably due to calcium pyrophosphate dihydrate deposition disease. The patients were a 53-year-old man and an 82-year-old woman with chondrocalcinosis in other joints and presence on computed tomography studies of the sacroiliac joints of sclerosis and irregularities of the joint margins with a thin linear calcific deposit within the joint. Both patients recovered fully under therapy with colchicine, analgesics and rest. These two cases suggest that acute sacroiliitis can be caused by calcium pyrophosphate dihydrate crystal deposition disease.
Purified membrane-bound alkaline phosphatase from rat osseous plate hydrolyzed pyrophosphate in the presence of magnesium ions, with a specific activity of 92.7 U/mg. Optimal apparent pH for pyrophosphatase activity was 8.0 and it remained unchanged on increasing the pyrophosphate concentration. In the absence of magnesium ions the enzyme had a Km = 88 microM and V = 36.7 U/mg for pyrophosphate and no inhibition by excess substrate was observed. Pyrophosphatase activity was rapidly destroyed at temperatures above 40 degrees C, but magnesium ions apparently protected the enzyme against denaturation. Sodium metavanadate (Ki = 1.0 mM) was a competitive inhibitor of pyrophosphatase activity, while levamisole (Ki = 8.2 mM) and theophylline (Ki = 7.4 mM) were uncompetitive inhibitors. Magnesium ions (K0.5 = 1.7 microM) stimulated pyrophosphatase activity, while cobalt (Ki = 48.5 microM) and zinc (Ki = 22.0 microM) ions were non-competitive inhibitors. Manganese and calcium ions had no effect on pyrophosphatase activity. The Mw of the pyrophosphatase protein was 130 kDa by gel filtration, but a value of 65 kDa was obtained by dissociative gel electrophoresis, suggesting that it was a dimer of apparently identical subunits. These results suggested that pyrophosphatase activity stems from the membrane-bound osseous plate alkaline phosphatase and not from a different protein.
Because skeletal images obtained with a commercial lyophilized preparation of 99mTc-Sn-pyrophosphate were occasionally degraded by liver and spleen uptake, a method for preparing 99mTc-Sn-pyrophosphate and a system for in-house quality assurance were developed. Control of the age of the stannous chloride solution and the final pH of the 99mTc-Sn-pyrophosphate eliminated the undesirable uptake. The quality and body distribution of the in-house preparation were compared to those of the radiopharmaceutical from a commercial kit.
Uncertainties as to the nature and implications of osteoarthritis and calcium pyrophosphate deposition disease (CPPD) in primates were subject to critical review through examination of 153 prosimians and 1,250 Old World non-prosimian primates. Osteoarthritis, calcium pyrophosphate deposition disease, and infectious arthritis/osteomyelitis were relatively rare phenomena, affecting only 1.7% and 2.5%, respectively, of free-ranging prosimians and other Old World non-prosimian primates. Frequency of infection in Indri and Presbytis appears to reflect a unique susceptibility or exposure. Papio may have a unique predisposition to CPPD. The dichotomy (frequency and joint distribution) between free-ranging and artificially restrained animals suggests caution in interpretation of osteoarthritis or CPPD in non-free-ranging animals.
A case of olecranon bursitis in an 81-year-old patient is presented. Analysis of the bursal fluid revealed positive birefringent crystals; radiographs showed calcifications in the distal triceps tendon. A bursectomy was performed. X-ray diffraction analyses demonstrated calcium pyrophosphate dihydrate patterns in a subcutaneous "tophus" and in a specimen of the tendon. On histologic examination, there was a bursitis with positive birefringent crystals on the bursa's inner surface; histologic images of "chondrocalcinosis" were observed in and around the tendon. It is concluded that bursitis may be part of the extraarticular manifestations of calcium pyrophosphate dihydrate crystal deposition disease.
Adenosine triphosphate pyrophosphohydrolase (ATPPPH) and neutral inorganic pyrophosphatase activities were assayed in synovial fluids (SF) from 37 patients with a variety of arthropathies. ATPPPH activity was detected in all fluids, but was highest in patients with chronic chondrocalcinosis; its activity in patients with osteoarthritis was higher than that in patients with rheumatoid arthritis, gout, or pseudogout. ATPPPH activity correlated positively with SF pyrophosphate concentration and negatively with SF white blood cell count. Pyrophosphatase activity did not correlate with diagnosis, pyrophosphate level, or white blood cell count.
OBJECTIVE: To identify the morphology of a mineral-forming of adult porcine hyaline articular cartilage digest and characterize the mineral it forms. METHODS: Electron microscopy, Fourier transform infrared (FTIR) spectroscopy, x-ray microanalysis, compensated polarized light microscopy, and biochemical studies including 14C-labeled UDPG pyrophosphohydrolase radiometric assay. RESULTS: This fraction of articular cartilage digest contained membrane-limited vesicles resembling growth plate cartilage matrix vesicles and formed mineral after only 24 hours in physiologic salt solution containing 1 mM ATP: The mineral contained inorganic pyrophosphate, 95% of which derived from ATP, and phosphate, 93% of which derived from inorganic phosphate in the medium. The FTIR spectrum of this mineral closely resembled the spectrum of standard calcium pyrophosphate dihydrate (CPPD) crystals. Compensated polarized light microscopy showed positively birefringent, rod-shaped crystals morphologically identical to CPPD. Ca:P ratios, defined by energy-dispersive microanalysis, were also consistent with CPPD. CONCLUSION: The articular cartilage vesicle fraction of porcine hyaline cartilage is capable of generating mineral that strongly resembles CPPD.
OBJECTIVE: The purpose of this study was to determine the effect of transforming growth factor beta 1 (TGF beta 1) on inorganic pyrophosphate (PPi) elaboration from articular tissues to better understand the pathophysiology of calcium pyrophosphate dihydrate (CPPD) crystal deposition in the joint. METHODS: PPi was measured in the media of adult porcine articular tissue in organ culture and monolayer cultures. RESULTS: TGF beta 1 strongly stimulated PPi elaboration by porcine fibrocartilage and hyaline cartilage. It modestly increased PPi elaboration by ligament, and had no effect on PPi elaborated by synovium. Of all cell types tested in cell culture, only chondrocytes responded to TGF beta 1 by significantly increasing PPi elaboration. CONCLUSION: TGF beta 1 stimulates PPi elaboration from hyaline cartilage, fibrocartilage, and ligament, indicating that there is in situ CPPD crystal formation in these tissues. The ability of tissues to respond to TGF beta 1 by increasing PPi elaboration correlates with the prevalence of CPPD crystal deposition found clinically. The unique response of chondrocyte monolayers to TGF beta 1 reinforces the key role of the chondrocyte in PPi elaboration in the joint. These findings support an etiologic role for responsiveness to TGF beta 1 in CPPD disease.
Riboswitches are highly structured RNA elements that control gene expression by binding directly to small metabolite molecules. Remarkably, many of these metabolites contain negatively charged phosphate groups that contribute significantly to the binding affinity. An example is the thiamine pyrophosphate-sensing riboswitch in the 5'-untranslated region of the E. coli thiM mRNA. This riboswitch binds, in order of decreasing affinity, to thiamine pyrophosphate (TPP), thiamine monophosphate (TMP), and thiamine, which contain two, one, and no phosphate groups, respectively. We examined the binding of TPP and TMP to this riboswitch by using (31)P NMR spectroscopy. Chemical-shift changes were observed for the alpha- and beta-phosphate group of TPP and the phosphate group of TMP upon RNA binding; this indicates that they are in close contact with the RNA. Titration experiments with paramagnetic Mn(2+) ions revealed strong line-broadening effects for both (31)P signals of the bound TPP; this indicates a Mg(2+) binding site in close proximity and suggests that the phosphate group(s) of the ligand is/are recognized in a magnesium ion-mediated manner by the RNA.
Pyrophosphate labeled with 99Tc was evaluated as an indicator of the calcification which occurs after implantation of polymeric materials in rats. Poly(2-hydroxyethyl methacrylate), both porous and homogeneous, had been implanted. 99Tc pyrophosphate was applied intravenously to rats in intervals from 14 days to 15 months. Scintigraphy, x-ray examination, and histology were carried out. The ratio of specific activity in the capsule around the implant to specific activity in the reference tissue was determined. The cumulation of activity was parallel with the development of calcification as revealed by histology. The method can be used not only in the screening test of new polymeric materials, but also in a quantitative determination of the degree of calcification in general.
The colorimetric assay for the activity of pyruvate dehydrogenase (EC 1.2.4.1) developed by Itokawa is based on a coupled reduction of ferricyanide and the formation of Prussian blue (Brian Research 94, 475-484). In this assay system, we found that the coenzyme, thiamine pyrophosphate, itself reduced ferricyanide independent of both the enzyme and the substrate. Similar effect was also observed with thiamine, but not with thiochrome. The reduction of ferricyanide by thiamine or thiamine pyrophosphate was blocked by trichloroacetic acid. Measurement of the activity of purified pyruvate dehydrogenase based on monitoring the reduction of ferricyanide as described by Schwartz et al (Biochem. Biophys. Res. Commun. 31, 495-500) has been widely used. Our findings clearly disprove the assumption that reduction of ferricyanide is dependent on pyruvate dehydrogenase and invalidate assays for thiamine-dependent dehydrogenases based on the reduction of ferricyanide.
To depict the three-dimensional distribution of 99mTc-pyrophosphate in the heart, emission computed tomography (ECT) was performed, following the conventional planar imaging, in 32 cases with suspected acute myocardial infarction (AMI). There were 23 cases with evidence of AMI, 7 with unstable angina (UA), and 2 with dilated cardiomyopathy (DCM). While the planar images showed discrete cardiac activity in only 11 of the 23 cases (48%) with AMI, the ECT images delineated focal myocardial uptake clearly in 20 of them (87%). On the other hand, the ECT images revealed cardiac blood-pool activity without significant myocardial uptake in all cases with UA and DCM in which the planar images showed diffuse activity. Thus, the ECT imaging of 99mTc-pyrophosphate seems to be a valuable technique for assessing the presence and localization of myocardial necrosis, especially in cases showing diffuse cardiac activity in the planar imaging.