Neuropeptide y inhibits pertussis toxin-catalyzed ADP-ribosylation in bovine adrenal chromaffin cell membranes

Evidence is presented that the neuropeptide Y receptor is directly coupled to an inhibitory G protein existing in cultured bovine adrenal chromaffin cell membranes. Pertussis toxin catalyzes the [³²P]ADP-ribosylation of a 41 kDa plasma membrane protein. 5′-Guanylylimidodiphosphate inhibited the [³²P]ADP labelling of this protein in a dose-dependent manner whereas GTP had no effect. Preincubation of the plasma membranes with high concentrations of neuropeptide Y followed by a brief exposure to a low concentration of 5′-guanylylimidodiphosphate significant;y inhibited ADP-ribosylation beyond that observed with 5′-guanylimidodiphosphate alone. These result suggest that the neuropeptide Y receptor in bovine adrenal chromaffin cells is directly coupled to a 41 kDa PTX substrate (presumably the α subunit of an inhibitory G protein).     

Binding of nucleotides to purified coupling factor-latent ATPase from Mycobacterium phlei.

Binding studies of various nucleotides to the purified coupling factor-latent ATPase from Mycobacterium phlei have been carried out using gel filtration, equilibrium dialysis, and ultrafiltration methods. The purified latent ATPase binds 3 mol of ADP per mol of the enzyme with an apparent dissociation constant of 68 muM. Binding of nucleotides occurred in the decreasing order: ADP, epsilon-ATP, epsilon-ADP, UDP, adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), IDP, and adenosine 5'-(alpha,beta-methylene)diphosphate (AdoP(CH2)P). AMP-P(NH)P inhibits both soluble (Ki = 77 muM) and membrane-bound latent ATPase activity. However, AMP-P(NH)P does not affect oxidative phosphorylation in membrane vesicles of M. phlei. AMP-P(NH)P exhibits one binding site per molecule of the enzyme with a dissociation constant of 71 muM. After trypsin treatment of the enzyme, the binding of ADP decreases 35%, while AMP-P(NH)P binding remains unchanged. Moreover, AMP-P(NH)P binding was not displaced by ADP. Studies with sulfhydryl agents showed that, in contrast to AMP-P(NH)P, binding of at least 1 mol of ADP requires the participation of sulfhydryl groups. The results indicate that AMP-P(NH)P and ADP do not share a common binding site and that the latent ATPase enzyme has separate sites for ATP hydrolysis and ATP synthesis.     

Energy-dependent endocytosis in erythrocyte ghosts. IV. Effects of Ca2+, Na+ + K+, and 5'-adenylylimidodiphosphate

The requirement of actual splitting of ATP for endocytosis in erythrocyte ghosts has been confirmed by use of the ATP analog, 5'-adenylylimidodiphosphate. (AMP-P(NH)P. This compound, in which the oxygen connecting the β and γ phosphorus atoms was replaced by an NH group, did not cause endocytosis nor was it a substrate for ATPase activity. AMP-P(NH)P was a competitive inhibitor both for the endocytosis and the Mg²⁺-ATPase activities. The $\text{K}{}_{\mathrm{<mtext>1</mtext>}}$ of AMP-P(NH)P for Mg²⁺-ATPase activity was 2.0 · 10^?4 M and, while the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of ATP for this activity was also 2.0 · 10^?4 M indicating nearly identical affinities of ATP and AMP-P(NH)P for the active site. ADP, or ADP plus orthophosphate, did not cause endocytosis, showing that endocytosis was not due to binding of the products of ATP hydrolysis. Sodium or potassium ion or ouabain had no effect on endocytosis, which eliminated the possibility of involvement of the Na⁺, K⁺ ATPase in the endocytosis process. Calcium could not be substituted for magnesium; rather it inhibited endocytosis at the concentration of 1 · 10^?3 M. EGTA relieved the inhibitory effect of Ca, which indicated that the binding of calcium to the membrane was reversible. These experimental results reaffirm the conclusion that ATP must be split to engender endocytosis under these conditions. Some characteristic parameters of the hemoglobin-fre porcine erythrocyte ghosts were studied in order to characterize the system more adequately.     

Insulin stimulates GDP release from G proteins in the rat and human liver plasma membranes

Plasma membranes (1–2 mg protein) prepared from the livers of adult male rats and human organ donors were incubated with 0.6 μM [α-³²P] guanosine triphosphate (GTP) in an adenosine triphosphate (ATP)-regenerating buffer at 37°C for 1 h; during this incubation, the [³²P]GTP is hydrolyzed and the nucleotide that is predominantly bound to the membranes is [³²P] guanosine diphosphate (GDP). [³²P]GDP release from the liver membranes was proportional to the protein concentration and increased as a function of time. At 5 mM, Ca²⁺, Mg²⁺, Mn²⁺, and Zn²⁺ maximally inhibited GDP release by 80–90%, whereas, 5 mM Cu²⁺ maximally stimulated the reaction by 100%. Therefore, cations were not included in the buffer used in the GDP release step. One μM Gpp(NH)p (5′-guanylylimidodiphosphate), a nonhydrolyzable analog of GTP, maximally stimulated [³²P]GDP release in the liver membranes by up to 30%. Although 10 nM Gpp(NH)p had no effect on GDP release, it appeared to stabilize the hormonal effect by blocking further GDP/GTP exchange. In the rat membranes, 1–100 nM glucagon (used as a positive control) stimulated [³²P]GDP release by about 17% (P < .05); similarly, 0.1–100 nM insulin stimulated [³²P]GDP release by 10–13% (P < .05). In the human membranes, 10 pM to 100 nM insulin stimulated [³²P]GDP release by 7–10%. In the rat membranes, 10 nM insulin stimulated [³²P]GDP release by 17 and 24% at 2 and 4 min, respectively (P < .05); in the human membranes, 10 nM insulin stimulated [³²P]GDP release by about 9% at 2 and 4 min. Normal rabbit IgG (used as a control for insulin receptor antibody) by itself stimulated the GDP release by rat and human membranes. However, the stimulation of the GDP release by insulin receptor antibody was consistently higher than that observed with normal rabbit IgG. Four to 15 μg of insulin receptor antibody stimulated [³²P]GDP release by 12–22% (P < .05) and 7–14% in rat and human membranes, respectively. These results indicate that ligand binding to the insulin receptor results in a functional interaction of the receptor with a guanine nucleotide-binding transducer protein (G protein) and activation of GTP/GDP exchange.     

Adenosine interactions with thyroid adenylate cyclase

Adenosine and certain adenosine analogues inhibit beef thyroid membrane adenylate cyclase. The inhibition has a rapid onset, is not directly on the catalytic or nucleotide regulatory sites, occurs with all activators tested (ITP, Gpp(NH)p, TSH, and F^?), and is seen also in mouse and human thyroid membranes. Addition of manganous ion, which activates adenylate cyclase, markedly enhances the inhibition by adenosine analogues. The order of potencies is: 2′,5′-dideoxyadenosine > 5′-deoxyadenosine > 2′-deoxy-3′-phosphoadenosine > 2′-deoxyadenosine > adenosine > adeninexyloside > adenine arabinoside. Purinemodified analogues are either inactive or stimulate slightly at high concentrations. This chemical specificity, the Mn²⁺ requirement, and the lack of reversal by theophylline, suggest that these membranes have little “R” site activity (stringent for the ribose moiety) and primarily contain a “P” site that has stringent purine requirement but permits changes in the ribose moiety. This site appears to be associated with the catalytic unit since it persists in solubilized adenylate cyclase.     

An exoribonuclease from Saccharomyces cerevisiae: effect of modifications of 5' end groups on the hydrolysis of substrates to 5' mononucleotides.

Using poly(A) as a substrate, an exoribonuclease has been purified from the high-salt wash of ribosomes of $\text{Saccharomyces cerevisiae}$. The product of the reaction of the exoribonuclease is 5′ AMP. Hydrolysis of [³H](pA)₃[¹⁴C](pA)_n shows that both labels are released at the same rate, suggesting that the enzyme acts in a processive manner. Removal of the terminal phosphate of poly(A) with alkaline phosphatase reduces the rate of hydrolysis by 80%. Treatment of the terminally dephosphorylated poly(A) with polynucleotide kinase restores the activity. Two 5′ capped mRNA's have been tested and they are hydrolyzed slowly, if at all, by the enzyme. In contrast, phage T4 mRNA, ribosomal RNA, and encephalomyocarditis viral RNA are hydrolyzed at greater than 50% of the rate of poly(A).     

Effect of melittin-induced membrane alterations on rat heart adenylate cyclase activity

Melittin, a surface-active, 26-amino acid polypeptide from bee venom, has been reported to alter a variety of membrane properties including stability, permeability, and fluidity, the latter having been shown to be altered in a biphasic manner. Melittin induced a biphasic alteration of rat heart microsomal adenylate cyclase activity, stimulating it at low concentration (<30 μg/ml) and inhibiting it at higher concentrations (100 μg/ml or higher). Melittin potentiated sodium fluoride and 5′-guanylylimidodiphosphate activation of adenylate cyclase below 40 μg/ml but it inhibited at high concentrations, except in the presence of high concentrations of 5′-guanylylimidodiphosphate (10^?4m). Basal and fluoride-activated adenylate cyclase exhibited no significant change in the K_m for ATP in the presence of melittin at <40 μg/ml, but the V was elevated. Potentiation by melittin of adenylate cyclase was observed at all fluoride, 5′-guanylylimidodiphosphate, and magnesium concentrations tested. The observed effects of melittin on rat heart adenylate cyclase are consistent with it acting by altering the properties of membrane lipids with which the enzyme is associated.     

Kinetic studies on rat liver and beef heart mitochondrial ATPase. Evidence for nucleotide binding at separate regulatory and catalytic sites.

Mitochondrial ATPases from rat liver and beef heart were used to study the effects of guanylylimidodiphosphate (GMP-P(NH)P) and adenylylimidodiphosphate (AMP-P(NH)P) on the kinetics of MgATP, MgITP, and MgGTP hydrolysis. AMP-P(NH)P was a noncompetitive inhibitor of hydrolysis of all substrates with the rat liver enzyme, whether activating anions were present or not. Also with the liver enzyme, AMP-P(NH)P caused only MgATP hydrolysis to appear to have positive cooperativity. With the beef heart enzyme, AMP-P(NH)P was a competitive inhibitor of ATPase activity and caused positive cooperativity; it gave noncompetitive patterns with GTP or ITP as substrates. In both enzyme systems, GMP-P(NH)P gave complex inhibition patterns with MgATP as the substrate, but was a competitive inhibitor of MgITP and MgGTP hydrolysis. These results are interpreted as indicating the existence of two types of nucleotide binding sites, with varying degrees of specificity and interaction on the ATPase molecules from both sources. It is postulated that MgATP and AMP-P(NH)P bind to regulatory site while MgATP, MgGTP, Mgitp, and GMP-P(NH)P bind to the catalytic site.     

Demonstration of adenylate cyclase activity in human red blood cell ghosts

The presence of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) activity was demonstrated in human erythrocyte ghosts and was found to be around 3 pmol adenosine ′,5′-monophosphatase (cyclic AMP) · 2 h^?1 · mg^?1 protein. This enzymatic activity is strongly stimulated by NaF and 5′-guanylimidodiphosphate, is slightly stimulated by epinephrine, norephrine, soproterenol, and prostaglandin E, and is inhibited by calcium. The hormone stimulation is not potentiated by 5′-guanylylimidodiphosphate.     

Multiple expressions of the activity of guanine nucleotide regulatory protein in human thyroid adenylate cyclase

Adenylate cyclase (ATP pyrophosphate-lyase, EC 4.6.1.1) in plasma membranes from human thyroid was highly responsive to thyrotropin. Pretreatment of thyroid plasma membranes with 5′-guanylylimidodiphosphate (Gpp(NH)p) in the presence of Mg²⁺ led to a temperature-dependent activation, which was seen neither in the absence of Mg²⁺ nor at 4 °C. By contrast, thyrotropin bound to its receptors regardless of the temperature and produced its maximal effect after 2 min of preincubation in the absence or presence of Mg²⁺. Furthermore, activation was seen after treatment with thyrotropin and Gpp(NH)p even carried out in the absence of Mg²⁺ or at 4 °C. However, the full activation by Gpp(NH)p required Mg²⁺, hormone, and elevated temperature. These observations suggest that there appears to be two types of nucleotide interaction responsible for the Gpp(NH)p activation in human thyroid membrane; one type seen in the absence of hormone may represent the system uncoupled from hormone receptor, while the fully coupled hormone-sensitive adenylate cyclase accounts for the second type of interaction which requires the presence of hormone.     

Structure of the linkage between adenovirus DNA and the 55,000 molecular weight terminal protein

The 5′ terminus of each complementary strand of adenovirus DNA isolated from virions is covalently linked to a protein with an apparent molecular weight of 55,000. We have determined the structure of the protein-DNA linkage. The 55,000 M_r protein, linked to a small [³²P]oligonucleotide, was isolated after DNase digestion of uniformly ³²P-labeled adenovirus 5 (Ad5) DNA-protein complex. The protein was digested with trypsin and the resulting [³²P] peptides were analyzed with the following results. (1) Acid hydrolysis released a single phosphorylated amino acid which was identified as O-phosphoserine in four separate electrophoretic or chromatographic systems; (2) treatment with snake venom phosphodiesterase yielded exclusively dAMP, dCMP and dTMP as expected (there are no guanylate residues in the first 25 nucleotides at the 5′ ends of Ad5 DNA); (3) prior treatment of the [³²P]peptide preparation with snake venom phosphodiesterase greatly reduced the yield of O-phosphoserine upon subsequent acid hydrolysis. These results suggest that Ad5 DNA is bound to the terminal protein by a phosphodiester linkage to the β-OH of a serine residue. This conclusion is supported by the finding that the DNA-protein linkage is readily hydrolyzed in alkali. In 50 mm-NaOH at 70 °C the half time for hydrolysis of the linkage is about ten minutes. After incubation of Ad5 DNA under these conditions we were able to label the 5′ termini with ³²P by sequential treatment with alkaline phosphatase and polynucleotide kinase. Digestion of the end-labeled DNA to 5′ mononucleotides yielded [³²P]dCMP. We conclude that the terminal protein is bound to Ad5 DNA by a phosphodiester linkage between the β-OH of a serine residue of the protein and the 5′-OH of the terminal deoxycytidine residue of the DNA.     

Conversion of 5′-Methylthioadenosine into S-adenosylmethionine by yeast cells

5′-Methylthio[U-¹⁴C]adenosine was used as a culture supplement for Candida utilitis. The resulting S-adenosylmethionine was hydrolyzed into its structural components. Virtually none of the label of the pentose was found in the carbohydrate part of the intracellular S-adenosylmethionine. Much of it was present in the four-carbon chain of the methionine part of the sulfonium compound. The U-¹⁴C)-labeled adenine of 5′-methylthio[U-¹⁴C]adenosine did not contribute to the labeling of the amino acid component of the sulfonium compound.     

Photolysis of water coupled to nitrate reduction by Nostoc muscorum subcellular particles.

The binding of tritiated guanylylimidodiphosphate ([³H]GMP-P(NH)P) to turkey erythrocyte ghosts was studied in parallel with the activation by GMP-P(NH)P of adenylate cyclase. The high affinity binding capacity for GMP-P(NH)P, 50 pmoles per mg protein, exceeds the estimated quantity of adenylate cyclase of 1 pmole per mg of protein. The rate of nucleotide binding is not affected by isoproterenol. Further, in the presence of the hormone the rate of binding is much slower than the rate of activation. Although the rate of dissociation of bound [³H]GMP-P(NH)P is negligible at 37°, it is increased dramatically by unlabeled GMP-P(NH)P, GTP, EDTA, ATP, AMP-P(CH₂)P, or p-aminophenylmercuric acetate. In contrast, the rate of decay of the GMP-P(NH)P-simulated state is not altered by these agents. Thus, the major fraction of GMP-P(NH)P binding to membranes is not relevant to cyclase activation.     

Enthalpy of nucleotides binding to myosin.

The enthalpies of binding adenosine 5'-diphosphate (ADP) and 5'-adenylyl imidodiphosphate [AMP-P(NH)P] to rabbit skeletal myosin have been measured in Pipes and Tris buffers at pH 7.8 and 15 degrees C. For ADP the enthalpy of binding was exothermic, whereas the enthalpy of binding AMP-P(NH)P, a nonhydrolyzable ATP analogue, was small and endothermic. For the reaction of ATP and myosin, the development of enthalpy was resolved into two phases: a fast endothermic phase, which is the summation of binding and hydrolysis, and a slow exothermic phase, which is associated with product-release steps. These results are discussed in terms of their implications for energy transduction.     

Partial purification and properties of a nucleoside hydrolase from Crithidia

An enzyme catalyzing the hydrolysis of nucleosides was found to occur in Crithidia fasciculata and was partially purified (30- to 40-fold) by treatment with either streptomycin sulfate or MnCl₂, ammonium sulfate fractionation, acidification and neutralization, passage through Sephadex G-200, and isoelectric focusing. The specific activity of these preparations was about 6 μmnoles of uridine hydrolyzed per mg protein per min. Specificity for the puriue or pyrimidine base was very broad; uridine gave the maximum rate of hydrolysis. Deoxyribosides were not hydrolyzed. The enzyme is relatively stable to heat and to acidification and can be stored frozen. Hydrolysis of uridine is inhibited by borate ions and by adenosine, inosine, and guanosine, but not by cytidine or xanthosine.     

Properties of rat liver plasma membrane adenylate cyclase after chromatography on O-diethylaminoethyl-cellulose and agarose-hexane-GTP: Sensitivity of partially purified and purified enzyme to Lubrol-PX, 5′-guanylylimidodiphosphate,and glucagon

Partially purified rat liver plasma membranes were enriched to yield a more glucagon-sensitive membrane fraction which was solubilized with Lubrol-PX. The supernate obtained after centrifugation at 165,000g was subjected to O-diethylaminoethyl anion exchange chromatography. An adenylate cyclase fraction was eluted and purified further by chromatography on agarose-hexane-GTP. The enzyme adsorbed to the affinity resin and was eluted with 0.5 m Tris-HCl. The protein isolated by chromatography on the affinity resin was homogenous by conventional acrylamide gel electrophoresis; one band was observed in sodium dodecyl sulfate. The purified enzyme was free of nucleotide phosphohydrolases found in the parent solubilized membrane preparation. The anion exchange product was not sensitive to glucagon; Lubrol-PX and 5′-guanylylimidodiphosphate [Gpp(NH)p] decreased the activity of this fraction. In the presence of detergent or guanyl nucleotide, glucagon, at 10^?6m, increased enzyme activity by 30 and 21%, respectively, to a statistically significant degree, but not above basal levels. Adenylate cyclase was also purified by subjecting the 165,000g supernate directly to agarose-hexane-GTP; agarose-hexane-ATP or agarose-hexane was not effective. The affinity-derived material was associated with 85 nmol of Lubrol-PX/mg of protein. When calculated on the basis of a molecular weight of 150,000 for detergent-free protein after gel filtration on Bio-Gel A-0.5 m, there was 13 mol of detergent/mol of the enzyme obtained by chromatography on the affinity resin. The direct affinity product was insensitive to glucagon and Gpp(NH)p; enzyme activity varied as a function of Lubrol concentration.     

Further chemical characterization and biological activities of fluorescent I,N6-ethenoadenosine derivatives

The fluorescent 1,N⁶-ethenoadenosine derivatives of adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, 3′:5′-cyclic adenosine monophosphate, adenosine and nicotinamide adenine dinucleotide have been prepared. Paper and thin layer chromatographic purification methods have been developed. Nuclear magnetic resonance and mass spectrum data indicate that only the purine ring has been modified.The 1,N⁶-ethenoadenosine triphosphate had about 70% of the activity of adenosine triphosphate as a substrate for total adenosine triphosphatase activity of hypophysectomized rat liver membranes. The 1,N⁶-ethenoadenosine diphosphate had about 86% of the activity of adenosine diphosphate as a substrate for adenosine diphosphatase of hypophysectomized rat liver membranes. The 1,N⁶-etheno derivative of nicotinamide adenine dinucleotide had about 8% of the activity of nicotinamide adenine dinucleotide as a substrate for nicotinamide adenine dinucleotide glycohydrolase and about 54% of the activity of nicotinamide adenine dinucleotide as a substrate for nicotinamide adenine dinucleotide pyrophosphatase of hypophysectomized rat liver membranes.K_m's for the ATPase, ADPase and yeast alcohol dehydrogenase using ε-ATP and ε-ADP and ε-NAD as substrates are presented.     

Energy-dependent endocytosis in erythrocyte ghosts. IV. Effects of Ca2+, Na+ +K+, and 5'-adenylylimidodiphosphate.

The requirement of actual splitting of ATP for endocytosis in erythrocyte ghosts has been confirmed by use of the ATP analog, 5'-adenylylimidodiphosphate, (AMP-P(NH)P). This compound, in which the oxygen connecting the beta and gamma phosphorus atoms was replaced by an NH group, did not cause endocytosis nor was it a substrate for ATPase activity. AMP-P(NH)P was a competitive inhibitor both for the endocytosis and the Mg2+-ATPase activities. The K1 of AMP-P(NH)P for Mg2+ ATPase activity was 2.0 - 10-4 M and, while the Km of ATP for this activity was also 2.0 - 10-4 M indicating nearly identical affinities of ATP and AMP-P(NH)P for the active site. ADP, or ADP plus orthophosphate, did not cause endocytosis, showing that endocytosis was not due to binding of the products of ATP hydrolysis. Sodium or potassium ion or ouabain had no effect on endocytosis, which eliminated the possibility of involvement of the Na+, K+ ATPase in the endocytosis process. Calcium could not be substituted for magnesium; rather it inhibited endocytosis at the concentration of 1 - 10-3 M. EGTA relieved the inhibitory effect of Ca, which indicated that the binding of calcium to the membrane was reversible. These experimental results reaffirm the conclusion that ATP must be split to engender endocytosis under these conditions. Some characteristic parameters of the hemoglobin-free porcine erythrocyte ghosts were studied in order to characterize the system more adequately.     

Characterization and Localization of Acid Phosphatase Activity of Trypanosoma gambiense*

SYNOPSIS. Properties and cellular location of acid phosphatase in Trypanosoma gambiense were studied. Activity was found in both the sediment (32,000 ×g) and the supernatant of homogenates. Cenrifugation in 0.3 M sucrose showed activity principally in the lowspeed fraction (4,000 ×g). One min of sonication released most of this activity. Several phosphomonoesters were hydrolyzed at acid H's. Enzymatic activity was relatively specific for pyrophosphate and p-nitrophenylphosphate at pH 3.6. At pH 5.2, purine and pyimidine nucleotide 5′-triphosphates as well as adenosine di- and ono-5′-phosphates were hydrolyzed nonspecifically. Activity with yrophosphate at pH 3.6 had a temperature optimum of 60-70 C while that for adenosine 5′-triphosphate (pH 5.2) was 50 C. These ctivities of the sediment required no metal co-factors and were inibited by Fe⁺⁺, inhibition at the lower pH being greater. Glucose 6-phosphate was hydrolyzed by the supernatant with maximum activity between pH 6.0 and 7.2 and a temperature optimum of 50 C. This pH range showed a broad plateau with 2 or 3 minor peaks. The hydrolysis of p-nitrophenylphosphate showed a similar pH curve. In glucose 6-phosphate hydrolysis, Mg⁺⁺ was a required co-factor but could be replaced by Ni⁺⁺ or Co⁺⁺. Ammonium sulfate fractionation precipitated most of the supernatant activity between 50 and 75% saturation. A modified Gomori technic produced spherical deposits of PbS thruout the cytoplasm of the intact cell. With the electron microscope, Pb phosphate deposition was observed in membrane-bound vesicles (i.e., lysosomes) approximately 100-150 mμ in diameter. These organelles were common in the region of the reservoir at the base of the flagellum. Acid phosphatase activity specific for glucose 6-phosphate as substrate was localized within this basal pocket.     

Inorganic polyphosphate hydrolysis catalyzed by skeletal muscular actomyosin complexes is uncoupled with motility

Hydrolysis of the triphosphate moiety of ATP, catalyzed by myosin, induces alterations in the affinity of the myosin heads for actin filaments via conformational changes, thereby causing motility of the actomyosin complexes. To elucidate the contribution of the triphosphate group attached to adenosine, we examined the enzymatic activity of heavy meromyosin (HMM) with actin filaments for inorganic tripolyphosphate (3PP) using a Malachite green method and evaluated using fluorescence microscopy the effects of 3PP on actin filament motility on HMM-coated glass slides. In the presence of MgCl₂, HMM hydrolyzed 3PP at a maximum rate of 0.016 s⁻¹ HMM⁻¹, which was four times lower than the hydrolysis rate of ATP. Tetrapolyphosphate (4PP) was hydrolyzed at a rate similar to that of 3PP hydrolysis. The hydrolysis rates of 3PP and 4PP were enhanced by roughly 10-fold in the presence of actin filaments. In motility assays, the presence of polyphosphates did not lead to the sliding movement of actin filaments. Moreover, in the presence of ATP at low concentrations, the sliding velocity of actin filaments decreased as the concentration of added polyphosphate increased, indicating a competitive binding of polyphosphate to myosin heads with ATP. These results suggested that the energy produced by standalone triphosphate hydrolysis did not induce the unidirectional motion of actomyosin and that the link between triphosphate and adenosine was crucial for motility.