Km Values For ATP Research Articles

Role of glycine 212 in the allosteric behavior of phosphofructokinase from Bacillus stearothermophilus.

Crystallographic studies indicate that the loop between alpha-helix 8 and beta-strand H (the 8H loop) which borders the effector site of Bacillus stearothermophilus phosphofructokinase (BsPFK) is involved in the allosteric mechanism of the enzyme [Schirmer, T., and Evans, P.R. (1990) Nature 343, 140-145]. The residue at one end of this loop, glycine 212, has been proposed to be a pivot about which the loop hinges. Using site-directed mutagenesis, glycine 212 was replaced with valine (G212V). Steady-state kinetic analysis and ligand binding studies on the altered and native PFKs showed that the G212V substitution resulted in discernible changes at the effector site. The mutated PFK required a 3-fold higher concentration of the allosteric inhibitor phosphoenolpyruvate than did the native enzyme to cause the same level of inhibition. The altered PFK had a 2-fold higher dissociation constant for the allosteric activator GDP than the wild-type enzyme. More importantly, whereas the native PFK was fully activated by 1 mM GDP from its PEP-inhibited T-state, the altered enzyme was only marginally activated. On the other hand, the G212V mutation resulted in no changes at the catalytic site of BsPFK. The catalytic rate constant kcat remained unchanged. The altered PFK had the same Km values for ATP and fructose-6-phosphate (Fru-6-P) as did the wild-type enzyme. Furthermore, starting from the same PEP-inhibited T-state, both enzymes gave identical sigmoidal responses to increasing Fru-6-P concentration, indicating that Fru-6-P can activate both to the R-state.

Mutational analysis of the autoinhibitory domain of calmodulin kinase II.

Calmodulin (CaM)-kinase II is inactive in the absence of Ca2+/CaM due to interaction of its autoinhibitory domain with its catalytic domain. Previous studies using synthetic autoinhibitory domain peptides (residues 281-302) identified several residues as important for inhibitory potency and suggested that His282 may interact with the ATP-binding motif of the catalytic domain. To further examine the autoinhibitory domain, site-specific mutants were expressed using the baculovirus/Sf9 cell system. The purified mutants had many biochemical properties identical to wild-type kinase, but mutants H282Q, H282R, R283E, and T286D had 10-20% constitutive Ca(2+)-independent activities, indicating that these residues are involved in the autoinhibitory interaction. The Ca(2+)-independent activities of the H282Q, H282R, and R283E mutants exhibited 10-fold lower Km values for ATP than the wild-type kinase. Wild-type and mutant kinases, except T286A and T286D, generated Ca2+ independence upon autophosphorylation in the presence of Ca2+/CaM, and those mutants having constitutive Ca2+ independence also exhibited enhanced Ca2+/CaM-independent autophosphorylation. This Ca(2+)-independent autophosphorylation resulted in a decrease in total kinase activity, but there was little increase in Ca(2+)-independent activity, consistent with autophosphorylation of predominantly Thr306 rather than Thr286. These results are consistent with an inhibitory interaction of His282 and possibly Arg283 with the ATP-binding motif of the catalytic domain, and they indicate that constitutively active CaM-kinase II cannot autophosphorylate on Thr286 in the absence of bound Ca2+/CaM. Based on these and other biochemical characterizations, we propose a molecular model for the interaction of a bisubstrate autoinhibitory domain with the catalytic domain of CaM-kinase II.

ATP-dependent arsenite transport in everted membrane vesicles of Escherichia coli.

Resistance to toxic oxyanions of arsenic and antimony in Escherichia coli results from active efflux of these anions out of the cell. Extrusion is an active process mediated by an ATP-dependent pump composed of two types of subunits, the integral membrane ArsB protein and the catalytic ArsA subunit. An in vitro assay for transport in everted membrane vesicles of E. coli was developed. Uptake of 73AsO2- by everted vesicles was time- and temperature-dependent and required both pump subunits. Transport required ATP; no other nucleotide, including GTP, CTP, UTP, or the nonhydrolyzable analog adenosine 5'-O-(thiotriphosphate), could substitute for ATP. Protonophores, ionophores, or inhibitors of other types of ion-motive ATPases did not inhibit arsenite uptake. The sulfhydryl reagent N-ethylmaleimide was a potent inhibitor of ATP-dependent arsenite accumulation in vesicles. The apparent Km values for ATP and arsenite were approximately 2 and 0.1 mM, respectively. Antimonite, the most potent activator of the ArsA ATPase, inhibited arsenite uptake with an apparent Ki of 10 microM.

Biochemical properties of Na+/K+-ATPase in axonal growth cone particles isolated from fetal rat brain

Axonal growth cones (AGC) isolated from fetal rat brain have an important specific activity of N+/K+-ATPase. Kinetic assays of the enzyme in AGC showed that Km values for ATP or K+ are similar to those reported for the adult brain enzyme. For Na+ the affinity (Km) was lower. Vmax for the three substrates was several times lower in AGC as compared to the adult value. We also observed two apparent inhibition constants of Na+/K+-ATPase by ouabain, one of low affinity, possibly corresponding to the α1 isoform and another of high affinity which is different to that described for the α2 isoform of the enzyme. These results support an important role for the sodium pump in the mantainance of volume and cationic balance in neuronal differentiating structures. The functional differences observed also suggest that the enzymatic complex of Na+/K+-ATPase in AGC is in a transitional state towards the adult configuration.

Genetic identification of an autoinhibitor in CDPK, a protein kinase with a calmodulin-like domain.

CDPKs are a family of calcium (Ca2+)-dependent protein kinases which are defined by a carboxyl-terminal calmodulin-like domain. Mutational analysis indicates that the junction domain, which joins the kinase and calmodulin-like domains, contains an autoinhibitor. CDPK isoform AK1 from Arabidopsis was expressed in Escherichia coli as a fusion protein sandwiched between glutathione S-transferase and six consecutive histidines at the N- and C-terminal ends, respectively. This fusion, called AK1-6H, was purified and displayed kinase activity which was stimulated up to 127-fold by Ca2+, with a typical specific activity of 2000 nmol min-1 mg-1, using syntide-2 as peptide substrate. A truncation which deletes the calmodulin-like domain, as in mutant delta C-6H, disrupts Ca2+ activation and leaves the enzyme with a basal level of activity. Delta C-6H could be activated 87-fold by preincubation with a purified polyclonal IgG which was raised against a junction domain fusion. A further deletion of the junction domain, as in mutant delta JC, results in a constitutively active enzyme. This indicates that the junction domain in delta C-6H can function as an autoinhibitor. Its function as an autoinhibitor in a full-length enzyme was confirmed by site-specific mutagenesis, as shown by mutant KJM23-6H, which had a six-residue substitution in the junction domain between A422 and A432. Both delta JC and KJM23-6H encoded Ca(2+)-independent enzymes which had specific activities greater than 70% that of a fully active AK1-6H and displayed equivalent Km values for ATP and syntide-2. Inhibition studies on delta JC, using peptides based on the autoinhibitory domains of Ca2+/calmodulin-dependent protein kinases, are consistent with a model where the junction domain contains a similar pseudosubstrate-type autoinhibitor.

Regeneration of catalytic activity of glutamine synthetase mutants by chemical activation: Exploration of the role of arginines 339 and 359 in activity

In order to understand the nature of ATP and L-glutamate binding to glutamine synthetase, and the involvement of Arg 339 and Arg 359 in catalysis, these amino acids were changed to cysteine via site-directed mutagenesis. Individual mutations (Arg-->Cys) at positions 339 and 359 led to a sharp drop in catalytic activity. Additionally, the Km values for the substrates ATP and glutamate were elevated substantially above the values for wild-type (WT) enzyme. Each cysteine was in turn chemically modified to an arginine "analog" to attempt to "rescue" catalytic activity by covalent modification; 2-chloroacetamidine (CA) (producing a thioether) and 2,2'-dithiobis (acetamidine)(DTBA) (producing a disulfide) were the reagents used to effect these chemical transformations. Upon reaction with CA, both R339C and R359C mutants showed a significant regain of catalytic activity (50% and 70% of WT, respectively) and a drop in Km value for ATP close to that for WT enzyme. With DTBA, chemically modified R339C had a greater kcat than WT glutamine synthetase, but chemically modified R359C only regained a small amount of activity. Modification with DTBA was quantitative for each mutant and each modified enzyme had similar Km values for both ATP and glutamate. The high catalytic activity of DTBA-modified R339C could be reversed to that of unmodified R339C by treatment with dithiothreitol, as expected for a modified enzyme containing a disulfide bond. Modification of each cysteine-containing mutant to a lysine "analog" was accomplished using 3-bromopropylamine (BPA).(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical Properties of a Novel 28-kDa Protein Tyrosine Kinase Partially Purified from the Particulate Fraction of Rat Spleen

In this report we present some of the biochemical properties of the enzyme, here called pp28(PTK), isolated from particulate fraction of rat spleen (1). The kinase is very susceptible for polyions as regulators of the enzymatic activity. The polyanions like dextran sulfate or heparin inhibited, and polycations such as spermidin, protamin, poly-L-lysine and some random polypeptides containing tyrosine besides a basic amino acid, stimulated the enzyme markedly. The kinase showed high sensitivity towards class IA salts. In the casein phosphorylation reaction the apparent Km value for ATP was 4 μM. An unusual property is associated with autophosphorylation which leads to a reduced activity towards external substrates. Some kinase inhibitors described in the literature were tested for their potency.

Substrate affinity of the protein tyrosine kinase pp60c-src is increased on thrombin stimulation of human platelets.

Human blood platelets contain high levels of non-receptor protein tyrosine kinases of the Src family, particularly pp60c-src, suggesting an important role for these enzymes in platelet physiology. Indeed, in response to various agonists of platelet function, a number of proteins become phosphorylated at tyrosine residues. However, no enzymic activation of an Src-related tyrosine kinase has yet been shown in platelets. In searching for the kinase(s) responsible, we found that all agonists tested that directly or indirectly activate protein kinase C in platelets (phorbol 12-myristate, 13-acetate, thrombin, vasopressin, collagen, calcium ionophore A23187) increased the overall activity of pp60c-src determined by IgG phosphorylation in an immunocomplex assay in the presence of low ATP concentrations. On the other hand, elevation of cyclic AMP directly by forskolin or indirectly by prostaglandin E1, or elevation of cyclic GMP by sodium nitroprusside did not significantly affect the activity of the enzyme. To substantiate the differences in enzyme activity, we determined Km and Vmax, values of pp60c-src from resting and thrombin-stimulated platelets. Thrombin treatment increased substrate affinity of pp60c-src as indicated by a 2- to 3-fold decrease in the Km values for ATP and the exogenous protein substrate casein. Vmax. values were only slightly altered under the assay conditions used. To further rule out modifications of pp60c-src in phosphorylation as a probable cause of the changed substrate affinity, we analysed tryptic phosphopeptides of immunoprecipitated, 32P-labelled pp60c-src of unstimulated and stimulated platelets. The platelet agonists listed above induced an increase in pp60c-src phosphorylation at Ser-12, which is the amino acid phosphorylated by protein kinase C. Surprisingly, we found that elevation of cyclic AMP did not affect 32P labelling of pp60c-src. On the basis of our data, we suggest that phosphorylation at Ser-12 might be one of the signal-triggering events that cause the increase in substrate affinity of pp60c-src.

Isolation of helicase alpha, a DNA helicase from HeLa cells stimulated by a fork structure and signal-stranded DNA-binding proteins

A DNA helicase, called DNA helicase alpha, was purified from HeLa cells to apparent homogeneity. The helicase and its single-stranded DNA-dependent ATPase activities cosedimented in glycerol gradients with two polypeptides of 110 and 90 kDa with a sedimentation coefficient of 7.4 S. The DNA helicase was markedly stimulated by DNA substrates with a 5'-tailed fork. A DNA substrate with a 3'-tailed fork structure was less stimulatory, although it was more active than substrates without a fork. The directionality of unwinding is 3'-->5' with respect to the single-stranded DNA to which the enzyme was bound. The helicase activity also required a single-stranded DNA-binding protein (SSB) for unwinding activity. The stimulation by SSBs was nonspecific; all SSBs tested, such as human SSB, bacteriophage T4 gene 32, and Escherichia coli SSB, stimulated the DNA helicase activity to a varying extent in the presence of a fork structure. With long duplex substrates (> 500 base pairs), the presence of a fork substantially stimulated the DNA helicase activity in the presence of E. coli SSB. Human SSB stimulated the DNA helicase activity to the greatest extent (> 10-fold) with a substrate containing a fork compared with substrates without a fork. DNA helicase activity required ATP hydrolysis and could be supported by all eight nucleoside triphosphates. The Km values for ATP and dATP in unwinding were 28 and 48 microM, respectively. In general, ribonucleoside triphosphates were better effectors than deoxyribonucleoside triphosphates. The properties of this DNA helicase make it a candidate for a DNA replicative helicase in human cells.

The role of an active-site lysyl residue of spinach phosphoribulokinase as explored by site-directed mutagenesis

Based on selective labeling by ATP analogues, Lys68 of the Calvin Cycle enzyme phosphoribulokinase (PRK) from spinach has been assigned to the active-site region [Miziorko et al. (1990), J. Biol. Chem. 265, 3642-3647]. The equivalent position is occupied by lysyl or arginyl residues in the PRK from both prokaryotic and eukaryotic sources, suggesting a requirement for a basic residue at this location. To examine this possibility, we have replaced Lys68 of the spinach enzyme with arginyl, glutaminyl, alanyl, or glutamyl residues by site-directed mutagenesis. All of the mutant enzymes retain substantial kinase activity; and even in the case of the radical substitution by glutamate, the Km values for ATP and ribulose 5-phosphate are not perturbed significantly. Glutamate at position-68 may destabilize tertiary structure, because the yield of this mutant protein from transformed E. coli is quite low compared to that of the other proteins in this series. Despite the active-site proximity of Lys68, our results show that this residue does not play a key role in catalysis or substrate binding.

Purification and characterization of Saccharomyces cerevisiae DNA damage-responsive protein 48 (DDRP 48).

A yeast protein was purified from wild type Saccharomyces cerevisiae (S. cerevisiae) to near homogeneity using an ethanolamine affinity chromatography procedure. The N-terminal amino acid sequencing and the amino acid composition analyses identified this protein as the product of the second open reading frame of S. cerevisiae DNA Damage-responsive gene 48 (DDR48) (Treger, J.M., and McEntee, K. (1990) Mol. Cell. Biol. 10, 3174-3184) The first methionine residue encoded by the translation starting codon was not present in the mature protein which is designated as DDRP 48. DDRP 48 was found to be a negatively charged and highly hydrophilic glycoprotein. The glycosidase cleavage analyses suggested that DDRP 48 was mainly N-link-glycosylated. The apparent molecular mass of DDRP 48 was estimated to be approximately 65 kilodaltons. DDRP 48 was found able to hydrolyze ATP and GTP yielding PPi. The Km values for ATP and GTP are 0.29 mM and 0.58 mM, respectively. The Western blot analysis demonstrated that DDRP 48 was expressed to various concentrations in different S. cerevisiae strains. Increased DDRP 48 abundance was observed after yeast cells carrying the wild type RAD 52 gene were exposed to either ethylmethane sulfonate or heat shock treatments. After similar DNA-damaging treatments, however, no significant inductions of DDRP 48 were found in a rad 52 mutant strain. These observations are consistent with the predictions resulting from previous studies on transcriptional regulation of the DDR 48 gene (Maga, J.A., McClanahan, T.A., and McEntee, K. (1986) Mol. & Gen. Genet. 205, 276-284; McClanahan, T., and McEntee, K. (1986) Mol. Cell. Biol. 6, 90-96).

Purification and characterization of lysosomal H(+)-ATPase. An anion-sensitive v-type H(+)-ATPase from rat liver lysosomes.

Lysosomal H(+)-ATPase was purified to homogeneity from rat liver lysosomes. It is a bafilomycin A1-sensitive Mg(2+)-ATPase, which reacts with antibodies against the 16- and 70-kDa subunits of vacuolar H(+)-ATPase (Nezu, J., Motojima, K., Tamura, H., and Ohkuma, S. (1992) J. Biochem. (Tokyo) 112, 212-219), and has been separated from both the N-ethylmaleimide (NEM)-sensitive/bafilomycin A1-insensitive Mg(2+)-ATPase (ATPase I) and the NEM-insensitive Mg2+/Ca(2+)-ATPase (ATPase II) (Hayashi, H., Arai, K., Sato, O., Shimaya, A., Sai, Y., and Ohkuma, S. (1992) Chem. Pharm. Bull. 40, 2783-2786). The purified enzyme had the subunit structure of vacuolar H(+)-ATPase, consisting of 110-, 70-, 56-, 42-, 39-, 34-, (32-,) and 16-kDa proteins. It had optimal activity at a pH of 7.0-8.0, with an apparently single Km value for ATP of 95 microM. It hydrolyzed ATP > or = dATP >> GTP, ITP >> UTP, but not CTP, and was inhibited by ADP. It demonstrated divalent cation specificity in the order of Mg2+, Mn2+ > Fe2+, Co2+ > Ca2+. Among various anions, Cl-, Br-, and F- activated ATPase activity, whereas NO3- inhibited activity. It was inhibited not only by bafilomycin A1 but also by NEM, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, quercetin, and dicyclohexylcarbodi-imide. The purified enzyme was incorporated into proteoliposomes where its proton pump activity was reconstituted. This suggested that the isolated enzyme maintains its H+ translocation activity. These findings suggest that the isolated enzyme is an anion-sensitive vacuolar type H(+)-ATPase that functions as a lysosomal proton pump.

Structural and functional studies of insulin receptors in human breast cancer.

We characterized the structure and the function of insulin receptors isolated from 10 human breast cancer specimens. We observed that the insulin receptor content, as determined by a specific radioimmunoassay, was four fold increased in human breast cancer tissue when compared to normal breast tissues. In both cancer and normal breast tissues, insulin receptor mRNA consisted of two major species of approximately 11.0 and 8.5 kilobases. The size of the insulin receptor alpha subunit was determined by 125I-insulin cross-linking followed by immunoprecipitation and polyacrylamide gel electrophoresis; a value of 135kDa was observed for receptors from both breast cancer and normal breast tissues. The functional binding ability of insulin receptors from cancer tissues was slightly lower as compared to normal tissue derived insulin receptor (% B/T = 2.22 +/- 0.50 per ng of insulin receptor as determined by radioimmunoassay vs. 2.96 +/- 0.49, mean +/- S.E.M.). The concentration of insulin that caused half maximal inhibition of 125I-insulin binding was very similar for both cancer and normal breast receptors (80pM). The size of the insulin receptor beta subunit as determined by receptor autophosphorylation was 95kDa. Basal and maximal insulin (100nM) stimulated receptor tyrosine kinase activity, in terms of both receptor autophosphorylation and phosphorylation of an exogenous substrate, was similar in malignant and normal breast tissue derived insulin receptor. Also, a very similar insulin stimulated Km value for ATP was showed by the tyrosine kinase of insulin receptors from breast cancer and normal breast tissue (11.1 and 10.8 microM ATP, respectively). However, in insulin receptors from breast cancer tissue the average tyrosine kinase sensitivity to insulin, as calculated on the exogenous substrate, was higher, although not significantly, with respect to normal breast tissue (ED50 at 0.28 +/- 0.09 and 1.08 +/- 0.33 nM insulin, respectively). A similarly different sensitivity to insulin was observed also for receptor autophosphorylation. In conclusion, this study demonstrates that breast cancer tissues have an increased number of structurally and functionally normal insulin receptors. In some breast cancer tissues, however, the sensitivity of the receptor tyrosine kinase activity to insulin is greatly increased. These data suggest that, in vivo, the mitogenic effect of insulin may play a role in the biology of certain breast cancers.

Three new DNA helicases fromSaccharomyces cerevisiae

At least six DNA helicases have been identified during fractionation of extracts from the yeast Saccharomyces cerevisiae. Three of those, DNA helicases B, C, and D, have been further purified and characterized. DNA helicases B and C co-purified with DNA polymerase delta through several chromatographic steps, but were separated from the polymerase by hydrophobic chromatography. DNA helicase D co-purified with Replication Factor C over seven chromatographic steps, and was only separated from it by glycerol gradient centrifugation in the presence of 0.2 M NaCl. All three helicases are DNA dependent ATPases with Km values for ATP of 190 microM, 325 microM, and 60 microM for DNA helicases B, C, and D, respectively. Their DNA helicase activities are comparable. They are 5'-3' helicases and have pH optima of 6.5-7 and Mg2+ optima of 1-2 mM. However, they differ in the nucleotide requirement for helicase action. Whereas all three helicases preferred ATP, dATP, UTP, CTP, and dCTP as cofactors, DNA helicase C also used GTP, but not dTTP. On the other hand, DNA helicase D used dTTP, but not GTP, and DNA helicase B used neither nucleotide as cofactor. These studies allowed us to conclude that DNA helicases B, C, and D are not only distinct enzymes, but also different from two previously identified yeast DNA helicases, the RAD3 protein and ATPase III.

Purification and properties of a from Potamon potamios skeletal muscle sarcoplasmic reticulum

1. 1. A 120-kDa (Ca2+ + Mg2+)-dependent ATPase was purified from the freshwater/land crab Potamon potamios muscle sarcoplasmic reticulum. 2. 2. The enzyme showed two Km values for ATP of 40 and 330 mM at 10–75 and > 75 μM ATP concentrations, respectively. Km values for calcium and magnesium were 2 and 294 μM, respectively. 3. 3. Optimal enzyme activity was observed at pH 7.5 and the Arrhenius plot showed a break at 25°C. 4. 4. An alternative method for the simultaneous purification of P. potamios(Ca2+ + Mg2+)- and (Na+ + K+)-dependent ATPase enzymes is also described.

Characterization of the phosphotransferase domain of casein kinase II by site-directed mutagenesis and expression in Escherichia coli.

The catalytic alpha subunit of casein kinase II contains the 11 conserved domains characteristic of all protein kinases. Domain II and VII are involved in nucleotide binding and phosphotransfer. Two residues of the alpha subunit, Val-66 (in domain II) and Trp-176 (in domain VII), were changed to Ala-66 and Phe-176, the residues present in more than 95% of the identified protein kinase sequences. These changes altered the selectivity of the alpha subunit for ATP and GTP. The Ala-66 mutant showed an increase in the Km value for GTP from 45 to 71 microM, while the Km value for ATP decreased from 13 to 9 microM. The Km value for ATP with the Phe-176 mutant showed a decrease from 13 to 7 microM. A double mutant of Ala-66/Phe-176 showed the combined effects, with a Km of 6 microM for ATP and 70 microM for GTP. Alteration of Trp-176 to Lys-176, an amino acid which is not present in the corresponding position of any known protein kinase, resulted in a lack of phosphotransferase activity. The mutations, Val-66 to Ala-66 and Trp-176 to Phe-176, also altered the interaction of the alpha subunit with the regulatory beta subunit. In contrast to the wild-type alpha subunit, which was stimulated 4-fold by addition of the beta subunit, the Ala-66 and Ala-66/Phe-176 mutants were not stimulated by the beta subunit, while the Phe-176 mutant was stimulated only 2.5-fold. All of the reconstituted holoenzymes were similar in molecular weight to the native holoenzyme. The stimulation of the phosphotransferase activity toward beta-casein B by spermine and polylysine, which is mediated by the beta subunit, was similar for holoenzymes reconstituted with either wild-type or mutant alpha subunits. Therefore, binding of the beta subunit appears to alter the active site of the alpha subunit directly or indirectly by inducing a conformational change. Ala-66 and Phe-176 mutations appear to change the structure of the alpha subunit sufficiently so that interaction of the subunits is altered and the stimulatory effect of the beta subunit is reduced or eliminated.

Translocation and rotation of microtubules caused by multiple species of Chlamydomonas inner-arm dynein

ABSTRACT Dynein was extracted from outer arm-less axonemes of the mutant odal and fractionated by high-pressure liquid chromatography on a MonoQ column into seven distinct subspecies (named a–g). Each subspecies contained one or two heavy chains and several mediumsized and light chains; by vanadate/UV-induced photocleavage and SDS-polyacrylamide gel electrophoresis, eight distinct heavy chains were identified. Analysis of the mutant axonemes indicated that the subspecies f (containing two heavy chains) is missing in the inner-arm mutant idal and the subspecies a, c and d are missing in the mutant ida4. Six subspecies (all but f supported microtubule translocation with the maximal rate ranging from 2 to 12 μm s−1 and the apparent Km for ATP ranging from about 10 to 100 μM. All the subspecies translocated microtubules with the plus end leading, indicating that all the inner-arm dyneins are minus end-directed motors. Five subspecies (all but b and f) displayed microtubule rotation during translocation at rates of up to about 10 Hz. Unexpectedly, the Km values for ATP for translocation and rotation did not always agree; because of this, the pitch of the movement was variable with some subspecies. These observations indicate that axonemes are equipped with several inner-arm subspecies and that torque generation is a feature common to many of them.

Induction and characterization of two types of ATPase on rat liver peroxisomes.

Clofibrate increased oligomycin-resistant ATPase activity in peroxisomes more than 17-fold (5.15 +/- 0.71 milliunits/mg protein) in rat liver. The activity was dependent on divalent cations (Mg2+ > Ca2+) with an optimum pH of 7.5. This activity was partially inhibited by N-ethylmaleimide (NEM), 4,4'-dithiocyanatostilbene-2,2'-disulfonic acid (DIDS), silicotungstic acid (STA), and high concentrations of N,N'-dicyclohexylcarbodiimide (DCCD). Proteinase K digestion of intact peroxisomes severely reduced the NEM-sensitive activity, but little affected the NEM-resistant activity. NEM-sensitive and -resistant ATPases showed Km values for ATP of 780 and 73 microM, respectively. The NEM-sensitive activity was inhibited completely by DIDS, 7-chloro-4- nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), tributyltin chloride (TBT), and quercetin, and partially by DCCD and STA, whereas the NEM-resistant activity was totally insensitive to these chemicals except for STA. These activities had unique requirements for divalent cations, anions, and substrates, respectively. They were partially separated by gel filtration chromatography and had molecular masses of 520 kDa (NEM-sensitive enzyme) and 450 kDa (NEM-resistant enzyme), respectively.

Ca(2+)-calmodulin-dependent protein kinases Ia and Ib from rat brain. II. Enzymatic characteristics and regulation of activities by phosphorylation and dephosphorylation.

In addition to physical properties (DeRemer, M. F., Saeli, R. J., and Edelman, A. M. (1992) J. Biol. Chem. 267, 13460-13465), enzymatic and regulatory characteristics indicate that calmodulin (CaM) kinase Ia and CaM kinase Ib are distinct entities. The Km values for ATP and site 1 peptide were similar between the two kinases, however, CaM kinase Ib is approximately 20-fold more sensitive to CaM than is CaM kinase Ia. The kinases also displayed differential sensitivities to divalent metal ions. For both kinases, site 1 peptide, synapsin I, and syntide-2 were highly preferred substrates relative to others tested. A 72-kDa protein from a heat-treated extract of rat pancreas was phosphorylated by CaM kinase Ib but not by CaM kinase Ia. CaM kinase Ia activity displayed a pronounced lag in its time course suggesting enzyme activation over time. Preincubation of CaM kinase Ia in the combined presence of Ca(2+)-CaM and MgATP led to a time-dependent increase in its site 1 peptide kinase activity of up to 15-fold. The extent of activation of CaM kinase Ia correlated with the extent of autophosphorylation. The enzyme retained full Ca(2+)-CaM dependence in the activated state which was rapidly reversible by treatment with protein phosphatase 2A catalytic subunit. Thus, the activation of CaM kinase Ia is a result of its Ca(2+)-CaM-dependent autophosphorylation. CaM kinase Ib was not activated by preincubation under autophosphorylating conditions yet lost approximately 90% of its activity toward either an exogenous substrate (site 1 peptide) or itself (autophosphorylation) after incubation with protein phosphatase 2A catalytic subunit. The deactivated state was not reversed by subsequent incubations under autophosphorylating conditions. Thus, CaM kinase Ib activity is dependent upon phosphorylation by a regulating kinase(s) which is resolved from CaM kinase Ib during purification of the latter.

Purification and characterization of (Ca2+-Mg2+)-ATPase in rat liver lysosomal membranes.

A (Ca(2+)-Mg2+)-ATPase associated with rat liver lysosomal membranes was purified about 300-fold over the lysosomal membranes with a 7% recovery as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included: preparation of lysosomal membranes, solubilization of the membrane with Triton X-100, WGA-Sepharose 6B, Con A-Sepharose, hydroxylapatite chromatography, and preparative polyacrylamide gel electrophoresis. The molecular mass, estimated by gel filtration with Sephacryl S-300 HR, was approximately 340 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular mass of 85 kDa. The isoelectric point of the purified enzyme was 3.6. The enzyme had a pH optimum of 4.5, a Km value for ATP of 0.17 mM and a Vmax of 71.4 mumol/min/mg protein at 37 degrees C. This enzyme hydrolyzed nucleotide triphosphates and ADP but did not act on p-nitrophenyl phosphate and AMP. The effects of Ca2+ and Mg2+ on the ATPase were not additive, thereby indicating that both Ca2+ and Mg(2+)-ATPase activities are manifested by the same enzyme. The (Ca(2+)-Mg2+)-ATPase differed from H(+)-ATPase in lysosomal membranes, since the enzyme was not inhibited by N-ethylmaleimide but was inhibited by vanadate. The effects of some other metal ions and compounds on this enzyme were also investigated. The N-terminal 18 residues of (Ca(2+)-Mg2+)-ATPase were determined.