Radiation inactivation of multispecific transport systems for bile acids and xenobiotics in basolateral rat liver plasma membrane vesicles.

The functional molecular mass of the cholate, phallotoxin, iodipamide, and ouabain transport proteins in isolated basolateral plasma membrane vesicles was determined by radiation inactivation. Purified basolateral plasma membrane vesicles were irradiated (-90 to -120 degrees C) with high energy electrons from a 10-MeV linear accelerator at doses from 0 to 30 megarads. After each dose, the initial uptake, the equilibrium binding, and the binding of the substrates at 4 degrees C were checked. The size of the transporting function was, for cholate, 107 +/- 8.9 kDa; for phallotoxin, 104 +/- 7 kDa; and for ouabain, 120 +/- 4.7 kDa. The target size for the binding proteins was 56 +/- 4.2, 57 +/- 5, and 47.2 +/- 1.95 kDa for cholate, phallotoxin, and taurocholate, respectively. In the case of iodipamide, the functional molecular mass for both the transport and binding proteins was 54 +/- 4.8 kDa.     

Disappearance of labelled IGF-2 from plasma of the ovine fetus in late gestation.

The role of IGF-2 in the fetus and its possible influence on fetal growth remains speculative. We investigated the size distribution of unsaturated binding sites for labelled oIGF-2 in ovine fetal plasma. In addition, the disappearance of each form of protein bound IGF-2 in the late gestation ovine fetus (125-135 days, n = 5) was estimated. One minute after injection into the fetal femoral vein, 125IoIGF- circulated in the fetal femoral artery bound primarily to a 50 kDa binding protein. Only a small amount of binding to a 150 kDa binding protein was seen with little to no free IGF-2 present. IGF-2 also circulated in association with a large molecular weight complex (ca. 250 kDa) presumed to be circulating receptor bound IGF-2. The half life of the 250 kDa form of IGF-2 was 385.9 +/- 65.4 min, for the 150 kDa form 308.0 +/- 65.0 min, for the 50 kDa form was 35.5 + 2.6 min and for the free form of IGF-2 was 1.6 +/- 0.6 min. There was no apparent movement of intact IGF-2 out of the fetal circulation into any of the fetal fluids or into the maternal circulation. Similarly there was no consistent placental uptake of IGF-2 from the fetal circulation.     

Glutamate dehydrogenase from liver of euthermic and hibernating Richardson's ground squirrels: evidence for two distinct enzyme forms.

Glutamate dehydrogenase (GDH) was purified to homogeneity from the liver of euthermic (37 degrees C body temperature) and hibernating (torpid, 5 degrees C body temperature) Richardson's ground squirrels (Spermophilus richardsonii). SDS-PAGE yielded a subunit molecular weight of 59.5+/-2 kDa for both enzymes, but reverse phase and size exclusion HPLC showed native molecular weights of 335+/-5 kDa for euthermic and 320+/-5 kDa for hibernator GDH. Euthermic and hibernator GDH differed substantially in apparent Km values for glutamate, NH4+, and alpha-ketoglutarate, as well as in Ka and IC50 values for nucleotide and ion activators and inhibitors. Kinetic properties of each enzyme were differentially affected by assay temperature (37 versus 5 degrees C). For example, the Km for alpha-ketoglutarate of euthermic GDH was higher at 5 degrees C (3.66+/-0.34 mM) than at 37 degrees C (0.10+/-0.01 mM), whereas hibernator GDH had a higher affinity for alpha-ketoglutarate at 5 degrees C (Km was 0.98+/-0.08 mM at 37 degrees C and 0.43+/-0.02 mM at 5 degrees C). Temperature effects on Ka ADP values of the enzymes followed a similar pattern; GTP inhibition was strongest with the euthermic enzyme at 37 degrees C and weakest with hibernator GDH at 5 degrees C. Entry into hibernation leads to stable changes in the properties of ground squirrel liver GDH that allow the enzyme to function optimally at the prevailing body temperature.     

Characterization of over-expressed alkaline phosphodiesterase I in tumour-derived fibroblasts from patients with neurofibromatosis

Alkaline phosphodiesterase I from cultured fibroblasts from patients with neurofibromatosis was partially purified and characterized following extraction with Triton X-100, and fractionation with high-performance liquid chromatography. Some properties were compared with the enzyme extracted from normal-appearing fibroblasts. The isoelectric points of both the tumour and normal-appearing cell enzymes were 6·0. The enzyme required Zn²⁺ for its activity, was heat labile, and nicked superhelical covalently closed circular ?X174 DNA. The activity was inhibited by GTP, DTT and EDTA. The native molecular weight of alkaline phosphodiesterase I was determined to be 430 000. No differences were found in properties of the tumour-derived and normal cell enzymes. On purification it was observed that the peak pattern of enzyme activity corresponded to that of 125 kDa protein, which was more abundant upon SDS-PAGE analysis in tumour cells than in normal cells. The most active fraction of isoelectric focusing, which was performed using disulfide cross-linked polyacrylamide gel, was used to produce an antibody. The bands of 125, 60 and 40 kDa were immuno-stained in tumour cell preparation. These results indicate that alkaline phosphodiesterase I, of which the molecular weight is probably 125 kDa, is over-expressed in tumour-derived fibroblasts from neurofibromatosis patients.     

Determination of the functional molecular size of vasopressin isoreceptors

The molecular size of vasopressin receptors in the intact membrane-bound state was determined by radiation inactivation (target size analysis). For the V1 receptor in rat liver a molecular size of (76 +/- 8) kDa was determined. For the V2 receptor in rat kidney and bovine kidney molecular sizes of (95 +/- 4) and (108 +/- 11) kDa were found. Statistical analysis gave evidence for size differences between rat liver and rat kidney receptors or differences between rat liver and bovine kidney receptors, but not between kidney receptors from different species. The results suggest that V1 and V2 receptors can be distinguished by functional properties as well as by their size.     

Human placental deoxyadenosine and deoxyguanosine phosphorylating activity

We studied deoxyadenosine and deoxyguanosine phosphorylating activities in human placental cytosol. The specific activities of nucleoside kinase enzymes in nanomoles per h per mg +/- SD were as follows: adenosine kinase, 30 +/- 14; deoxyadenosine kinase, 12 +/- 2; deoxycytidine kinase, 0.30 +/- 0.04; and deoxyguanosine kinase, 27 +/- 16. Three major activities were resolved by ion exchange and affinity chromatography: deoxyguanosine-deoxycytidine kinase, deoxycytidine-deoxyadenosine kinase, and adenosine-deoxyadenosine kinase. Two other activities contained significant quantities of deoxyadenosine kinase. Deoxyguanosine-phosphorylating activity eluted as a single peak in association with deoxycytidine kinase. This deoxyguanosine-deoxycytidine kinase had an apparent molecular weight of 54,000, a Stokes radius of 31 A, and apparent Km values of 10, 130, and 14 microM for deoxyguanosine, deoxycytidine, and ATP, respectively. Four peaks of deoxyadenosine phosphorylating activity were resolved by affinity chromatography with AMP-Sepharose 4B. Adenosine-deoxyadenosine kinase had an apparent molecular weight of 38,000, a Stokes radius of 27.4 A, and apparent Km values of 0.4, 510, and 75 microM for adenosine, deoxyadenosine, and ATP, respectively. Attempts to distinguish whether adenosine-deoxyadenosine kinase was one enzyme with these two activities or two separate enzymes suggested that the former was the case. Deoxycytidine-deoxyadenosine kinase had apparent Km values of 0.7, 670, and 12 microM for deoxycytidine, deoxyadenosine, and ATP, respectively. Its apparent molecular weight was estimated to be 49,000 and its Stokes radius 30 A. Two other minor peaks of deoxyadenosine-phosphorylating activity had characteristics different from either deoxycytidine kinase or adenosine kinase-associated deoxyadenosine kinase. Our studies indicate that human placental cytosol contains a complex mixture of nucleoside kinase enzymes.     

Functional size analysis of pyrophosphatase from Rhodospirillum rubrum determined by radiation inactivation.

A radiation inactivation technique was employed to determine the functional size of pyrophosphatase (PPase) from the chromatophores of Rhodospirillum rubrum. The activities of hydrolysis and synthesis reactions of pyrophosphatase and its coupled proton translocation decayed in a simple exponential function with the increase of radiation dosages. D37 values of 5.2 +/- 0.7 and 5.8 +/- 0.8 Mrads were obtained for pyrophosphate hydrolysis and its associated proton translocation yielding molecular masses of 167.7 +/- 30.7 and 156.3 +/- 26.6 kDa, respectively. Similarly, a D37 value of 4.4 +/- 0.6 Mrads was measured for the acid-base induced pyrophosphate synthesis resulting in a radiation sensitive size of 196.3 +/- 31.9 kDa.     

Isolation and characterization of a calcium-binding protein derived from mRNA termed p9Ka, pEL-98, 18A2, or 42A by the newly synthesized vasorelaxant W-66 affinity chromatography.

W-66 (N-(2-aminoethyl)-N-[2-(4-chlorocinnamylamino) ethyl]-5-isoquinolinesulfonamide), a newly synthesized isoquinolinesulfonamide, was shown to have a potent vasodilatory action and calmodulin (CaM)-antagonizing action. Using the W-66 affinity chromatographic technique, we purified two Ca(2+)-binding proteins from the EGTA-soluble fraction of bovine aorta. One was CaM and the other was an acidic protein with a molecular mass of 11 kDa. It was tentatively named "calvasculin." Calvasculin was a dimeric protein. Equilibrium dialysis showed that 1 mol of calvasculin (dimer) bound to 1.98 +/- 0.30 mol of Ca2+ in the presence of 10(-3) M Ca2+. Calvasculin failed to activate Ca2+/CaM-dependent enzymes such as myosin light chain kinase, Ca2+/CaM-dependent phosphodiesterase, or Ca2+/CaM-dependent protein kinase II and to inhibit the CaM stimulation of these enzymes. The partial amino acid sequence of calvasculin revealed a high homology to the predicted protein derived from mRNA, named pEL-98, 18A2, 42A, or p9Ka. We also examined the physicochemical and biochemical properties of calvasculin. Using the antibody specific for calvasculin, we obtained evidence that calvasculin is present in abundance in bovine aorta but not in brain, lung, heart, or testis.     

Comparison of properties of the cellular and extracellular phosphodiesterases induced by cyclic adenosine 3',5'-monophosphate in Dictyostelium discoideum

The kinetic properties and susceptibilities to various agents of intracellular (particulate and soluble) and extracellular phosphodiesterases [EC 3.1.4.17] of Dictyostelium discoideum induced by cyclic adenosine 3',5'-monophosphate (cyclic AMP) were studied and compared. Intracellular particulate phosphodiesterase was obtained by solubilization of the light mitochondrial fraction with Emulgen. The Michaelis constants of this enzyme were 4.5 +/- 0.7 and 10 +/- 0.7 microM, while those of the intracellular soluble phosphodiesterase were 4.6 +/- 0.3 and 13 +/- 2.8 microM. However, the Michaelis constant of the extracellular phosphodiesterase was 6.8 +/- 0.9 microM, differing from the values of the two intracellular enzymes. Susceptibilities of the enzyme activity to various agents (theophylline, caffeine, dithiothreitol, glutathione, etc.) were essentially the same among these three phosphodiesterases. In the presence of 10 mM ethylenediaminetetraacetate, the activities of the particulate and the soluble enzymes were both decreased to about 60%, while that of the extracellular enzyme remained at 90%. The inhibition constants of cyclic inosine monophosphate for the cellular enzymes (35 and 100 microM for the particulate enzyme, and 37 and 90 microM for the soluble one) were considerably different from the value for the extracellular enzyme (48 microM). These results suggest that the characteristics of these three phosphodiesterases are substantially similar, but that the affinity of the intracellular (particulate and soluble) enzymes for the substrate is somewhat different from that of the extracellular enzyme.     

Functional molecular mass of the 14C-azidobenzamidotaurocholic acid binding proteins in hepatocellular bile acid transport systems.

The apparent target size of 14C-azidobenzamidotaurocholate binding proteins in basolateral rat liver plasma membranes (blPm) was determined by analysis of the radiation induced decrease of the binding of this photoreactive taurocholate analog to blPm. Radiation causes a dose-dependent mono-exponential reduction of binding of ABATC to the protein subunits with molecular masses of 48-50 and 52-54 kDa in SDS-PAGE. The minimal functional molecular mass of the 48-50 and 52-54 kDa ABATC binding proteins was determined to be 99 +/- 8.2 and 93.2 +/- 7 kDa, respectively.     

Membrane-bound Na,K-ATPase: target size and radiation inactivation size of some of its enzymatic reactions

Frozen samples of membrane-bound pig kidney Na,K-ATPase were subjected to target size analysis by radiation inactivation with 10-MeV electrons at -15 degrees C. The various properties investigated decreased monoexponentially with radiation dose, and the decay constants, gamma, were independent of the presence of other proteins and of sucrose concentrations above 0.25 M. The temperature factor was the same as described by others. Irradiation of four proteins of known molecular mass, m, showed that gamma for protein integrity was proportional to m with a proportionality factor about 20% higher than that conventionally used. By this standard curve, glucose-6-phosphate dehydrogenase activity used as internal standard gave a radiation inactivation size of 110 +/- 5 kDa, very close to m = 104-108 kDa for the dimer, as expected. For Na+/K+-transporting ATPase the following target sizes and radiation inactivation size values were very close to m = 112 kDa for the alpha-peptide: peptide integrity of alpha, 115 kDa; unmodified binding sites for ATP and vanadate, 108 kDa; K+-activated p-nitrophenylphosphatase activity, 106 kDa. There was thus no sign of dimerization of the alpha-peptide or involvement of the beta-peptide. In contrast, optimal Na+/K+-transporting ATPase activity had a radiation inactivation size = 189 +/- 7 kDa, and total nucleotide binding capacity corresponded to 72 +/- 3 kDa. These latter results will be extended and discussed in a forthcoming paper.     

Soluble NTPDase: An additional system of nucleotide hydrolysis in rat blood serum

The participation of a nucleoside triphosphate diphosphohydrolase in the nucleotide hydrolysis by rat blood serum was evaluated. Nucleoside triphosphate diphosphohydrolase and phosphodiesterase are enzymes possibly involved in ATP and ADP hydrolysis. The specific activity of the phosphodiesterase activity (using thymidine 5'-monophosphate p-nitrophenyl ester as substrate) was 4.92 +/- 0.73 (mean +/- SD, n = 10) nmol p-nitrophenol.min(-1).mg(-1) protein and the specific activities for ATP and ADP were 1.31 +/- 0.37 (mean +/- SD, n = 7) and 1.36 +/- 0.25 (mean +/- SD, n = 5) nmol Pi.min(-1).mg(-1) protein, respectively. A competition plot demonstrated that ATP and ADP hydrolysis occurs at the same active site. The effect of suramin and phenylalanine on ATP, ADP and thymidine 5'-monophosphate p-nitrophenyl ester hydrolysis was investigated. The results were opposite considering the hydrolysis of ATP and ADP and that of the substrate marker for the enzyme phosphodiesterase. These results are indicative of the presence of, at least, two enzymes participating in the serum nucleotide hydrolysis. The presence of cAMP did not affect the hydrolysis velocity of ATP and ADP, while thymidine 5'-monophosphate p-nitrophenyl ester hydrolysis was inhibited by cAMP by approximately 47%, suggesting that the hydrolysis of the ATP and ADP, under our assay conditions, occurs at a different site from the phosphodiesterase site. Both enzyme activities, in the rat blood serum, may be involved in the modulation of the nucleotide/nucleoside ratio in the circulation, serving an in vivo homeostatic and antithrombotic function. In addition, the phosphodiesterase may act on DNA or RNA liberated upon tissue injury and/or cell death.     

Radiation target sizes of the Na,K-ATPase and p-aminohippurate transport system in the basolateral membrane of renal proximal tubule

Basolateral membrane vesicles made from rabbit kidney proximal tubules were frozen and irradiated with a high energy electron beam and the effects of irradiation on Na,K-ATPase activity, p-aminohippurate (PAH) transport, the membrane diffusion barrier and vesicle volume were measured. The vesicle volume and diffusion barrier were not significantly changed by radiation exposure. Na,K-ATPase activity was inactivated as a simple exponential function of radiation dose. Target size analysis of the data yielded a molecular size of 267 +/- 17 kDa, consistent with its existence as a (alpha beta)2 dimer. The carrier-mediated PAH uptake by basolateral membrane vesicles was also inactivated as a function of radiation dose. A target molecular size of 74 +/- 16 kDa was calculated for the PAH transport system. This study is the first measurement of the functional size of the organic acid transport system based directly on flux measurements.     

Two W-containing formate dehydrogenases (CO2-reductases) involved in syntrophic propionate oxidation by Syntrophobacter fumaroxidans.

Two formate dehydrogenases (CO2-reductases) (FDH-1 and FDH-2) were isolated from the syntrophic propionate-oxidizing bacterium Syntrophobacter fumaroxidans. Both enzymes were produced in axenic fumarate-grown cells as well as in cells which were grown syntrophically on propionate with Methanospirillum hungatei as the H2 and formate scavenger. The purified enzymes exhibited extremely high formate-oxidation and CO2-reduction rates, and low Km values for formate. For the enzyme designated FDH-1, a specific formate oxidation rate of 700 U.mg-1 and a Km for formate of 0.04 mm were measured when benzyl viologen was used as an artificial electron acceptor. The enzyme designated FDH-2 oxidized formate with a specific activity of 2700 U.mg-1 and a Km of 0.01 mm for formate with benzyl viologen as electron acceptor. The specific CO2-reduction (to formate) rates measured for FDH-1 and FDH-2, using dithionite-reduced methyl viologen as the electron donor, were 900 U.mg-1 and 89 U.mg-1, respectively. From gel filtration and polyacrylamide gel electrophoresis it was concluded that FDH-1 is composed of three subunits (89 +/- 3, 56 +/- 2 and 19 +/- 1 kDa) and has a native molecular mass of approximately 350 kDa. FDH-2 appeared to be a heterodimer composed of a 92 +/- 3 kDa and a 33 +/- 2 kDa subunit. Both enzymes contained tungsten and selenium, while molybdenum was not detected. EPR spectroscopy suggested that FDH-1 contains at least four [2Fe-2S] clusters per molecule and additionally paramagnetically coupled [4Fe-4S] clusters. FDH-2 contains at least two [4Fe-4S] clusters per molecule. As both enzymes are produced under all growth conditions tested, but with differences in levels, expression may depend on unknown parameters.     

On the presence of two non-specific nucleotide-sugar-hydrolysing enzymes in rat liver.

Evidence is presented for the occurrence of two different non-specific nucleotide-sugar hydrolases in rat liver and other rat tissues. These two enzymes (I and II) were separated by chromatography on a 5'-AMP-aminohexyl-Sepharose column. Enzyme I is most probably identical with phosphodiesterase I (EC 3.1.4.1). Enzyme II appeared to be identical with an enzyme described in literature as 'CMP-sialic acid hydrolase' [Kean & Bighouse (1974) J. Biol. Chem. 249, 7813-7823], since almost all activity with CMP-N-acetylneuraminate as substrate was recovered in this enzyme fraction. CMP-N-acetylneuraminate was a poor substrate for Enzyme I, whereas deoxythymidine-5'-p-nitrophenyl phosphate and all nucleoside-diphosphosugars tested were good substrates for both Enzyme I and II. Therefore it is suggested that CMP-N-acetylneuraminate is used as an additional substrate to discriminate between the activities of Enzyme I and II in homogenates or membrane preparations. The various substrates appeared to be competitive inhibitors of each other, suggesting that, in each enzyme preparation, only one enzyme is responsible for the hydrolysis of the various substrates. The dissimilar properties of the two enzymes are substantiated by studying the subunit molecular masses (Enzyme I, 125 kDa; Enzyme II, 50-55 kDa), the sensitivity towards Triton X-100, Sarkosyl and sodium dodecyl sulphate and towards trypsin treatment. It is discussed whether the alpha-N-acetylglucosamine phosphodiesterase described by Varki & Kornfeld [(1981) J. Biol. Chem. 256, 9937-9943] is identical with one of the nucleotide-sugar hydrolases described here.     

Characterization of soluble cyclic AMP phosphodiesterases and partial purification of a major form in human leiomyoma of the uterus

Human leiomyoma of the uterus contained seven forms of cyclic AMP phosphodiesterase in the crude cytosol as revealed by a specific activity stain on non-denaturing polyacrylamide gel electrophoresis. The enzymes from human myometrium and normal uterus showed an identical activity pattern. Ferguson plot analysis showed four different molecular weight species of Mr 229,000 +/- 4,000, 186,000 +/- 4,000, 174,000 +/- 4,000 and 162,000 +/- 4,000. The Mr 174,000 species comprised four differently charged forms. Sucrose density gradient centrifugation of the crude cytosol revealed the presence of three molecular weight species sedimenting at 11.8S, 8.1S and 3.6S. The Michaelis constant (Km) for the band 1 form which displayed linear kinetics was 5 microM and the band 2 form which produced non-linear kinetics had Km values of 5.8 and 37 microM.     

Glycolytic enzymes of Trypanosoma brucei. Simultaneous purification, intraglycosomal concentrations and physical properties

We have developed a method for the simultaneous purification of hexokinase, glucosephosphate isomerase, phosphofructokinase, fructose-1,6-bisphosphate aldolase, triosephosphate isomerase, D-glyceraldehyde-phosphate dehydrogenase, phosphoglycerate kinase, glycerol-3-phosphate dehydrogenase and glycerol kinase from Trypanosoma brucei in yields varying over 8-55%. Crude glycosomes were prepared by differential centrifugation of cell homogenates. Subsequent hydrophobic interaction chromatography on phenyl-Sepharose resulted in six pools containing various mixtures of enzymes. These pools were processed via affinity chromatography (immobilized ATP), hydrophobic interaction chromatography (octyl-Sepharose) and ion-exchange chromatography (CM- and DEAE-cellulose) which resulted in the purification of all nine enzymes. The native enzyme and subunit molecular masses, as determined by gel filtration and gel electrophoresis under denaturing conditions, were compared with those of their homologous counterparts from other organisms. Trypanosomal hexokinase is a hexamer and differs in subunit composition from the mammalian enzymes (monomers) as well as in subunit size (51 kDa versus 96-100 kDa, respectively). Phosphofructokinase only differs in subunit size (51 kDa for T. brucei versus 80-90 kDa for mammals) but had identical subunit composition (tetrameric). The others all have the same subunit composition as their mammalian counterparts. Except for triosephosphate isomerase, all Trypanosoma enzymes have subunits which are 1-5 kDa larger in size. Together these nine enzymes contribute 3.3 +/- 1.6% to the total cellular protein of T. brucei and at least 90% to the total glycosomal protein. A comparison of calculated intraglycosomal concentrations of the enzymes with the glycosomal metabolite concentrations shows that in the case of aldolase, glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase, the concentration of active sites is of the same order of magnitude as that of their reactants. A common feature of the glycosomal glycolytic enzymes (with the exception of glucosephosphate isomerase) is that they are highly basic proteins with pI values between 8.8 and 10.2, values which are 1-4 higher than in the case of their mammalian cytosolic counterparts and 3-6 higher than in the case of the various unicellular organisms. It is suggested that both the larger subunit size and the basic character of the T. brucei glycolytic proteins are involved in the routing of the enzymes from their site of biogenesis (the cytosol) towards their site of action (the glycosome).     

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is a reliable internal standard for radiation-inactivation studies of membranes in the frozen state

The target size of four soluble enzymes (beta-galactosidase, pyruvate kinase, alcohol dehydrogenase, and glucose-6-phosphate dehydrogenase) in the presence or absence of subcellular membrane fractions has been determined by the radiation-inactivation method using samples in the frozen state. For each of the four enzymes, full activity was recovered after freezing and thawing in the absence of radiation. We found minimal (less than 20%) binding of the enzymes to either submitochondrial vesicles or sarcoplasmic reticulum vesicles. Under the conditions tested, beta-galactosidase, pyruvate kinase, and alcohol dehydrogenase exhibited target sizes which varied according to the experimental conditions, i.e., the buffer selected and also the presence or absence of membrane preparations. For these tetrameric enzymes, the target sizes were generally comparable to either a monomer or a dimer. By contrast, the target size of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was found to be essentially invariant when frozen in a variety of buffers and in the presence or absence of either cryoprotectant (sucrose or glycerol) or different membrane preparations. The target size from 19 separate determinations gave an average value of 104 +/- 16 kDa, which is comparable to the molecular weight of the enzyme (104 kDa). We conclude that glucose-6-phosphate dehydrogenase from L. mesenteroides is a reliable internal standard for radiation-inactivation studies of membrane preparations in the frozen state.     

Express method of isolation of mammalian phenylalanine-tRNA-synthetase and preparation of monoclonal antibodies against this enzyme

A rapid and efficient procedure for isolating homogeneous beef liver phenylalanyl-tRNA synthetase (EC.6.1.1) was developed that enables to purify the enzyme 5000 fold and to achieve the activity of 8 e.a.u. per mg of protein. The molecular mass of the native enzyme was estimated to be 260 kDa, for alpha subunit - 59 kDa, and for beta - 72 kDa. Two cellular clones were derived by means of hybridization of immunised splenocytes with myeloma cells. They secrete monoclonal antibodies, designated P6 and P1 2, that bind to human placental and bovine liver phenylalanyl-tRNA synthetases but not to the same enzymes from E. coli and T. thermophilus. P6 and P1 2 antibodies do not affect the aminoacylation capacity of human or bovine phenylalanyl-tRNA synthetases. By immunoblotting, it was shown that P6 antibodies recognize the alpha subunit of the enzyme.     

A calcium-binding protein from rabbit lung cytosol identified as the product of growth-regulated gene (2A9) and its binding proteins

Using Ca(2+)-dependent affinity chromatography on a synthetic compound (W-77)-coupled Sepharose 4B column, we purified two different Ca(2+)-binding proteins from rabbit lung extracts. The molecular weights of these proteins were estimated to be 17 kDa (calmodulin) and 10 kDa, respectively. The partial amino acid sequence of the 10-kDa protein revealed that it has two EF-hand structures. In addition, the 10-kDa protein was highly homologous (91%) to the product of growth-regulated gene, 2A9 (calcyclin). The Ca(2+)-binding property of the 10-kDa protein was observed by a change in the uv difference spectrum. Equilibrium dialysis showed that 1 mol of the 10-kDa protein bound to 2.04 +/- 0.05 mol of Ca2+ in the presence of 10(-4) M Ca2+. However, the protein failed to activate calmodulin-dependent enzymes such as Ca2+/CaM kinase II, myosin light chain kinase, and phosphodiesterase. We found that a 50-kDa cytosolic protein of the rabbit lung, intestine, and spleen bound to the 10-kDa protein, in a Ca(2+)-dependent manner. The distribution of calcyclin and calcyclin binding proteins was unique and seems to differ from that of calmodulin and calmodulin-binding proteins. Thus, calcyclin probably plays a physiological role through its binding proteins for the Ca(2+)-dependent cellular response.