Changes in AMP deaminase activities in the hearts of diabetic rats

AMP deaminase from normal and diabetic rat hearts was separated on cellulose phosphate and quantitated by HPLC. From soluble fractions three different AMP deaminase activities, according to KCl elution from cellulose phosphate and percent of total activity were: 170 mM (85%), 250 mM (8%) and 330 mM (7%) KCl. The AMP deaminase activity which eluted with 170 mM KCl was resolved to two distinct peaks by HPLC anionic exchange. After 4 weeks of diabetes the heart enzyme profile change to: 170 mM (10%), 250 mM (75%) and 330 mM (15%). Once purified the four activities were kinetically distinct: 170 mM KCl cytosolic, AMP Km = 1.78, stimulated by ATP, GTP, NADP and strongly inhibited by NAD; 170 mM KCl mitochondria AMP Km = 17.9, stimulated by ATP, ADP; 250 mM KCl isozyme, AMP Km = 0.66, stimulated by ADP; and 330 mM KCl isozyme, AMP Km = 0.97, inhibited by ATP, NAD(P).     

Interaction of the antiestrogen [3H]H1285 with the two forms of the molybdate-stabilized calf uterine estrogen receptor

The high affinity antiestrogen [3H]H1285 bound to the cytosol calf uterine estrogen receptor dissociated very slowly (t 1/2 approx 30 h at 20 degrees C) and did not demonstrate a change in dissociation rate in the presence of molybdate, which is characteristic of [3H]estradiol-receptor complexes. [3H]H1285-Receptor complexes sediment at approx 6S on 5-20% sucrose density gradients containing 0.3M KCl with or without 10 mM molybdate. This is in contrast to [3H]estradiol-receptor complexes which sedimented at approx 4.5S without molybdate and at approx 6S with molybdate. These results suggest a physicochemical difference in the estrogen receptor when occupied by antiestrogens versus estrogens. We recently reported that the cytoplasmic uterine estrogen receptor, when bound by estradiol and prepared in 10 mM molybdate, eluted from DEAE-Sephadex columns as Peak I (0.21 M KCl) & Peak II (0.25 M KCl). However, [3H]H1285 bound to the estrogen receptor eluted only as one peak at 0.21 M KCl, also suggesting that the initial interaction of antiestrogens with the estrogen receptor is different. We have extended these studies and report that H1285 can compete with [3H]estradiol for binding to both forms of the estrogen receptor and [3H]H1285 can bind to both forms if the unoccupied receptor is first separated by DEAE-Sephadex chromatography. However, if the receptor is first bound by unlabeled H1285, eluted from the column and post-labeled by exchange with [3H]estradiol, only one peak is measured. Thus, it appears that H1285 binding alters the properties of the receptor such that all receptor components seem to elute as one form. These partially purified [3H]H1285-receptor complexes obtained from DEAE-Sephadex columns sedimented as 5.5S in sucrose density gradients in contrast to the sedimentation values for the [3H]estradiol-receptor components eluting as Peak I (4.5S) and Peak II (6.3S). These differences in the physicochemical characteristics of the estrogen receptor when bound by estrogen versus antiestrogens may be related to some of the biological response differences induced by these ligands.     

A kinetic study of sugarcane sucrose synthase.

The kinetic data on sugarcane (Saccharum spp. hybrids) sucrose synthase (SuSy, UDP-glucose: D-fructose 2-alpha-D-glucosyltransferase, EC 2.4.1.13) are limited. We characterized kinetically a SuSy activity partially purified from sugarcane variety N19 leaf roll tissue. Primary plot analysis and product inhibition studies showed that a compulsory order ternary complex mechanism is followed, with UDP binding first and UDP-glucose dissociating last from the enzyme. Product inhibition studies showed that UDP-glucose is a competitive inhibitor with respect to UDP and a mixed inhibitor with respect to sucrose. Fructose is a mixed inhibitor with regard to both sucrose and UDP. Kinetic constants are as follows: Km values (mm, +/- SE) were, for sucrose, 35.9 +/- 2.3; for UDP, 0.00191 +/- 0.00019; for UDP-glucose, 0.234 +/- 0.025 and for fructose, 6.49 +/- 0.61. values were, for sucrose, 227 mm; for UDP, 0.086 mm; for UDP-glucose, 0.104; and for fructose, 2.23 mm. Replacing estimated kinetic parameters of SuSy in a kinetic model of sucrose accumulation with experimentally determined parameters of the partially purified isoform had significant effects on model outputs, with a 41% increase in sucrose concentration and 7.5-fold reduction in fructose the most notable. Of the metabolites included in the model, fructose concentration was most affected by changes in SuSy activity: doubling and halving of SuSy activity reduced and increased the steady-state fructose concentration by about 42 and 140%, respectively. It is concluded that different isoforms of SuSy could have significant differential effects on metabolite concentrations in vivo, therefore impacting on metabolic regulation.     

Characterization of steroid hormone receptors with ion-exchange fast protein liquid chromatography

Androgen, estrogen and progesterone receptors have been characterized with anion exchange Fast Protein Liquid Chromatography (FPLC) on a Mono Q column (Pharmacia). In the presence of sodium molybdate androgen receptors in cytosols from rat prostate, rat epididymis and calf uterus eluted as a single sharp peak at 0.32 M NaCl with recoveries of approx 90%. The molybdate-stabilized form of the androgen receptor from rat prostate was purified about 75-fold. The receptor containing FPLC-peak fractions sedimented in high salt (0.4 M KCl) linear sucrose gradients at 3.6 S (prostate) and at 4.6 S (epididymis and calf uterus) respectively. Multiple forms of the androgen receptor were present in cytosols from rat prostate prepared in the absence of sodium molybdate, probably due to proteolytic breakdown of the native form. Calf uterine estradiol and progesterone receptors prepared in the presence of sodium molybdate (20 mM) eluted from the Mono Q column at 0.32 M NaCl. The molybdate-stabilized forms of the oestradiol and progesterone receptors were purified approx 70-fold and 30-fold respectively. In the absence of molybdate the estradiol receptor dissociated into two major forms eluting at 0.23 M NaCl and 0.37 M NaCl. After heat induced transformation (30 min at 25 degrees C) of the estradiol receptor one major peak was eluted at 0.42 M NaCl, indicating a change in the surface charge of the estradiol receptor as a result of the 4 S to 5 S transformation. It is concluded that the FPLC anion exchange system is a powerful, fast tool for characterization and partial purification of steroid receptors. In addition this technique could be applied as a rapid procedure for the quantitative estimation of steroid receptors in small biological samples.     

Inhibition of Sucrose:Sucrose Fructosyl Transferase by Cations and Ionic Strength

Fructans are storage carbohydrates found in many temperate grasses. The first enzyme in the biosynthetic pathway of most fructans is sucrose:sucrose fructosyl transferase (SST). In this report, we demonstrate that K+ and ionic strength noncompetitively inhibit the activity of SST from wheat (Triticum aestivum L.) stems. The Ki for this inhibition is high, 122 mM, but in the range of concentrations of K+ found in the tissue (205-314 mM). Addition of KCl to the assay system had no effect on the pH optimum (5.5) or the Km for sucrose (266 mM) but reduced the Vmax. At equivalent ionic strengths, inhibition by choline chloride was about half that of KCl, indicating that inhibition by ionic strength might be responsible for approximately 50% of the KCl inhibition. Inhibition by LiCl and (NH4)2SO4 was similar to that by choline chloride. Soluble invertase activity found in the SST preparations was less sensitive to KCl and more sensitive to choline chloride than was SST. SST from barley (Hordeum vulgare L.) stems and leaves, as well as SST from leaves of orchardgrass (Dactylis glomerata), was also inhibited by KCl. SST from onion (Allium cepa L.) bulbs and asparagus (Asparagus officinalis L.) stems was not inhibited by KCl; thus, inhibition of activity by KCl is not a universal characteristic of SST from all sources.     

Characterization of two independent modes of action of ATP on human erythrocyte sugar transport

Intracellular ATP has been reported either to stimulate [Jacquez, J.A. (1983) Biochim. Biophys. Acta 727, 367-378] or to inhibit [Hebert, D. N., & Carruthers, A. (1986) J. Biol. Chem. 261, 10093-10099] human erythrocyte sugar transport. This current study provides a rational explanation for these divergent findings. Protein-mediated 3-O-methyl-alpha-D-glucopyranoside (3OMG) uptake by intact human red blood cells (lacking intracellular sugar) at ice temperature in isotonic KCl containing 2 mM MgCl2, 2 mM EGTA, and 5 mM Tris-HCl, pH 7.4 (KCl medium), is characterized by a Km(app) of 0.4 +/- 0.1 mM and a Vmax of 114 +/- 20 mumol L-1 min-1. Lysis of red cells in 40 volumes of EGTA-containing hypotonic medium and resealing in 10 volumes of KCl medium increase the Km(app) and Vmax for uptake to 7.1 +/- 1.8 mM and 841 +/- 191 mumol L-1 min-1, respectively. Addition of ATP (4 mM) to the resealing medium restores Michaelis and velocity constants for zero-trans 3OMG uptake to 0.42 +/- 0.11 mM and 110 +/- 15 mumol L-1 min-1, respectively. Addition of CaCl2 to extracellular KCl medium (calculated [Ca2+]o = 101 microM) reduces the Vmax for zero-trans 3OMG uptake in intact cells and ATP-containing ghosts by 79 +/- 4% and 61 +/- 9%, respectively. Intracellular Ca2+ (15 microM) reduces the Vmax for 3OMG uptake by ATP-containing ghosts by 38 +/- 12%. In nominally ATP-free ghosts, extracellular (101 microM) and intracellular (11 microM) Ca2+ reduce the Vmax for 3OMG uptake by 96 and 94%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular forms of guanine aminohydrolase (author's transl)

Guanine aminohydrolase (E.C. 3.5.4.3) has been purified 11-fold from the supernatant fraction of guinea-pig liver homogenates in 0.25 M sucrose (centrifuged at 50,000 X g) through thermic denaturation at 60 degrees C and ammonium sulphate fractionation (30--60% saturation). The enzyme in the homogenates and purified preparations exhibited two Km values. In both preparations four enzymatic electrophoretic bands have been detected. Purified guanine aminohydrolase is chromatographically resolved on DEAE-sephadex in three components whose active forms appeared separately on their pherograms. The enzymatic form eluted at lower ionic strength has the least anodic mobility, is inhibited by guanine (4 X 10(-5) M) and presents only one Km value (1.5 X 10(-5) M). The enzymatic form eluted at greater ionic strength exhibits the highest anodic mobility, is also inhibited by guanine (7 X 10(-5) M) and its Km value seems to be 6.3 X 10(-6) M. Molecular weight of enzymatics forms determined by Sephadex G-200 chromatography, is 120,000 +/- 5,000. The preceding results, correlated with the chromatographic homogeneity of guanine aminohydrolase, purified in Sephadex G-100, suggests that the four molecular forms of the native enzyme may be considered as isozymes.     

Hydrolysis of cis and trans isomers of retinyl palmitate by retinyl ester hydrolase of pig liver

Relative retinyl ester hydrolase activities of pig liver homogenates (n = 4) toward 9,13-cis-, 13-cis-, 9-cis-, and all-trans-retinyl palmitate were 6.8 +/- 0.5 (SE), 5.7 +/- 0.5, 2.4 +/- 0.1, and 1, respectively. The range of apparent Km values for the four isomers was 142 to 268 microM, and the pH optima were 8-9 in all cases. Peak activities of retinyl ester hydrolase activities in pig liver cytosol toward 13-cis- and all-trans-retinyl palmitate were found in the 20 to 40% and in the 60 to 80% saturated ammonium sulfate (AS) fractions, respectively. By use of size-exclusion chromatography in 2 M KCl, hydrolase activity eluted at volumes corresponding to greater than 2000, 180, and 15 kDa from the 20-40% AS fraction, and at 180 kDa from the 60-80% AS fraction. On the basis of molecular size, different substrate specificities, detergent effects, and susceptibilities to inhibition by phenylmethylsulfonyl fluoride, we conclude that at least three distinct retinyl ester hydrolases are present in pig liver cytosol.     

A possible role of inorganic phosphate as a regulator of oxidative phosphorylation in combined urea synthesis and gluconeogenesis in perfused rat liver. A phosphorus magnetic resonance spectroscopy study

Metabolic control of oxidative metabolism was studied in perfused rat liver by means of phosphorus magnetic resonance spectroscopy. Oxygen consumption, ATP, and Pi were measured with different rates of gluconeogenesis and urea synthesis by varying concentrations of the substrates in the perfusate. Five levels of oxygen consumption (VO2) were obtained: an average control value of 1.94 +/- 0.14 and 2.93 +/- 0.25, 3.29 +/- 0.46, 3.85 +/- 0.26, and 4.18 +/- 0.56 mumol/min/g liver (mean +/- S.D., n = 6). The corresponding ATP concentrations were 2.51 +/- 0.20, 2.39 +/- 0.08, 2.24 +/- 0.09, 2.13 +/- 0.12, and 1.91 +/- 0.13 mM. Pi increased stoichiometrically with the decrease in ATP. Free Pi (Pif) was calculated as NMR-visible Pi in control plus -delta ATP (1.94 mM + (-delta ATP]. The kinetic relationship of oxidative phosphorylation as a function of Pif followed a Michaelis-Menten type of equation: VO2 = 5.55/(1 + 0.24/[( Pif] - 1.81]. The observed Km value for Pi of 0.24 mM approximates the reported Km value in isolated mitochondria of 1 mM. The free Pi concentration of 1.94 mM is in the range of the Km value, while the free ADP concentration of 200 microM exceeds the Km value of 20 microM. Therefore, it is suggested that Pi play a major role in the regulation of mitochondrial oxidative phosphorylation in combined urea synthesis and gluconeogenesis.     

Non-additive counteraction of KCl-perturbation of lactate dehydrogenase by trimethylamine N-oxide

Added KCl increases the apparent Michaelis constant (Km) of pyruvate for porcine muscle-type lactate dehydrogenase (100 mM KCl, 83%; 200 mM KCl, 188%). The effects of 100 mM KCl were fully reversed by 375 mM trimethylamine N-oxide (TMAO). TMAO (375-750 mM) partially reversed the effects of 200 mM KCl. TMAO as the sole solute, at concentrations up to 750 mM, had no effect on Km. This is atypical because compensatory osmolytes such as TMAO characteristically counteract protein perturbation in an additive manner.     

Effect of sucrose concentration on the invertase activity of intact yeast cells (S. cerevisiae)

Summary The kinetic parame6ers for soluble (Km=7.4mM and, V_max=2.6 U/mL) and cell wall bound invertase (Km=15.4mM and V_max=3.2 U/L) were determined. The invertase activity for both forms varied by around 10% against sucrose concentration ranging from 80 g/L to 200 g/L, whereas the transferase activity increased markedly with increasing sucrose concentration.     

A method for isolation of undegraded free and membrane-bound ribosomes from rat lactating mammary gland

Mammary gland polysomes are difficult to isolate from the lactating rat using methods developed for other species and tissues, most likely due to high calcium-stimulated ribonuclease activity in that tissue. A new method, utilizing ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) to bind calcium, yields highly aggregated polysomes from lactating rat mammary gland. Fresh mammary tissue is pulverized under liquid nitrogen. Free and membrane-bound polysomes are isolated by differential centrifugation in solutions containing 100 mM KCl, 100 mM MgCl2, 75 mM EGTA, 500 micrograms/ml heparin and 50 mM Tris buffer, pH 8.2 at 5 degrees C. Bound polysomes are released from the endoplasmic reticulum using Triton X-100 and deoxycholate. Polysome profiles are obtained on linear sucrose gradients and scanned at 254 nm. The method gives quantitative recovery of homogenate total RNA. To demonstrate that the method can be used to study nutritional effects on mammary gland polysome aggregation, lactating rats were fasted 22-66 h and then refed a stock diet for 71-95 h. Refeeding increased the percentage of polysomes (trimers or larger) in the bound fraction from 84 +/- 1 to 93 +/- 1% (P less than 0.001) and in the free fraction from 42 +/- 2 to 55 +/- 3% (P less than 0.001).     

In vitro inhibition of stable 1,3-beta-D-glucan synthase activity from Neurospora crassa.

Glucan synthase activity of Neurospora crassa was isolated by treatment of protoplast lysates with 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4, containing 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 200 mM inorganic phosphate, 10 microM GTP, 1 mM DTT, 10 mM sodium fluoride, and 600 mM glycerol. Resulting activity was partially purified by sucrose gradient density sedimentation. Approximately 70% of enzyme activity in the sucrose gradient peak fraction was soluble and enzyme activity was purified 7.3-fold. Partially purified enzyme activity had a half-life of several weeks at 4 degrees C, and a Km(app) of 1.66 +/- 0.28 mM. Inhibitors (Cilofungin, papulacandin B, aculeacin A, echinocandin B, sorbose and UDP) of 1,3-beta-D-glucan synthase activity were tested against crude particulate and detergent treated enzyme fractions and the Ki(app) of each inhibitor determined. It seems likely that this stable preparation of glucan synthase activity may be useful for in vitro enzyme screens for new glucan synthase inhibitors.     

Nuclear Magnetic Resonance Studies on the Effects of Decreased External Sodium on Guinea Pig Cerebral Cortex Slices and the Permeabilities of Various Sodium Substitutes

Decreasing the external sodium concentration ([Na+]e) to 10 mM in the presence of 280 mM sucrose had no significant effect on phosphocreatine (PCr) or on intracellular pH (pHi) as assessed using 31P nuclear magnetic resonance spectroscopy. Zero [Na+]e in the presence of 300 mM sucrose caused a fall in PCr levels to 50% of control values, and the pHi fell to 6.85 from a control value of 7.30. 1H nuclear magnetic resonance spectroscopy confirmed that the sucrose had not entered the tissue. The decreases in PCr content and in pHi, known to occur on depolarization using 40 mM external potassium concentration ([K+]e), were further decreased in the presence of 10 mM [Na+]e), to 51.4 +/- 4.0 and 6.80 +/- 0.10% of control values, respectively. The free intracellular magnesium concentration was significantly increased from a control value of 0.37 +/- 0.10 mM to 0.66 +/- 0.13 mM (p less than 0.001), when [Na+]e was decreased to 10 mM, but was not further affected by high [K+]e or zero Na+. Membrane permeabilities of the sodium substitutes N-methyl-D-glucamine (NMG), tris(hydroxymethyl)aminomethane (Tris), tetramethylammonium (TMA), and choline were assessed using 1H nuclear magnetic resonance spectroscopy. In the presence of 10 mM [Na+]e, NMG, TMA, and choline (all at 140 mM) were taken up and remained within the tissue for at least 2 h, but no uptake of Tris (140 mM) or sucrose (above) could be detected. Tissue lactate levels (from the lactate/N-acetyl aspartate ratio) increased in the presence of the substitutes that were taken up, although no change in pH was detected.     

The effect of extracellular potassium on the intracellular potassium ion activity and transmembrane potentials of beating canine cardiac Purkinje fibers

We used open tip microelectrodes containing a K+-sensitive liquid ion exchanger to determine directly the intracellular K+ activity in beating canine cardiac Purkinje fibers. For preparations superfused with Tyrode's solution in which the K+ concentration was 4.0 mM, intracellular K+ activity (ak) was 130.0+/-2.3 mM (mean+/-SE) at 37 degrees C. The calculated K+ equilibrium potential (EK) was -100.6+/- 0.5 mV. Maximum diastolic potential (ED) and resting transmembrane potential (EM) were measured with conventional microelectrodes filled with 3 M KCl and were -90.6+/-0.3 and -84.4+/-0.4 mV, respectively. When [K+]o was decreased to 2.0 mM or increased to 6.0, 10.0, and 16.0 mM, ak remained the same. At [K+]o=2.0, ED was -97.3+/-0.4 and Em - 86.0+/-0.7 mV; at [K+]o=16.0, ED fell to -53.8+/-0.4 mV and Em to the same value. Over this range of values for [K+]o, EK changed from - 119.0+/-0.3 to -63.6+/-0.2 mV. These values for EK are consistent with those previously estimated indirectly by other techniques.     

A kinetic isotope effect study and transition state analysis of the S-adenosylmethionine synthetase reaction

The biosynthesis of S-adenosylmethionine occurs in a unique enzymatic reaction in which the synthesis of the sulfonium center results from displacement of the entire polyphosphate chain from MgATP. The mechanism of S-adenosylmethionine synthetase (ATP:L-methionine s-adenosyltransferase) from Escherichia coli has been characterized by kinetic isotope effect and substrate trapping measurements. Replacement of 12C by 14C at the 5' carbon of ATP yields a primary Vmax/Km isotope effect (12C/14C) of 1.128 +/- 0.003 in the absence of added monovalent cation activator (K+). At saturating K+ concentrations (10 mM) the primary isotope effect diminishes slightly to 1.108 +/- 0.003, indicating that the step in the mechanism involving bond breaking at the 5' carbon of MgATP has a small commitment to catalysis at conditions near Vmax. No alpha-secondary 3H isotope effect from [5'-3H]ATP was detected, (1H/3H) = 1.000 +/- 0.002, even in the absence of KCl. There was no significant primary sulfur isotope effect from [35S]methionine at KCl concentrations from 0 to 10 mM. Substitution of the methyl group of methionine with tritium yielded a beta-secondary isotope effect (CH3/C3H3) = 1.009 +/- 0.008 independent of KCl concentration. The reaction of selenomethionine and [5'-14C]ATP gave a primary isotope effect of 1.097 +/- 0.006, independent of KCl concentration. Substrate trapping experiments demonstrated that the step in the mechanism involving bond making to sulfur of methionine does not have a significant commitment to catalysis at 0.25 mM KCl, therefore intrinsic isotope effects were observed. Substrate trapping experiments indicated that the step involving bond breaking at carbon 5' of MgATP has a 10% commitment to catalysis at 0.25 mM KCl. The isotope effects are interpreted in terms of an Sn2-like transition state structure in which bonding of the C5' is symmetric with respect to the departing tripolyphosphate group and the incoming sulfur of methionine. With selenomethionine as substrate an earlier transition state is implicated.     

Determination of depolarisation- and agonist-evoked calcium fluxes on skeletal muscle cells in primary culture

Changes in intracellular calcium concentration ([Ca2+]i) evoked by prolonged depolarisation (120 mM KCl) or by the application of 15 mM caffeine were measured on skeletal muscle cells in primary culture. The extrusion rate (PVmax) of calcium from the myoplasm was determined, which in turn enabled the calculation of the calcium flux (Fl) underlying the measured calcium transients. PVmax was found to increase during differentiation, from 107 +/- 10 microM/s at the early myotube stage to 596 +/- 36 microM/s in secondary myotubes. This was paralleled by a decrease in resting [Ca2+]i from 99 +/- 4 to 51 +/- 2 nM. The depolarisation-evoked Fl rose to peak and then ceased despite the continuous presence of KCl. In contrast, the caffeine-induced Fl showed a peak and a clear steady-level with a peak-to-steady ratio of 5.6 +/- 1.2. Removal of external calcium suppressed the depolarisation--induced flux by 88 +/- 5% indicating that both an influx and a release from the SR underlie the K(+)-evoked calcium transients. Subsequent applications of caffeine resulted in essentially identical fluxes indicating an efficient refilling of the internal stores. Moreover, if a depolarisation-induced calcium transient preceded the second caffeine-evoked release, the latter was significantly larger than the first suggesting that much of the calcium that entered was stored in the SR rather than extruded.     

Molecular forms of cholecystokinin in human plasma during infusion of bombesin

Bombesin is a tetradecapeptide with stimulatory actions on several gastrointestinal functions. Infusion of bombesin (60 pmol/kg. 20 min) into 7 normal subjects induced significant increases in plasma cholecystokinin (CCK) as measured with 2 sequence-specific radioimmunoassays. Employing antibody 1703, specific for carboxyl-terminal CCK-peptides containing at least 14 amino acid residues, plasma CCK concentrations rose from 0.8 +/- 0.2 pmol/l to 9.9 +/- 1.7 pmol/l (p less than 0.005), while using antibody T204, specific for the sulfated tyrosine region of CCK, plasma CCK levels increased from 2.9 +/- 0.5 pmol/l to 12.4 +/- 1.3 pmol/l (p less than 0.005). Plasma samples obtained from 3 subjects during bombesin infusion were fractionated by Sephadex column chromatography. Fractionation revealed 4 molecular forms of CCK: peak I eluted in the void volume and comprised 0-7% of CCK-like immunoreactivity, peak II eluted at 35% and comprised 8-41% of CCK-like immunoreactivity, peak III eluted at 50% and comprised 44-61% of CCK-like immunoreactivity, and peak IV eluted at 75% and comprised 15-27% of CCK-like immunoreactivity. Radioimmunoassay with a carboxyl-terminal CCK-antibody fully cross-reacting with gastrin did not reveal additional molecular forms of CCK. Since both the carboxyl-terminus and the sulfated tyrosine region are required for biological activity of CCK, it is likely that all these molecular forms of CCK possess biological activity.     

HPLC analysis of estrogen receptor by a multidimensional approach

Previously we demonstrated the polymorphism of estrogen receptors (ER) in cytosol of various tissues based upon properties of size, shape and surface charge. This study describes the application of a multidimensional approach utilizing HPLC for characterization of ER. Cytosols from human uterus and endometrial carcinomas were characterized sequentially by high performance size exclusion chromatography (HPSEC) on Spherogel TSK-3000 SW, and high performance ion-exchange chromatography (HPIEC) using SynChropak AX-1000 anion exchange columns. Using HPSEC, specific estrogen binding was exhibited by a 30 A isoform and by one appearing after the V0 (approximately 60 A) in human uterus. However, in endometrial carcinoma other smaller binding components with Stoke's radii of less than 20 A were observed also. In buffers containing 400 mM KCl, predominantly a 28-30 A species was observed by HPSEC. Further characterization of the 28-30 A isoform from low and high salt elution from HPSEC was accomplished with an AX-1000 column. With either condition, 2 forms were eluted on HPIEC, 1 in the column wash (retention time 8-9 min), and the other at 50-70 mM phosphate. The elution profile of the larger species (approximately 60 A by HPSEC) on the ion-exchange column was time dependent. Immediate analysis (within 15 min) showed a profile similar to that of the original cytosol which contained minor components eluting in wash buffer and at 50-70 mM phosphate and a major isoform at 180 mM phosphate. However delayed analysis (after 2 h) of the 60 A isoform showed a similar profile (components in buffer wash and at 50-70 mM phosphate) obtained with the 30 A species. This time dependent change was not observed for the 30 A species or for the original cytosol. Estrogen receptors in cytosol sedimented at 10S and 4S in low ionic strength gradients and at 4S in sucrose gradients containing 400 mM KCl. The 28-30 A and 60 A species recovered from HPSEC sedimented at 3.5S. This multidimensional approach indicates that native estrogen receptors dissociated into a number of smaller molecular isoforms, which were distinguishable by different surface charge properties.     

Purification and characterization of squid brain myosin.

Myosin was extracted from frozen squid brain and purified by a modification of the procedure of Pollard et al. (Pollard, T.D., Thomas, S.M., and Niederman, R. (1974) Anal. Biochem. 60, 258-266). Myosin was eluted from Bio-Gel A-15m column as a single peak of (K+-EDTA)-activated ATPase ((K+-EDTA)-ATPase) activity with an average partition coefficient (Kav) of 0.22. In sodium dodecyl sulfate-acrylamide gel electrophoresis, the purified myosin showed a predominant band with similar electrophoretic mobility as the heavy chain of rabbit skeletal muscle myosin, and two less intense bands near the bottom of the gel. No actin band was seen. The properties of the (K+-EDTA)-ATPase activity were: (a) the time course of the reaction was biphasic at 25 degrees but linear at 32 degrees; (b) the optimum rate of reaction was obtained between 0.3 and 0.8 M KCl; (c) the pH optimum was between 8.0 and 9.0; (d) the reaction was specific for ATP with an apparent Km of 0.19 mM. ATPase activity in 0.06 M KCl and 5 mM MgCl2 was increased about 1.5 times by a 10-fold excess of rabbit skeletal muscle F-actin and about 5 times by a 40-fold excess. The actin activation was inhibited slightly by the addition of 0.2 mM CaCl2 and completely by the addition of 10 mM CaCl2. Myosin formed arrowhead patterns with rabbit skeletal muscle F-actin as observed by electron microscopy of negatively stained samples. It also aggregated in bipolar filaments which attached to decorated actin filaments at different angles, as well as formed cross-connections and ladder-like patterns between actin filaments. These two forms of interactions between myosin and actin were abolished by treatment with MgATP.