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Biomedical subjects

T F Walseth

Publications and source records attributed to T F Walseth.

At least 73 records · Page 4Linked to original sources
PubMed1987

Pyridine nucleotide metabolites stimulate calcium release from sea urchin egg microsomes desensitized to inositol trisphosphate.

Inositol trisphosphate (IP3) was previously shown to release Ca2+ from a nonmitochondrial store in sea urchin eggs. In this study, egg homogenates and purified microsomes were monitored with either fura 2 or Ca2+-sensitive minielectrodes to determine whether other stimuli would induce Ca2+ release. Pyridine nucleotides (whose concentrations are known to change at fertilization) were found to release nearly as much Ca2+ as did IP3. Average releases/ml of homogenate were: 0.6 microM IP3, 10.9 nmol of Ca2+; 50 microM NADP, 7.3 nmol of Ca2+; and 100 microM NAD, 6.5 nmol of Ca2+ (n = 6). Specificity was demonstrated by screening a series of other phosphorylated metabolites, and none was found to reproducibly release Ca2+. Calcium release induced by IP3 or NADP was immediate, whereas a lag of 1-4 min occurred with NAD. This lag before NAD-induced Ca2+ release led to the discovery that a soluble egg factor (Mr greater than 100,000) converts NAD into a highly active metabolite that releases Ca2+ without a lag. The NAD metabolite (E-NAD) was purified to homogeneity by high pressure liquid chromatography and produced half-maximal Ca2+ release at about 40 nM. Injection of E-NAD into intact eggs produced both an increase in intracellular Ca2+ (as assayed with indo-1) and a cortical reaction. Following Ca2+ release by each of the active agents (IP3, NAD, and NADP), the homogenates resequestered the released Ca2+ but were desensitized to further addition of the same agent. A series of desensitization experiments showed that homogenates desensitized to any two of these agents still responded to the third, indicating the presence of three independent Ca2+ release mechanisms. This is further supported by experiments using Percoll density gradient centrifugation in which NADP-sensitive microsomes were partially separated from those sensitive to IP3 and NAD.

Animals↗
PubMed1987

Inositol trisphosphate stimulates calcium release from peeled skeletal muscle fibers.

The effects of inositol phosphates (tris (InsP3), bis (InsP2), mono (InsP)) on rabbit adductor magnus and soleus muscles were determined using mechanically peeled fibers (sarcolemma removed). Isometric force generation of each fiber was continuously monitored and was used along with 45Ca to detect calcium release from internal fiber stores. All experiments were conducted at a physiological Mg2+ concentration (10(-3) M) of the bathing solutions. The inositol phosphates did not directly activate the contractile apparatus. At bath concentrations of 100-300 microM, only InsP3 was capable of stimulating Ca2+ release. In contrast, 1 microM InsP3 maximally and selectively stimulated Ca2+ release when microinjected into the myofilament lattice. Calcium releasing effects of InsP2 and InsP were manifested at 10 microM when they were microinjected. The end-to-end internal Ca2+ release and subsequent fiber force generation stimulated by the locally applied microinjected InsP3 suggests that the InsP3-induced Ca2+ release mechanism may involve propagation, but not via the Ca2+-induced Ca2+ release, since procaine did not inhibit this response. These findings support the possibility that InsP3 plays a role in skeletal muscle excitation-contraction coupling.

Animals↗
PubMed1987

The dynamics of cGMP metabolism in neuroblastoma N1E-115 cells determined by 18O labeling of guanine nucleotide alpha-phosphoryls.

The rates of phosphodiesterase-promoted hydrolysis of cGMP and cAMP have been measured in intact neuroblastoma N1E-115 cells by determining rates of 18O incorporation from 18O-water into the alpha-phosphoryls of guanine and adenine nucleotides. The basal rate of guanine nucleotide alpha-phosphoryl labeling ranged from 180 to 244 pmol X mg protein-1 X min-1. Sodium nitroprusside (SNP) caused a sustained 3.4-fold increase in this 18O-labeling rate in conjunction with 28- and 50-fold increases in cellular cGMP concentration at 3 and 6 min, respectively. This 18O-labeling rate (795 pmol X mg protein-1 X min-1) corresponded with the sum of the low (1.7 microM) and high (34 microM) Km phosphodiesterase activities assayable in cell lysates which exhibited a combined maximum velocity of 808 pmol X mg protein-1 X min-1 to which the high Km species contributed 84%. This information and the characteristics of the profile of 18O-labeled molecular species indicate that cGMP metabolism was restricted to a very discrete cellular compartment(s) of approximately 12% of the cell volume. Carbachol (1 mM) produced a transient increase (6-fold) in cellular cGMP concentration and a transient increase (90%) in the rate of 18O labeling of alpha-GTP during the first minute of treatment which translates into 30 additional cellular pools of cGMP hydrolyzed in this period. IBMX (1 mM) produced a relatively rapid increase in cellular cGMP (3- to 5-fold) and cAMP (2-fold) concentrations and a delayed inhibition of 18O labeling of guanine and adenine nucleotide alpha-phosphoryls without further elevation of cyclic nucleotide levels. These results indicate that besides inhibiting cyclic nucleotide hydrolysis, IBMX also imparts a time-dependent inhibitory influence on the generation of cyclic nucleotides. The data obtained show that measurement of 18O labeling of guanine and adenine nucleotide alpha-phosphoryls combined with measurements of cyclic nucleotide steady state levels provides a means to assess the rates of cyclic nucleotide synthesis and hydrolysis within intact cells and to identify the site(s) of action of agents that alter cellular cyclic nucleotide metabolism.

1-Methyl-3-isobutylxanthine↗
PubMed1987

Receptor-mediated adenylate cyclase-coupled mechanism for PGE2 inhibition of insulin secretion in HIT cells.

Prostaglandin E2 (PGE2) inhibits glucose-induced insulin secretion, and inhibitors of PGE2 synthesis augment this event. However, there has been confusion regarding prostaglandin regulation of insulin secretion, partly because no mechanism has been demonstrated for the inhibitory action of PGE2 on beta-cell function. These studies were performed with a clonal cell line of glucose-responsive beta-cells (HIT cells) to determine whether PGE2 effects on insulin secretion are receptor mediated and, if so, whether the postreceptor effects are mediated by inhibitory regulatory components (Ni) of adenylate cyclase. Saturable [3H]PGE2 binding to HIT cells was demonstrated. This binding was dissociable and specific for prostaglandins of the E series. Scatchard analyses of binding data indicated a single class of sites with a Kd of approximately 1 X 10(-9) M. Guinea pig islets were also demonstrated to have a single class of binding sites with a similar Kd but only 22% as many binding sites (0.060 vs. 0.013 pmol/mg protein, HIT cells vs. guinea pig islet). HIT cells were demonstrated to synthesize PGE2, and this synthesis was inhibitable by acetylsalicylic acid. Accumulation of cAMP by HIT cells was inhibited in a concentration-dependent manner by PGE2 with an IC50 of approximately 1 X 10(-9) M. Insulin secretion by HIT cells during static incubations with 11.1 mM glucose was also inhibited by PGE2 in a concentration-dependent manner with an IC50 of 1 X 10(-9) M. PGE2 was more potent than epinephrine but less potent than somatostatin in this regard. Maximum inhibition of glucose-induced insulin secretion was 26, 37, and 29% of control values for somatostatin, PGE2, and epinephrine, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylate Cyclase Toxin↗
PubMed1986

Light-induced increases in cGMP metabolic flux correspond with electrical responses of photoreceptors.

The metabolism of photoreceptor cGMP and the relationship of its light-sensitive regulation to rhodopsin photoisomerization and to the photoreceptor electrical response was examined in isolated, intact rabbit retinas. The dynamics of cGMP metabolism were assessed by measuring the rate of 18O incorporation from 18O-water into the alpha-phosphoryls of the guanine nucleotides. The photoreceptor electrical response was determined by measuring the aspartate-isolated mass receptor potential. Basal cGMP flux in dark-adapted retinas was 33 pmol cGMP X mg protein-1 X s-1 which translates into a metabolic rate in the rod outer segment (ROS) of 1.7 mM/min in ATP equivalents. Photic stimulation increased this flux as much as 4.5-fold. With continuous illumination, increasing intensity caused increments in cGMP metabolic flux to a maximum of 4.5-fold, with corresponding increases in the electrical response over the same 3-log unit intensity range. Tight coupling between activation of guanylate cyclase and phosphodiesterase was indicated by either no changes in cGMP steady state concentrations or relatively small fluctuations represented by increases of 50% at lower light intensities and a 12% decrease at one of the highest intensities. A stoichiometry of about 10,000 molecules of cGMP generated and hydrolyzed per photon absorbed was calculated for the lowest light intensity when the increment in cGMP metabolic flux per photon was maximal. Flashing light caused an increase in flux in proportion to frequency up to 1 Hz and a nearly proportional increase in the voltage time integral of the electrical response up to 0.5 Hz. This indicates that the temporal resolution, or "on"/"off" rate, of the cGMP metabolic response was as fast or faster than the temporal resolution of the electrical response. The concentration of cGMP remained relatively stable in spite of the marked acceleration of cGMP flux that occurred over the 32-fold range of frequencies tested. Taken together these results show that the light-accelerated rate of cGMP synthesis tightly coupled to hydrolysis becomes a primary energy-utilizing system in the photoreceptor and represents a response that fulfills certain of the fundamental criteria required of a metabolic event playing an essential role in phototransduction.

Animals↗
PubMed1984

Anion-exchange chromatography of proteins on AG MP-1 using high-performance liquid chromatography equipment.

That the macroporous anion-exchange resin AG MP-1 can be used with HPLC equipment and common aqueous buffers for the chromatography of proteins is shown. The utility of this system is illustrated by the partial purification and complete resolution of the three protein synthesis elongation factors from each other, starting with a crude extract of Escherichia coli. The factors were purified 10- to 30-fold in a yield of 50 to 90% with a single 60-min chromatographic program of increasing NaCl concentration. Other proteins from various biological sources were purified with similar results. Thus, it appears that AG MP-1 is useful, at least in some applications, for the rapid, reproducible, and economical purification of proteins using HPLC equipment.

Animals↗
PubMed1983

Magnitude of increase in retinal cGMP metabolic flux determined by 18O incorporation into nucleotide alpha-phosphoryls corresponds with intensity of photic stimulation.

The property of cyclic nucleotide phosphodiesterases to catalyze 3'-P--O bond cleavage and the insertion of a single nonexchangeable atom of 18O from [18O]water into the phosphoryl of the 5'-nucleotide product has been utilized as a means for measuring the hydrolytic flux of cGMP and cAMP in isolated dark-adapted intact rabbit retinas. Without illumination 18O labeling of guanine nucleotide (GTP and GDP) alpha-phosphoryls proceeds linearly for at least 80 s at a rate of 3.3 nmol of 18O/s.g of retina (wet weight). This rate is estimated to be approximately 8 times greater in the rod outer segment layer where over 90% of retinal cGMP metabolic components reside. Photic stimulation during a 20-s incubation was provided by intermittent flashes of light representing 800 ms of total illumination. Light stimuli over a range of intensities of greater than 3 log units commencing with a minimally detectable intensity produce graded increments in the rate of 18O incorporation into guanine nucleotide alpha-phosphoryls to a maximum increase of 5-fold. On the basis of only the 800-ms period of illumination this maximum increase is 125-fold. Steady state levels of retinal cGMP are not altered appreciably over this greater than 3 log range of light intensities but a light stimulus exceeding this intensity range causes an approximate 50% decrease in retinal cGMP concentration and a relative decline in the maximal rate of 18O labeling of guanine nucleotide alpha-phosphoryls. No light-related increases were detected in 18O incorporation into adenine nucleotide alpha-phosphoryls nor the gamma-phosphoryls of GTP or ATP or Pi. These observations indicate that light stimuli over greater than 3 log of light intensity produce incremental increases in cGMP metabolic flux that result from comparable increases in the rates of both cGMP generation and cGMP hydrolysis. It is postulated that increases in cGMP metabolic flux rather than changes in cGMP steady state levels are integral to phototransduction by a mechanism that involves the coupling of cGMP synthesis and/or hydrolysis to either the release of calcium from disc membranes or the inhibition of Na+ conductance by the photoreceptor membrane. This is suggested to occur by an energy-linked process and/or the generation of protons.

Adenine Nucleotides↗
PubMed1980

18O-Labeling of guanosine monophosphate upon hydrolysis of cyclic guanosine 3':5'-monophosphate by phosphodiesterase.

The hydrolysis of cGMP by phosphodiesterase was conducted in [18O]water to determine the site of bond cleavage and the stoichiometry of 18O incorporation into 5'-GMP. Three different forms of phosphodiesterase including a calmodulin-calcium-dependent enzyme in its basal and activated states were examined. The hydrolysis of cGMP catalyzed by each of the forms of phosphodiesterase proceeded with incorporation of 1 18O atom recoverable in the phosphate moiety of each molecule of 5'-GMP generated. No molecular species of phosphate deriving from the 5'-GMP generated containing two or three 18O were detectable. These results indicate that the phosphodiesterase-catalyzed hydrolysis of cGMP proceeds by nucleophilic substitution at phosphorus resulting in P-O bond cleavage. The stoichiometry of 18O incorporation indicates that the reaction proceeds without phosphate-water oxygen exchange when the hydrolytic reaction is catalyzed by diverse forms of phosphodiesterase in the basal or activated state. These considerations of the phosphodiesterase reaction help to establish the validity of monitoring the rate of enzyme-catalyzed hydrolysis of cGMP as a function of the rate of 18O-labeling of the phosphate of 5'-GMP when the reaction proceeds in a medium of predetermined 18O enrichment.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1979

The enzymatic preparation of [alpha-(32)P]nucleoside triphosphates, cyclic [32P] AMP, and cyclic [32P] GMP.

A method has been developed for the enzymatic preparation of alpha-(32)P-labeled ribo- and deoxyribonucleoside triphosphates, cyclic [(32)P]AMP, and cyclic [(32)P]GMP of high specific radioactivity and in high yield from (32)Pi. The method also enables the preparation of [gamma-(32)P]ATP, [gamma-(32)P]GTP, [gamma-(32)P]ITP, and [gamma-(32)P]-dATP of very high specific activity and in high yield. The preparation of the various [alpha-(32)P]nucleoside triphosphates relies on the phosphorylation of the respective 3'-nucleoside monophosphates with [gamma-(32)P]ATP by polynucleotide kinase and a subsequent nuclease reaction to form [5'-(32)P]nucleoside monophosphates. The [5'-(32)P]nucleoside monophosphates are then converted enzymatically to the respective triphosphates. All of the reactions leading to the formation of [alpha-(32)P]nucleoside triphosphates are carried out in the same reaction vessel, without intermediate purification steps, by the use of sequential reactions with the respective enzymes. Cyclic [(32)P]AMP and cyclic [(32)P]GMP are also prepared enzymatically from [alpha-(32)P]ATP or [alpha-(32)P]GTP by partially purified preparations of adenylate or guanylate cyclases. With the exception of the cyclases, all enzymes used are commerically available. The specific activity of (32)P-labeled ATP made by this method ranged from 200 to 1000 Ci/mmol for [alpha-(32)P]ATP and from 5800 to 6500 Ci/mmol for [gamma-(32)P]ATP. Minor modifications of the method should permit higher specific activities, especially for the [alpha-(32)P]nucleoside triphosphates. Methods for the use of the [alpha-(32)P]nucleoside phosphates are described for the study of adenylate and guanylate cyclases, cyclic AMP- and cyclic GMP phosphodiesterase, cyclic nucleotide binding proteins, and as precursors for the synthesis of other (32)P-labeled compounds of biological interest. Moreover, the [alpha-(32)P]nucleoside triphosphates prepared by this method should be very useful in studies on nucleic acid structure and metabolism and the [gamma-(32)P]nucleoside triphosphates should be useful in the study of phosphate transfer systems.

Adenosine Triphosphate↗
PubMed1979

Production, purification, and properties of dextransucrase from Leuconostoc mesenteroides NRRL B-512F.

The production of dextransucrase from Leuconostoc mesenteroides NRRL B-512F was stimulated 2-fold by the addition of 0.005% of calcium chloride to the medium; levansucrase levels were unaffected. Dextransucrase was purified by concentration and dialysis of the culture supernatant with a Bio-Fiber 80 miniplant, and by treatment with dextranase followed by chromatography on Bio-Gel A-Fm. A 240-fold purification, with a specific activity of 53 U/mg, was obtained. Contaminating enzyme activities of levansucrase, invertase, dextranase, glucosidase, and sucrose phosphorylase were decreased to non-detectable levels. Poly(acrylamide)-gel electrophoresis of the purified enzyme showed only two protein bands, both of which had dextransucrase activity. These bands also gave a carbohydrate stain, indicating that the dextransucrase could be a glycoprotein. Acid hydrolysis, followed by paper chromatography, of the purified enzyme showed that the major carbohydrate was mannose. Concanavalin A completely removed dextransucrase activity from solution, confirming the mannoglycoprotein character of the enzyme. Dextransucrase activity was not altered by the addition of 0.008-4 mg/ml of dextran, but its storage stability was increased by the addition of 4 mg/ml of dextran. As previously shown by others, the activity of dextransucrase was decreased by EDTA, and was restored by the addition of calcium ions. Zinc, cadmium, lead, mercury, and copper ions were inhibitory to various degrees.

Calcium Chloride↗
PubMed1978

The mechanism of acceptor reactions of Leuconostoc mesenteroides B-512F dextransucrase.

Reactions of dextransucrase and sucrose in the presence of sugars (acceptors) of low molecular weight have been observed to give a dextran of low molecular weight and a series of oligosaccharides. The acceptor reaction of dextransucrase was examined in the absence and presence of sucrose by using D-[14C]glucose, D-[14C]fructose, and 14C-reducing-end labeled maltose as acceptors. A purified dextransucrase was preincubated with sucrose, and the resulting D-fructose and unreacted sucrose were removed from the enzyme by chromatography of columns of Bio-Gel P-6. The enzyme, which migrated at the void volume was collected and referred to as "charged enzyme". The charged enzyme was incubated with 14C-acceptor in the absence of sucrose. Each of the three acceptors gave two fractions of labeled products, a high molecular weight product, identified as dextran, and a product of low molecular weight that was an oligosaccharide. It was found that all three of the acceptors were incorporated into the products at the reducing end. Similar results were obtained when the reactions were performed in the presence of sucrose, but higher yields of labeled products were obtained and a series of homologous oligosaccharides was produced when D-glucose or maltose was the acceptor. We propose that the acceptor reaction proceeds by nucleophilic displacement of glucosyl and dextranosyl groups from a covalent enzyme-complex by a specific, acceptor hydroxyl group, and that this reaction effects a glycosidic linkage between the D-glucosyl and dextranosyl groups and the acceptor. We conclude that the acceptor reactions serve to terminate polymerization of dextran by displacing the growing dextran chain from the active site of the enzyme; the acceptors, thus, do not initiate dextran polymerization by acting as primers.

Chromatography, Paper↗