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H R Kaslow

Publications and source records attributed to H R Kaslow.

At least 37 records · Page 2Linked to original sources
PubMed1989

Radiometric assays for glycerol, glucose, and glycogen.

We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus (1971, J. Biol. Chem. 246, 3885-3894) for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with [32P]ATP and glycerokinase, residual [32P]ATP is hydrolyzed by heating in acid, and free [32P]phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays.

Adipose Tissue↗
PubMed1988

Regulation of glycogen synthase in muscle and adipose tissue during fasting and refeeding.

Using immunoblot analysis, we examined the electrophoretic mobility of glycogen synthase from rat skeletal muscle and adipose tissue. Extracts from muscle freeze clamped in situ contained at least three forms of synthase with different electrophoretic mobilities. Extracts from adipose tissue also contained multiple forms but lacked the form with greatest mobility found in the muscle extracts. Phosphorylation at multiple sites of glycogen synthase is known to deactivate the enzyme and retard its electrophoretic mobility in sodium dodecyl sulfate gels. These results suggest that there is very little or no dephosphorylated glycogen synthase in adipose tissue and that phosphorylated forms of glycogen synthase synthesize adipose tissue glycogen. Relative to control, it is known that fasting decreases and refeeding increases glucose incorporation into glycogen in rat epididymal adipose tissue but not skeletal muscle incubated in vitro in the presence of insulin. Fasting did not change the electrophoretic pattern of muscle synthase but decreased the relative amount of adipose tissue forms with greater mobility. Refeeding increased above control the relative amount of adipose tissue synthase with greater mobility. These results indicate that changes in the phosphorylations that retard mobility contribute to the effects of fasting and refeeding on adipose tissue glycogen metabolism.

Adipose Tissue↗
PubMed1987

Fructose effect to suppress hepatic glycogen degradation.

The effect of fructose on glycogen degradation was examined by measuring the flux of 14C from prelabeled glycogen in perfused rat livers. During 2-h refeeding of 24-h-fasted rats, newly synthesized hepatic glycogen was labeled by intraperitoneal injection of [U-14C] galactose (0.1 mg and 0.02 microCi/g of body weight). The livers of refed rats were then perfused in a nonrecirculating fashion for an initial 30 min with glucose alone (10 mM) for the following 60 min with glucose (10 mM) without (n = 5) or with fructose (1, 2, or 10 mM; n = 5 for each). When livers were exposed to fructose, release of label into the perfusate immediately declined and remained markedly suppressed through the end of perfusion (p less than 0.05). The suppression was dose-dependent; at steady state (50-70 min), label release was suppressed 45, 64, and 72% by 1, 2, and 10 mM fructose, respectively (p less than 0.0001). Suppression was not accompanied by significant changes in the activities of glycogen synthase or phosphorylase assessed in vitro. These results suggest the existence of allosteric inhibition of phosphorylase in the presence of fructose. Fructose 1-phosphate (Fru-1-P) accumulated in proportion to fructose (0.11 +/- 0.01 without fructose, 0.86 +/- 0.03, 1.81 +/- 0.18, and 8.23 +/- 0.60 mumol/g of liver with 1, 2, and 10 mM fructose, respectively; p less than 0.0001). Maximum inhibition of label release was 82%; the Fru-1-P concentration for half inhibition was 0.57 mumol/g of liver, well within the concentration of Fru-1-P attained during refeeding. We conclude that fructose enhances net glycogen accumulation in liver by suppressing glycogenolysis and that the suppression is presumably caused by allosteric inhibition of phosphorylase by Fru-1-P.

Animals↗
PubMed1987

Sulfhydryl-alkylating reagents inactivate the NAD glycohydrolase activity of pertussis toxin.

The combination of ATP, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate), and DTT (dithiothreitol) is known to promote the expression of the NAD glycohydrolase activity of pertussis toxin, which resides in the toxin's S1 subunit. By monitoring changes in electrophoretic mobility, we have found that ATP and CHAPS act by promoting the reduction of the disulfide bond of the S1 subunit. In addition, ATP, CHAPS, and DTT allowed sulfhydryl-alkylating reagents to inactivate the NAD glycohydrolase activity. In the presence of iodo[14C]acetate, the combination of ATP, CHAPS, and DTT increased 14C incorporation into only the S1 subunit of the toxin, indicating that alkylation of this subunit was responsible for the loss of activity. If iodoacetate is used as the alkylating reagent, alkylation can be monitored by an acidic shift in the isoelectric point of the S1 peptide. Including NAD in alkylation reactions promoted the accumulation of a form of the S1 peptide with an isoelectric point intermediate between that of native S1 and that of S1 alkylated in the absence of NAD. This result suggests that NAD interacts with one of the two cysteines of the S1 subunit. In addition, we found the pH optimum for the NAD glycohydrolase activity of pertussis toxin is 8, which may reflect the participation of a cysteine in the catalytic mechanism of the toxin.

Adenosine Triphosphate↗
PubMed1987

Structure-activity analysis of the activation of pertussis toxin.

Bordetella pertussis, the causative agent of whooping cough, releases pertussis toxin in an inactive form. The toxin consists of an A protomer containing one S1 peptide subunit and a B oligomer containing several other peptide subunits. The toxin binds to cells via the B oligomer, and the S1 subunit is activated and expresses ADP-ribosyltransferase and NAD glycohydrolase activities. Treatment of purified toxin with dithiothreitol (DTT) in vitro increases both activities. ATP and the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) synergistically reduce the A0.5 (activation constant) for DTT from greater than 100 mM to 200 microM. We studied the structure-activity relationships of activators of the toxin. In the presence of CHAPS (1%) and DTT (10 mM) the following compounds increased the NAD glycohydrolase activity of the toxin with the following A0.5's in microM and fraction of the ATP effect in parentheses: ATP, 0.2 (1.0); ADP, 6 (0.8); UTP, 15 (0.7); GTP, 35 (0.6); pyrophosphate, 45 (0.7); triphosphate, 60 (0.6); tetraphosphate, greater than or equal to 170 (greater than or equal to 0.4). Thus, the polyphosphate moiety is sufficient to stimulate the toxin, and the adenosine moiety confers upon ATP its extraordinary affinity for the toxin. Phospholipid and detergents could substitute for CHAPS in the activation of the toxin. Glutathione substituted for DTT with an A0.5 of 2 mM, a concentration within the range found in eucaryotic cells. Thus, membrane lipids and cellular concentrations of glutathione and ATP are sufficient to activate pertussis toxin without the need for a eucaryotic enzymatic process.

ADP Ribose Transferases↗
PubMed1986

Stimulation of the thiol-dependent ADP-ribosyltransferase and NAD glycohydrolase activities of Bordetella pertussis toxin by adenine nucleotides, phospholipids, and detergents.

Pertussis toxin catalyzed ADP-ribosylation of the guanyl nucleotide binding protein transducin was stimulated by adenine nucleotide and either phospholipids or detergents. To determine the sites of action of these agents, their effects were examined on the transducin-independent NAD glycohydrolase activity. Toxin-catalyzed NAD hydrolysis was increased synergistically by ATP and detergents or phospholipids; the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) was more effective than the nonionic detergent Triton X-100 greater than lysophosphatidylcholine greater than phosphatidylcholine. The A0.5 for ATP in the presence of CHAPS was 2.6 microM; significantly higher concentrations of ATP were required for maximal activation in the presence of cholate or lysophosphatidylcholine. In CHAPS, NAD hydrolysis was enhanced by ATP greater than ADP greater than AMP greater than adenosine; ATP was more effective than MgATP or the nonhydrolyzable analogue adenyl-5'-yl imidodiphosphate. GTP and guanyl-5'-yl imidodiphosphate were less active than the corresponding adenine nucleotides. Activity in the presence of CHAPS and ATP was almost completely dependent on dithiothreitol; the A0.5 for dithiothreitol was significantly decreased by CHAPS alone and, to a greater extent, by CHAPS and ATP. To determine the site of action of ATP, CHAPS, and dithiothreitol, the enzymatic (S1) and binding components (B oligomer) were resolved by chromatography. The purified S1 subunit catalyzed the dithiothreitol-dependent hydrolysis of NAD; activity was enhanced by CHAPS but not ATP. The studies are consistent with the conclusion that adenine nucleotides, dithiothreitol, and CHAPS act on the toxin itself rather than on the substrate; adenine nucleotides appear to be involved in the activation of toxin but not the isolated catalytic unit.

Adenine Nucleotides↗
PubMed1985

L-type glycogen synthase. Tissue distribution and electrophoretic mobility.

We previously reported (Kaslow, H.R., and Lesikar, D.D.FEBS Lett. (1984) 172, 294-298) the generation of antisera against rat skeletal muscle glycogen synthase. Using immunoblot analysis, the antisera recognized the enzyme in crude extracts from rat skeletal muscle, heart, fat, kidney, and brain, but not liver. These results suggested that there are at least two isozymes of glycogen synthase, and that most tissues contain a form similar or identical to the skeletal muscle type, referred to as "M-type" glycogen synthase. We have now used an antiserum specific for the enzyme from liver, termed "L-type" glycogen synthase, to study its distribution and electrophoretic mobility. Immunoblot analysis using this antiserum indicates that L-type glycogen synthase is found in liver, but not skeletal muscle, heart, fat, kidney, or brain. In sodium dodecyl sulfate-polyacrylamide gels of crude liver extracts prepared with protease inhibitors, rat L-type synthase was detected with electrophoretic mobility Mapp = 85,000. In contrast, the M-type enzyme in crude skeletal muscle extracts with protease inhibitors was detected with Mapp = 86,000 and 89,000. During purification of L-type synthase, apparent proteolysis can generate forms with increased electrophoretic mobility (Mapp = 75,000), still recognized by the antiserum. These M-type and L-type antisera did not recognize a protein with Mapp greater than phosphorylase. The anti-rat L-type antisera recognized glycogen synthase in blots of crude extracts of rabbit liver, but with Mapp = 88,000, a value 3,000 greater than that found for the rat liver enzyme. The anti-rat M-type antisera failed to recognize the enzyme in blots of crude extracts of rabbit muscle. Thus, in both muscle and liver, the corresponding rat and rabbit enzymes are structurally different. Because the differences described above persist after resolving these proteins by denaturing sodium dodecyl sulfate electrophoresis, these differences reside in the structure of the proteins themselves, not in some factor bound to the protein in crude extracts.

Animals↗
PubMed1985

Adenine nucleotides directly stimulate pertussis toxin.

Both cholera toxin and pertussis toxin catalyzed ADP-ribosylation of purified bovine brain tubulin. The effect of cholera toxin was evident in the absence or presence of nucleotides. In contrast, pertussis toxin required adenine nucleotides for its ADP-ribosylating activity. ATP, ATP gamma S, App(NH)p, deoxy-ATP, and ADP all supported pertussis toxin-catalyzed ADP-ribosylations in the absence or presence of EDTA, suggesting that nucleotide hydrolysis was not involved. Adenine nucleotides also promoted pertussis toxin-catalyzed ADP-ribosylation of heat-treated bovine serum albumin. This result suggests that adenine nucleotides directly affect pertussis toxin. ATP stimulation of pertussis toxin-catalyzed hydrolysis of NAD to ADP-ribose supports this hypothesis.

Adenine Nucleotides↗
PubMed1984

Isozymes of glycogen synthase.

Antisera to rat skeletal muscle glycogen synthase failed to recognize liver glycogen synthase by electroblot analysis. The antisera recognized the enzyme in skeletal muscle, heart, fat, kidney, and brain. The results support the hypothesis that there are at least two isozymes of glycogen synthase, and that most tissues contain a form similar or identical to the skeletal muscle type. There is a virtual absence of the muscle-type enzyme in adult rat liver.

Adipose Tissue↗
PubMed1984

Fasting and diabetes alter adipose tissue glycogen synthase.

In a previous report (J. Biol. Chem. 254: 4678-4683, 1979), we showed that fasting blunted the ability of insulin to promote glucose incorporation into glycogen in vitro. In addition, we showed that glycogen synthase activity was altered in two ways: the concentration of glucose 6-P causing half-maximal activation increased, and positive cooperativity appeared in the glucose 6-P activation of the enzyme. We now show that streptozotocin-diabetes causes the same changes in glucose incorporation and glycogen synthase activity. We show that these changes in glycogen synthase activity persist during enzyme purification; thus it is likely the changes are a result of a structural alteration of the enzyme. Because glycogenolysis of a glycogen particle from rabbit skeletal muscle also caused the appearance of positive cooperativity, we propose that both phosphorylation and glycogenolysis are involved in the appearance of positive cooperativity.

Adipose Tissue↗
PubMed1983

A direct radioassay for pyruvate kinase activity.

Pyruvate kinase catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate. A direct radioassay for this enzyme using [14C]PEP as substrate has been developed. The product, [14C]pyruvate, can be separated from the substrate rapidly and easily by applying the mixture to a hydroxyapatite column, and eluting the [14C]pyruvate directly into a scintillation vial. The [14C]PEP is bound to the column which can be regenerated and used indefinitely. The assay is sensitive, rapid, and particularly well suited for the simultaneous assay of large numbers of samples.

Animals↗
PubMed1983

Identification by direct photoaffinity labeling of an altered phosphodiesterase in a mutant S49 lymphoma cell.

Extracts of a mutant S49 lymphoma cell line, termed K30a, hydrolyze cAMP and cGMP at rates much faster than do wild type S49 extracts. This elevated phosphodiesterase activity, called K-PDE, elutes as a single peak of activity on DEAE-cellulose columns (Brothers, V. M., Walker, N., and Bourne, H. R. (1982) J. Biol. Chem. 257, 9349-9355). Direct photoaffinity labeling of K30a extracts with [32P]cGMP results in radiolabeling of a unique polypeptide, not observed in wild type extracts, which migrates in sodium dodecyl sulfate polyacrylamide gels with an Mr = 106,000. The 106-kDa band was identified as the catalytic K-PDE polypeptide based on the following observations: competitive inhibitors and substrates of K-PDE inhibit photolabeling of the 106-kDa band, indicating that [32P] cGMP photolabels the enzyme at its catalytic site; on DEAE-cellulose chromatography the polypeptide that is susceptible to photolabeling co-elutes with K-PDE activity; the 106-kDa band is detectable in extracts of WT X K30a hybrids (where WT denotes wild type) in amounts proportional to the K-PDE activity in the hybrids, but is undetectable in wild type. The hybrid phenotype strongly suggests that the K30a phenotype is not due to mutations that affect either a diffusible regulator of transcription or an enzyme that modifies K-PDE. Although wild type cells contain a minor cGMP phosphodiesterase activity distinct from the major cAMP phosphodiesterase, the wild type cGMP phosphodiesterase is not susceptible to radiolabeling with [32P]cGMP; this rules out the possibility that the K30a phenotype is caused by overexpression of a wild type phosphodiesterase. We conclude that the K30a mutation produced expression of a new species of phosphodiesterase molecule that is not detectably expressed in the parental S49 wild type cell line.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1981

Cholera toxin can catalyze ADP-ribosylation of cytoskeletal proteins.

Cholera toxin catalyzes transfer of radiolabel from [32P]NAD+ to several peptides in particulate preparations of human foreskin fibroblasts. Resolution of these peptides by two-dimensional gel electrophoresis allowed identification of two peptides of Mr = 42,000 and 52,000 as peptide subunits of a regulatory component of adenylate cyclase. The radiolabeling of another group of peptides (Mr = 50,000 to 65,000) suggested that cholera toxin could catalyze ADP-ribosylation of cytoskeletal proteins. This suggestion was confirmed by showing that incubation with cholera toxin and [32P]NAD+ caused radiolabeling of purified microtubule and intermediate filament proteins.

Adenosine Diphosphate Ribose↗
PubMed1981

Fibroblast defect in pseudohypoparathyroidism, type I: reduced activity of receptor-cyclase coupling protein.

Erythrocytes of many patients with pseudohypoparathyroidism, type I (PHP-I), exhibit reduced activity of the N protein, a guanine nucleotide-binding regulatory component of hormone-sensitive adenylate cyclase. We compared N and adenylate cyclase activities and the accumulation of cAMP in fibroblasts propagated from skin biopsies of six normal subjects and seven PHP-I patients. N activities were reduced by approximately 40% in fibroblasts as well as erythrocytes of five PHP-I patients. N activities in fibroblasts from two PHP-I patients with normal erythrocyte N activities were within the normal range. These results are consistent with the hypothesis that N deficiency is generalized in tissues of most PHP-I patients and is the primary defect responsible for their resistance to metabolic effects of hormones that work by stimulating adenylate cyclase. Fibroblast N deficiency was not associated with decreases in hormone-stimulated adenylate cyclase or cAMP accumulation in fibroblasts, probably because these activities involve many potentially regulable cellular components in addition to the N protein.

Adenylyl Cyclases↗
PubMed1981

Human mutation affecting hormone-sensitive adenylate cyclase.

Hormone-sensitive adenylate cyclase contains a recently discovered protein component that is required for stimulation of cyclic AMP synthesis by hormones and guanine nucleotides. We measured this protein in erythrocyte membranes of ten patients with pseudohypoparathyroidism (PHP), using assays of its biochemical activity and of its susceptibility to radiolabeling in the presence of 32P-NAD and cholera toxin. By both assays, the protein was reduced by 50% in erythrocytes of 4 PHP patients, as compared with normal and hypoparathyroid subjects. These 4 subjects, in contrast to the 6 PHP patients (5 in one family) whose erythrocytes contained apparently normal amounts of the cyclase component, exhibited the full spectrum of skeletal abnormalities found in PHP. We conclude that partial deficiency of the guanine nucleotide regulatory protein is a biochemical marker for a subset of PHP patients. If present in other tissues, this deficiency could explain the resistance of target organs in PHP to parathormone and other hormones that work via cyclic AMP.

Adenylyl Cyclases↗