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R G Kemp

Publications and source records attributed to R G Kemp.

At least 55 records · Page 3Linked to original sources
PubMed1988

Phosphofructokinase in the rat nervous system: regional differences in activity and characteristics of axonal transport.

The distribution of phosphofructokinase (PFK) in gray and white matter regions of the rat nervous system was evaluated. Determinations of PFK activity revealed that cell body enriched regions (sensorimotor cortex) had a significantly higher level of activity than axonal regions (sciatic nerve, dorsal roots, and optic nerve). The level of PFK activity was also significantly higher in central axons (optic nerve) than in peripheral axons (sciatic nerve). Differences in PFK activity could be largely attributed to differences in tissue content of the enzyme rather than to differences in the types of PFK isozymes present. Cortex contained significantly larger amounts of PFK relative to total protein than did peripheral nerve. However, purification of PFK revealed that all three of the PFK isozymes, C (86 kd), A (84 kd), and B (80 kd), were present in both cortex and sciatic nerve. Both SDS/PAGE and immunoblotting studies using PFK isozyme-specific antibodies demonstrated that the relative proportions of the three PFK isozymes were similar in cell body and axonal regions of the nervous system. The PFK-C and PFK-A isozymes each comprised about half the total and only small amounts of the PFK-B isozyme were present in both regions. However, immunoprecipitation experiments suggested that quantitatively different proportions of the possible PFK hybrids (tetramers) may be distributed between axonal and cell body regions. The transport of PFK was examined in this study and PFK was identified in slow component b (SCb) of axonal transport. SCb moves at a rate of 2-4 mm/day in rat axons and is known to contain several other enzymes of intermediary metabolism as well as actin. The finding that PFK, the rate limiting enzyme in glycolysis, is present in SCb lends support to the hypothesis that glycolytic enzymes are not freely diffusing proteins in axons but, instead, are present as organized assemblies that have long-term, yet flexible, associations with structural elements of the cytoplasm.

Animals↗
PubMed1987

Identification of highly reactive cysteinyl and methionyl residues of rabbit muscle phosphofructokinase.

The reactivity of the 16 thiol groups of rabbit skeletal muscle phosphofructokinase has been studied extensively over the past 20 years. Several of these thiols show high reactivity with a variety of reagents, display differential reactivity in the presence of allosteric ligands and substrates, and appear to be important to function because their modification changes activity and regulatory properties. In the present study, the location in the primary structure of several highly reactive thiol groups has been established by reaction with [14C]iodoacetate. In the course of these studies, 2 methionyl residues that are located at or near proposed ligand-binding sites are readily carboxymethylated by iodoacetate. In addition to confirming the presence of the most reactive thiol group at sequence position 88, a thiol protected from reaction by the presence of fructose-6-P and cyclic AMP has been found at position 169. Cysteine 169 is close to a residue important to the binding of fructose-6-P in the homologous structure from Bacillus stearothermophilis phosphofructokinase. The modification of Cys-169 brings about extensive, but not total, loss of activity. Another cysteine, at position 232, was found to be highly reactive also. Substrate provided partial protection against carboxymethylation at this position. Carboxymethylation of enzyme restricted to methionines 74 and 173 brought about no changes in the total activity or in the ATP inhibition profile of the enzyme. This is significant since position 74 was projected on the basis of the homologous procaryotic structure to be important in the binding of nucleotide to the allosteric site.

Allosteric Site↗
PubMed1987

Amino acid sequence at the citrate allosteric site of rabbit muscle phosphofructokinase.

Previously, this laboratory has demonstrated [Colombo, G., & Kemp, R. G. (1976) Biochemistry 15, 1774-1780] that under appropriate conditions the citrate inhibitory binding site of rabbit skeletal muscle phosphofructokinase can be covalently modified by using pyridoxal phosphate and sodium borohydride. In the current study, phosphofructokinase was modified by [3H]pyridoxal phosphate and sodium borohydride with or without the addition of citrate to protect the ligand binding site. The modified proteins were digested with trypsin, and the peptides were separated by high-pressure liquid chromatography. A comparison of the tryptic chromatographic profiles showed that while the label was broadly distributed among nine peaks in the elution profile of the enzyme modified in the presence of the protective ligand, a single peptide contained 70% of the total radioactivity of the enzyme modified in the absence of citrate. This peptide was presumed to contain at least part of the citrate inhibitory site of the enzyme. The sequence of the peptide was determined and shown to match with positions 528-536 of phosphofructokinase with the modified residue being Lys-529. A comparison of the sequence with that of procaryotic phosphofructokinase indicated that a homologous residue in the enzyme from Bacillus stearothermophilis is critical to an allosteric site. A second peptide that was the most abundant labeled peptide in the digest of the enzyme modified in the presence of citrate was found to be identical with the second most abundant peptide of the digest from the unprotected enzyme. This peptide corresponded to residues 681-692 with the lysine at position 684 being the site of phosphopyridoxylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Site↗
PubMed1987

Desensitization of muscle phosphofructokinase to ATP inhibition by removal of a carboxyl-terminal heptadecapeptide.

Brief exposure of rabbit skeletal muscle phosphofructokinase to Staphylococcus aureus V8 protease results in the release from the enzyme of two carboxyl-terminal peptides from the enzyme that together comprise 17 amino acids. The rate of proteolysis was increased in the presence of activators of the enzyme, ammonium sulfate and AMP, and was decreased in the presence of allosteric inhibitors, MgATP and citrate. No change was observed in the maximal velocity of the modified enzyme or in its affinity for substrates when assayed under noninhibitory conditions. Equilibrium binding studies indicated no change in the affinity of the modified enzyme for its allosteric activator, AMP. On the other hand, the proteolyzed enzyme exhibited markedly reduced inhibition by ATP and by citrate. ATP inhibition was observed only at very high concentrations of ATP. Fructose-6-P saturation curves of the modified enzyme were nearly hyperbolic. The interaction coefficient deduced from the slope of a Hill-type plot was 1.2 under conditions that yielded a coefficient of 3.0 with native phosphofructokinase. Binding studies verified a decrease in affinity for ATP for at least one of the ATP binding sites. Because kinetic studies showed no effect on the Km for ATP, it was concluded that the affinity was decreased at the MgATP inhibitory site only. Proteolytic removal of the terminal 8 residues from the enzyme produced no striking change in regulatory properties, thus focusing the critical region to the sequence His-Ala-His-Leu-Glu-His-Ile-Ser-Arg. It is suggested that the three histidine residues clustered in the carboxyl terminus may contribute to the binding of MgATP to the inhibitory site.

Adenosine Monophosphate↗
PubMed1987

Isolation and sequence determination of an active site peptide of rabbit muscle pyruvate kinase.

Rabbit muscle pyruvate kinase was inactivated by 2', 3'-dialdehyde ADP with the incorporation of one molecule of reagent per enzyme subunit. The inactivated protein was digested with trypsin after reduction and carboxymethylation. The labeled peptide was isolated by gel filtration and further purified by HPLC. The peptide was sequenced both by liquid-phase and gas-phase automatic Edman degradation. A 34-residue peptide was obtained. This peptide is identical to a tryptic peptide labeled with trinitrobenzenesulfonate, isolated and sequenced by Johnson et al. (Biochem. Biophys. Res. Commun. (1979) 90, 525-530) from bovine muscle pyruvate kinase. Available evidence suggests that dialdehyde ADP labels the enzyme at the same lysine in position 25 of the peptide, as found by Johnson et al. The high homology between the isolated peptide and regions of other pyruvate kinases from low to high eukaryotes supports the idea that this peptide is related to the enzyme active site.

Adenosine Diphosphate↗
PubMed1985

Isolation and characterization of phosphofructokinase C from rabbit brain.

Phosphofructokinase from rabbit brain consists of hybrids of the A, B, and C isozymes. Phosphofructokinase C was isolated from a purified mixture of such hybrids in a 2-step procedure. In the first step, phosphofructokinase B was removed by chromatography on DEAE-Sephadex. In the second step, subunits of phosphofructokinases A and C were separated by dissociation at pH 5.0 followed by chromatography on carboxymethylcellulose. The separated isozymes were then reassociated by neutralization. Phosphofructokinase C was structurally distinct from phosphofructokinases A (obtained from muscle or brain) and B (obtained from liver) as shown by one-dimensional chymotryptic and staphylococcal V8 protease fingerprints of all three isozymes. In addition, phosphofructokinase C cross-reacted weakly or not at all with antisera raised against phosphofructokinase B or phosphofructokinase A. Phosphofructokinase C was also kinetically distinct from the A and B isozymes. The C isozyme was more sensitive than the A isozyme but less sensitive than the B isozyme to inhibition by ATP, was less sensitive than the A isozyme but more sensitive than the B isozyme to inhibition by citrate, and was less sensitive than either of the other two isozymes to activation by inorganic phosphate, AMP, and fructose 2,6-bisphosphate. The self-association properties of phosphofructokinase C differed from those of the A and B isozymes in that at pH 8.0, the C isozyme did not form oligomers larger than a tetramer under conditions where the other two isozymes did. Thus the properties of phosphofructokinase C are in general quite distinct from those of the other two phosphofructokinase isozymes.

Adenosine Monophosphate↗
PubMed1985

Exogenous ATP enhances calcium influx in intact thymocytes.

Recent observations have indicated that exogenous adenosine triphosphate (ATP) may influence lymphocyte functions such as proliferation and cytoxicity. Here we report a novel activity of extracellular ATP--it specifically increases Ca2+ uptake in murine lymphocytes. ATP added to thymocytes increases the rate of [45Ca2+] uptake by up to 20-fold. The increased rate is seen with ATP concentrations as low as 500 microM and is half-maximal at approximately 2 mM ATP. The magnitude of stimulation by ATP is dependent on Mg2+ concentration, and ATP-Mg2+ complex is probably the true activator. Of the high-energy phosphate-containing compounds tested, including deoxy-ATP, only GTP showed a modest stimulation of calcium uptake. ADP, AMP, cyclic AMP, and adenosine did not significantly increase calcium uptake. Cellular integrity as indicated by trypan blue exclusion and ethidium bromide/acridine orange staining was unaffected by ATP. Ca2+ influx is the major mode of action of ATP in raising intrathymocyte Ca2+ levels, because neither the Ca2+ efflux nor the [45Ca2+]-Ca2+ exchange was significantly altered in the presence of ATP. Verapamil, a Ca2+ channel blocking agent, could not prevent the ATP effect, suggesting that ATP may be acting by a mechanism other than the voltage-dependent Ca2+ channel. An analysis of intracellular and extracellular ATP levels by chemiluminescence assay indicated no significant ATP entry into intact lymphocytes. Also, ATP added to the medium containing thymocytes was destroyed (approximately 50% by 20 min). The nonhydrolyzable ATP analogs, AMPPCP and AMPPNP, were unable to stimulate a significant amount of Ca2+ uptake, suggesting the involvement of a cell surface phosphotransferase activity. This was supported by the demonstration of a threefold to fivefold increase in the labeling of protein and phospholipid fractions obtained from intact thymocytes exposed to [gamma 32P]ATP for 30 min. Ca2+ is believed to play an important role in a variety of lymphocyte functions, including mitogenesis and natural killer cell activity. The data herein thus provide a potential mechanism for the action of exogenous ATP on these lymphocyte functions.

Adenosine Triphosphate↗
PubMed1984

Isozyme composition and phosphorylation of brain phosphofructokinase.

Rabbit brain phosphofructokinase was purified to homogeneity by a rapid procedure involving affinity chromatography and gel filtration. The enzyme consists of hybrids of the three phosphofructokinase subunit types C, A, and B. The molecular weights of these subunits are 86,000, 84,000, and 80,000, respectively; they are present in brain phosphofructokinase in a ratio of approximately 5:4:1.5. The enzyme as isolated from rabbit brain contains 0.16-0.18 mol phosphate per mole of subunit; another 0.4-0.5 mol phosphate per mole subunit can be incorporated in vitro in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase. The initial rate of phosphorylation is increased by fructose 2,6-bisphosphate or AMP and decreased by citrate or high concentrations of ammonium sulfate. All three subunit types are phosphorylated in vitro, and the phosphorylation site on each subunit is sensitive to cleavage by trypsin at a terminal region of each subunit. However, these sites show different relative rates of phosphorylation in vitro in the presence of ammonium sulfate. In vitro phosphorylation of brain phosphofructokinase had no affect on specific activity, inhibition by ATP, or activation by fructose 2,6-bisphosphate.

Animals↗
PubMed1984

Hepatic fructose 2,6-bisphosphate in rats with peritonitis and septic shock.

The possible role of inhibited gluconeogenic enzymes in rat liver during preterminal peritonitis septic shock was investigated. There was no difference in maximal activity of the enzymes phosphofructokinase and fructose biphosphatase in septic and control, fasted rats. Rats with sepsis showed a decrease in hexose monophosphates and an increase in fructose biphosphate. There was an unexpected increase in fructose 2,6-bisphosphate despite the hyperglucagonemic state of sepsis. This suggested a dissociation in the coordination of extracellular hormonal and intracellular effector mechanisms in the control of glucose metabolism during the preterminal phase of septic shock. This dissociation may be responsible for the metabolic dyshomeostasis in septic shock.

Animals↗
PubMed1983

Binding of hexose bisphosphates to muscle phosphofructokinase.

On the basis of kinetic activation assays, the apparent affinity of muscle phosphofructokinase for fructose 2,6-bisphosphate was about 9-fold greater than that for fructose 1,6-bisphosphate, which in turn was about 10 times higher than that for glucose 1,6-bisphosphate. Equilibrium binding experiments showed that both fructose bisphosphates bind to phosphofructokinase with negative cooperativity; the affinity for fructose 2,6-bisphosphate was about 1 order of magnitude greater than the affinity for fructose 1,6-bisphosphate. Binding of fructose 2,6-bisphosphate to phosphofructokinase was antagonized by fructose 1,6-bisphosphate and glucose 1,6-bisphosphate and vice versa. Both fructose bisphosphates promoted aggregation of the enzyme to higher polymers as indicated by sucrose density gradient centrifugation. Other indicators of phosphofructokinase conformation such as thiol reactivity and maximum activation of in vitro phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase gave identical results in the presence of fructose 2,6-bisphosphate, fructose 1,6-bisphosphate, or glucose 1,6-bisphosphate, indicating a common conformation is produced by all three ligands. It is concluded that the sugar bisphosphates bind to a single site on the enzyme.

Animals↗
PubMed1983

Allosteric regulatory properties of muscle phosphofructokinase.

We have reviewed the allosteric regulatory properties of skeletal muscle phosphofructokinase and recent results on the phosphorylation of this enzyme. The number and affinities of various ligand binding sites are described, and a simple three state model is presented to explain the kinetic and ligand-binding properties of the enzyme. Data describing a lack of fit to a concerted transition model are presented. The widespread occurrence of partial phosphorylation of phosphofructokinase at a specific site near the carboxyl terminus is documented, as well as the lack of significant kinetic consequences of such phosphorylation.

Adenosine Triphosphate↗
PubMed1983

Lectin- and ionophore-stimulated Ca2+ influx in murine lymphocytes: inhibition by disialoganglioside.

Gangliosides suppress lymphocyte mitogenesis when added exogenously to the cells. On the premise that the mechanism of ganglioside action may be an interference with primary induction events, mitogen-induced 45Ca2+ influx in murine lymphocytes was studied. Disialoganglioside (GD1a) at physiopathological concentrations inhibits concanavalin A-induced 45Ca2+ uptake as well as blast transformation. The suppressive action of GD1a is both concentration dependent (50% suppression at 13 microM) and very rapid (within 1 min). GD1a is not cytotoxic nor does it significantly alter the rate of Ca2+ efflux. The uptake studies were extended to A23187, a compound with mitogenic and specific divalent cation ionophore activities. Ca2+ uptake by lymphoid cells from AKR/J, Swiss, and CBA mice is stimulated by A23187; and GD1a, in a dose-dependent manner, inhibits the ionophore-induced 45Ca2+ influx. Pretreatment of thymocytes with GD1a renders the cells greatly insensitive to the subsequent ionophore activity of A23187. The results suggest that exogenous gangliosides may function as an inhibitor of some of the mitogen-triggered early events, including Ca2+ metabolism, and thus influence the immunological behavior of intact lymphoid cells.

Animals↗
PubMed1982

Properties of phospho and dephospho forms of muscle phosphofructokinase.

Rabbit muscle phosphofructokinase, phosphorylated either in vivo or in vitro, can be dephosphorylated by bovine intestinal alkaline phosphatase. Small but consistent differences in the allosteric regulatory properties of the phospho and dephospho forms are seen with the pattern in the direction of the phospho form being more inhibited. In vitro phosphorylated phosphofructokinase is more sensitive to allosteric inhibition by ATP and citrate and less sensitive to activation by AMP, glucose 1,6-bisphosphate, and inorganic phosphate than untreated or dephosphorylated phosphofructokinase. Fructose 2,6-bisphosphate activates muscle phosphofructokinase with a K alpha less than 1 microM. The phospho-enzyme is less sensitive to activation by the bisphosphate. On the other hand, there are no differences in the sedimentation properties under a variety of conditions or in the stability of phosphorylated and dephosphorylated phosphofructokinases as shown by sucrose density gradient centrifugation and low pH inactivation, respectively.

Adenosine Monophosphate↗
PubMed1982

Age-related changes in 5'-nucleotidase and alkaline phosphatase activities in mouse thymocytes.

Enzymatic activities of thymocytes isolated from Swiss albino mice were studied at various ages from immediately post weaning until 100 weeks of age, approaching the life expectancy of these animals. Between 5 and 10 weeks of age, the activities of lactate dehydrogenase and alkaline phosphatase decreased to a level that was maintained throughout the remainder of the aging profile. Neutral beta-glycerophosphatase (pH 7.5) activity in a thymus membrane preparation was similar in all age groups. The activity of membrane-bound 5'-nucleotidase, that is, AMP-hydrolyzing activity inhibited by 100 microM alpha, beta-methyleneadenosine 5'-diphosphate, progressively increased as a function of age, indicating thymocyte population changes occurring very late in life. In thymocytes of the oldest mice examined (100 weeks of age), 5'-nucleotidase specific activity was approximately ten-fold greater than the activity found in 5-week-old mice. Thus, membrane-bound 5'-nucleotidase activity in thymocytes increased markedly as a function of age in Swiss albino mice; yet several other enzymatic activities, including alkaline phosphatase, remained relatively unchanged in mature mice.

Aging↗