Purification and characterization of the AMP-activated protein kinase. Copurification of acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA reductase kinase activities

1. We have purified the AMP-activated protein kinase 4800-fold from rat liver. The acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase kinase activities copurify through all six purification steps and are inactivated with similar kinetics by treatment with the reactive ATP analogue fluorosulphonylbenzoyladenosine. 2. The final preparation contains several polypeptides detectable by SDS/polyacrylamide gel electrophoresis, but only one of these, with an apparent molecular mass of 63 kDa, is labelled using [14C]fluorosulphonylbenzoyladenosine. This is also the only polypeptide in the preparation that becomes significantly labelled during incubation with [gamma 32P]ATP. This autophosphorylation reaction did not affect the AMP-stimulated kinase activity. 3. In the absence of AMP the purified kinase has apparent Km values for ATP and acetyl-CoA carboxylase of 86 microM and 1.9 microM respectively. AMP increases the Vmax 3-5-fold without a significant change in the Km for either protein or ATP substrates. 4. The response to AMP depends on the ATP concentration in the assay, but at a near-physiological ATP concentration the half-maximal effect of AMP occurs at 14 microM. Studies with a range of nucleoside monophosphates and diphosphates, and AMP analogues showed that the allosteric activation by AMP was very specific. ADP gave a small stimulation at low concentrations but was inhibitory at high concentrations. 5. These results show that the AMP-activated protein kinase is the major HMG-CoA reductase kinase detectable in rat liver under our assay conditions and that it is therefore likely to be identical to previously described HMG-CoA reductase kinase(s) which are activated by adenine nucleotides and phosphorylation. The AMP-binding and catalytic domains of the kinase are located on a 63-kDa polypeptide which is subject to autophosphorylation.     

Phosphorylation of bovine hormone-sensitive lipase by the AMP-activated protein kinase. A possible antilipolytic mechanism

Hormone-sensitive lipase is phosphorylated at a single site (site 2) in vitro by the AMP-activated protein kinase, without any direct effect on the activity of the enzyme. The amino acid sequence around this site has been determined. Ca2+/calmodulin-dependent protein kinase II also phosphorylates hormone-sensitive lipase predominantly at this site, whilst cyclic-GMP-dependent protein kinase phosphorylates exclusively the regulatory site (site 1) which is also phosphorylated by cyclic-AMP-dependent protein kinase. Phosphorylation of site 2 has been found to inhibit subsequent phosphorylation and activation of hormone-sensitive lipase by the cyclic-AMP-dependent and cyclic-GMP-dependent protein kinases, indicating that site-2 phosphorylation may have an antilipolytic role in vivo.     

Negative interactions between phosphorylation of acetyl-CoA carboxylase by the cyclic AMP-dependent and AMP-activated protein kinases

We have reported previously that cyclic AMP-dependent protein kinase phosphorylates two sites on acetyl-CoA carboxylase (site 1: Arg-Met-Ser(P)-Phe, and site 2: Ser-Ser(P)-Met-Ser-Gly-Leu), while the AMP-activated protein kinase also phosphorylates site 1, plus site 3 (Ser-Ser-Met-Ser(P)-Gly-Leu), the latter being two residues C-terminal to site 2. We now report that prior phosphorylation of site 2 by cyclic AMP-dependent protein kinase prevents the subsequent phosphorylation of site 3 and the consequent large decrease in Vmax produced by the AMP-activated protein kinase. Similarly, prior phosphorylation of site 3 by the AMP-activated protein kinase prevents subsequent phosphorylation of site 2 by cyclic AMP-dependent protein kinase.     

Variation in heat shock proteins within tropical and desert species of poeciliid fishes

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30- kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.      

Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.     

Disintegration of Dung Pats from Cattle Treated with the Ivermectin Anthelmintic Bolus,or the Biocontrol Agent Duddingtonia flagrans

An experiment was performed during the grazing seasons of 1998, 1999 and 2000 to study the influence of the antiparasitic drug ivermectin and the nematophagous fungus Duddingtonia flagrans on cattle dung disintegration. The faeces originated from groups of animals that were part of a separate grazing experiment where different control strategies for nematode parasite infections were investigated. Each group consisted of 10 first-season grazing cattle that were either untreated, treated with the ivermectin sustained-release bolus, or fed chlamydospores of D. flagrans. Faeces were collected monthly on 4 occasions and out of pooled faeces from each group, 4 artificial 1 kg dung pats were prepared and deposited on nylon mesh on an enclosed pasture and protected from birds. The position of the new set of pats was repeated throughout the 3 years of the study. Each year, the dung pats were weighed 4, 6, 8 and 10 weeks after deposition and immediately afterwards replaced to their initial positions.

Results showed that there was no difference in faecal pat disintegration between groups. However, the time-lag between deposition and complete disintegration of the faeces varied significantly between deposition occasions. Dung pats disappeared within 2 weeks (visual observation) when subjected to heavy rainfall early after deposition, whereas an extended dry period coincided with faeces still remaining 12 months after deposition.

     

Artificial selection on sexual aggression: Correlated traits and possible trade-offs

Forced copulation is an extreme form of sexual aggression that can affect the evolution of sex-specific anatomy, morphology, and behavior. To characterize mechanistic and evolutionary aspects of forced copulation, we artificially selected male fruit flies based on their ability to succeed in the naturally prevalent behavior of forced matings with newly eclosed (teneral) females. The low and high forced copulation lineages showed rapid divergence, with the high lineages ultimately showing twice the rates of forced copulation as the low lineages. While males from the high lineages spent more time aggressively pursuing and mounting teneral females, their behavior toward non-teneral and heterospecific females was similar to that of males from the low lineages. Males from the low and high lineages also showed similar levels of male-male aggression. This suggests little or no genetic correlations between sexual aggression and non-aggressive pursuit of females, and between male aggression toward females and males. Surprisingly however, males from the high lineages had twice as high mating success than males from the low lineages when allowed to compete for consensual mating with mature females. In further experiments, we found no evidence for trade-offs associated with high forced mating rates: males from the high lineages did not have lower longevity than males from the low lineages when housed with females, and four generations of relaxed selection did not lead to convergence in forced mating rates. Our data indicate complex interactions among forced copulation success and consensual mating behavior, which we hope to clarify in future genomic work.