Rate zonal sedimentation of proteins in one hour or less.

Rate zonal sedimentation gives information about the shape and size of proteins, and is useful for investigating protein-protein interactions. However, rate zonal sedimentation experiments typically last approximately 1 day. In contrast, this report describes a rate zonal sedimentation method requiring 1 h or less. This was accomplished by centrifuging small density gradients (200 microliters) prepared with sucrose or OptiPrep in a fixed-angle rotor at high relative centrifugal force. By using small gradient volumes, the sample dilution that occurs with larger gradients and with many chromatographic techniques was also avoided. For a variety of proteins, plots of S20,w versus distance sedimented during centrifugation in a TLA 120.2 rotor were linear. As a practical application, sedimentation of the heterotrimeric stimulatory G protein and its dissociated alpha-subunit were determined. The results were similar to those obtained with 17- to 22-h centrifugations in an SW 50.1 rotor and agreed with previously published values. Long periods of centrifugation might preclude the study of some unstable proteins or the investigation of protein-protein interactions whose affinities are to low to survive the lengthy centrifugations required to carry out traditional rate zonal sedimentation experiments. A rate zonal sedimentation technique that rivals many chromatographic methods in celerity will help to circumvent these problems.

Does subunit dissociation necessarily accompany the activation of all heterotrimeric G proteins?

Heterotrimeric (alpha beta gamma) G proteins mediate a variety of signal transduction events in virtually every cell of every eukaryotic organism. The predominant hypothesis is that dissociation of the alpha-subunit from the G beta gamma-subunit complex necessarily accompanies the activation of these proteins, and that the alpha-subunit is primarily responsible for regulating the response of effector molecules. However, there is increasing evidence that both the alpha-subunit and the beta gamma-subunit complex function in regulating effector activity. Furthermore, data for some G proteins suggest that they function as activated heterotrimers rather than as dissociated subunits.

Chloride effects on Gs subunit dissociation. Fluoroaluminate binding to Gs does not cause subunit dissociation in the absence of chloride ion.

The stimulatory guanine nucleotide binding protein (Gs) is heterotrimeric ( alpha beta gamma), and mediates activation of adenylyl cyclase by a ligand-receptor complex. The alpha subunit of Gs (Gsalpha) has a guanine nucleotide binding site, and activation occurs when tightly bound GDP is displaced by GTP. Together, GDP and fluoroaluminate (AlF4-) form a transition state analog of GTP that activates Gs. The work of other investigators suggests that AlF4- causes subunit dissociation when it activates Gs. We have observed that in solution AlF4- did not cause Gs subunits to dissociate unless NaCl was also present. The effect of NaCl was concentration dependent (10-200 mM). Omitting F-, Al3+, or Mg2+ prevented the NaCl-induced dissociation of Gs subunits. Na2SO4 could not substitute for NaCl in causing subunit dissociation, but KCl could, suggesting that the anion was responsible for the effect. Gs subunit reassociation occurred when the concentration of Cl- was reduced even though the concentrations of AlF4- and Mg2+ were maintained. The absence of Cl- did not prevent AlF4- binding to Gsalpha. We have concluded that AlF4-, a ligand which is capable of activating G proteins, can bind to Gs in solution without causing subunit dissociation.

GTP binding to Gs does not promote subunit dissociation.

The stimulatory G protein (Gs) mediates activation of adenylyl cyclase. Gs is a heterotrimeric protein (alpha beta gamma) that is activated when guanosine triphosphate (GTP) or a non-hydrolyzable GTP analogue displaces tightly bound guanosine diphosphate (GDP) from the guanine nucleotide-binding site of the alpha-subunit (Gs alpha). Divalent cations such as magnesium are also required for Gs activation. Subunit dissociation can accompany Gs activation and is thought to be critical for this process. We investigated the effects of MgCl2 and various purine nucleotides on Gs-subunit dissociation and activation. Subunit dissociation was assayed by measuring the amount of G protein beta-subunit that was co-precipitated by Gs alpha-specific antiserum. Gs activation was determined by its ability to reconstitute adenylyl cyclase activity in S49 cyc-membranes that lack Gs alpha. High concentrations of MgCl2 caused bound GDP to dissociate from Gs and inactivated the protein unless high concentrations of GDP or GTP were present in solution. MgCl2 caused a concentration-dependent dissociation of Gs subunits. GTP gamma S (a non-hydrolyzable GTP analogue) shifted the MgCl2 concentration-response curve for subunit dissociation to lower concentrations of MgCl2, suggesting that GTP gamma S promoted subunit dissociation. On the other hand, GDP and GTP were equally effective in shifting the curve to higher concentration of MgCl2. These results suggest that GTP, the compound that activates Gs in vivo, was no more effective at promoting Gs subunit dissociation than was GDP.

Cell-free synthesis of functional type IV adenylyl cyclase.

Type IV adenylyl cyclase was synthesized in a cell-free coupled transcription and translation system. Radiolabeled type IV adenylyl cyclase was specifically immune precipitated with anti-ACIV antibodies. The molecular weight of in vitro translated type IV adenylyl cyclase was 110,000, similar to that for type IV adenylyl cyclase produced in the baculovirus system [B. Gao and A. G. Gilman, (1991) Proc. Natl. Acad. Sci. USA 88, 10178-10182]. Dimyristoyl phosphatidylcholine was required for efficient stimulation of activity by both forskolin and GS, with a maximum specific activity of 700 +/- 100 nmol cAMP.min-1.mg-1 attained with both effectors combined. Both bovine brain GS and in vitro translated GS alpha activated in vitro translated type IV adenylyl cyclase; however, G beta gamma only enhanced stimulation in the presence of in vitro translated GS alpha. Forskolin maximally activated at concentrations from 200 to 400 microM in the absence or presence of GS. The in vitro translated product was very stable as production of cAMP by forskolin/GS activated type IV adenylyl cyclase was linear for up to 90 min at 30 degrees C.

Regulation of glycogen synthase activity in isolated rat adipocytes by levamisole.

The effect of levamisole on glycogen synthase activity in isolated adipocytes was studied. The addition of levamisole to these cells resulted in an acute concentration-dependent increase in glycogen synthase activity. In contrast, epinephrine, dibutyryl cyclic AMP (DcAMP) and cysteamine decreased glycogen synthase activity. The stimulatory effect of levamisole on the activity of the enzyme was not affected by the presence of epinephrine but was diminished when either DcAMP or cysteamine was present. The results of this study suggest that levamisole increases adipocyte glycogen synthase activity by a mechanism that can be reversed by the elevation of cAMP.

Regulation of pyruvate dehydrogenase activity in rat fat pads and isolated hepatocytes by levamisole.

The effect of levamisole on the catalytic activity of the pyruvate dehydrogenase (PDH) complex in rat adipose tissue segments and isolated hepatocytes was studied. Levamisole, an anthelmintic and immunotherapeutic agent with concentration-dependent oxidant/antioxidant properties, increased the catalytic activity of the pyruvate dehydrogenase complex in these systems. The activity of this enzyme complex may be regulated in part by protein-thiol modification. The treatment of rat adipose tissue segments and isolated hepatocytes with and without levamisole (0.1-3.0 mM) for 30 min resulted in a reversible increase in the catalytic activity of this enzyme complex. Levamisole at 1.5 mM and 2.0 mM were as potent as insulin (1.0 mU/ml) in adipose tissue and as dichloroacetate (2.0 mM) in isolated hepatocytes, respectively, in increasing the catalytic activity of the PDH complex. The stimulatory effects seen with levamisole were not accompanied by decreases in adenosine triphosphate (ATP) levels. The results of the present investigation suggest that the pyruvate dehydrogenase complex in adipose tissue and liver may be useful models for studying the mechanism of action of levamisole.

Regulation of glucose transport in isolated adipocytes by levamisole.

When isolated rat adipocytes were incubated with increasing concentrations of levamisole (0.5-5 mM), basal glucose oxidation decreased by almost 50% and insulin-stimulated glucose oxidation decreased by 90%. The decrease in glucose oxidation correlated with an inhibition of glucose transport, since levamisole at 5.0 mM decreased basal 3-O-methylglucose transport by 60% and insulin-stimulated transport by 80%. Diamide-stimulated glucose transport was also inhibited approximately 80% by 5.0 mM levamisole. Levamisole at concentrations up to 5.0 mM had no effect on phosphofructokinase activity. The present results suggest that levamisole inhibits glucose utilization by inhibiting glucose transport in a concentration-dependent manner.