Evidence for the existence of three or more slow phases in the refolding of ribonuclease A and some characteristics of the phases

The slow refolding kinetics of RNase A have been analyzed, by using a nonlinear least-squares program for deconvoluting the kinetic phases and applying statistical tests for quality of fit. It is found that a minimum of three slow phases are required to fit the kinetic data properly, and this is true whether the method of detection is absorbance of fluorescence. Since the number of phases and the relaxation times for each phase are independent of the method of detection, it is concluded that the same three rate-limiting processes are seen by absorbance and fluorescence. These phases correspond to the XY, CT, and ct phases described in our earlier studies. The fact that fluorescence-detected kinetics are somewhat slower than absorbance-detected kinetics is a trivial effect due not to differences in relaxation times but to the fact that the amplitude of the CT phase is enhanced in fluorescence measurements, at the expense of the faster XY phase, because of intrinsic fluorescence changes associated with the isomerization of proline-93. By use of a new double-jump technique [Schmid, F.X., Grafl, R., Wrba, A., & Beintema, J.J. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 872], it is shown that proline-93 isomerizes as the rate-limiting step in only one of the three phases, the CT phase, and that this phase involves only 25-30% of the RNase molecules. There is still no indication as to the molecular events that occur in the large, ammonium sulfate dependent XY phase, which is the pathway for formation of the nativelike intermediate.     

Effect of Clostridium perfringens Beta Toxin on Blood Pressure of Rats

Guanethidine treatment or adrenal medullectomy significantly inhibited the elevation in blood pressure induced by Clostridium perfringens beta toxin, and the combination of the two drastically reduced the pressure rise, to less than 19% of that in control rats. When rats were pretreated with tetrodotoxin or hexamethonium, the toxin-evoked rise was significantly inhibited. Elevation in blood pressure induced by the toxin in spinal rats tended to be less than that in control rats. When investigated by a microscopical technique, arteriolar constriction in the mesenteric vasculature was observed after the blood pressure elevation induced by the toxin reached a maximum. Blood flow in the skin decreased with an increase in blood pressure following intravenous injection of the toxin. It is concluded that beta toxin acts on the autonomic nervous system and produces arterial constriction.     

Hydrolysis of dansyl-peptide substrates by leucine aminopeptidase: origin of dansyl fluorescence changes during hydrolysis

The origin of the fluorescence changes observed in stopped-flow experiments of the hydrolysis of three 5-(dimethylamino)naphthalene-1-sulfonyl-(dansyl) peptide substrates by porcine kidney cytosol leucine aminopeptidase has been investigated. The substrates used all have the potential to accept energy from aromatic residues of the enzyme via resonance energy transfer when they are bound as enzyme-substrate complexes, indicating that fluorescence changes due to the buildup and decay of such intermediates are possible. However, the fluorescence of these substrates differs from that of the products, and direct excitation of their dansyl groups during hydrolysis can also be responsible for the observed fluorescence changes due to changes in the concentrations of free substrate and product. The dansyl fluorescence changes observed with excitation wavelengths near 280 nm are not accompanied by quenching of the enzyme fluorescence, as would be expected if there were enzyme-to-substrate energy transfer. The magnitude of the maximal fluorescence change at a fixed concentration of substrate is also independent of the enzyme concentration. Furthermore, the excitation profile for the fluorescence changes shows that they arise from direct excitation of the dansyl group. Thus, there is no energy transfer in these reactions, and the fluorescence changes observed arise from direct excitation of the dansyl group and reflect the instantaneous concentration of substrate. This behavior contrasts sharply with that for the reaction of carboxypeptidase A with dansyl-Gly-Tyr, which has been studied as a positive control for an energy-transfer system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a novel 14 kDa bile acid-binding protein from rat ileal cytosol

A 14 kDa polypeptide in rat ileal cytosol has been identified as the major intestinal cytosolic bile acid-binding protein (I-BABP) by photoaffinity labeling with the radiolabeled 7,7-azo derivative of taurocholate (7,7-azo-TC). To further characterize I-BABP, the protein was purified by lysylglycocholate Sepharose 4B affinity and DE-52 anion-exchange chromatography. The purified I-BABP contained a single 14 kDa band on SDS-PAGE. The 14 kDa protein showed a 26-fold increase in binding affinity for [3H]7,7-azo-TC compared to cytosolic protein. Immunoblotting of protein fractions separated by affinity chromatography showed that neither liver fatty acid binding protein (L-FABP) nor intestinal fatty acid binding protein (I-FABP) bind to the affinity column and that the 14 kDa protein which bound to the column and was subsequently eluted with detergent did not cross-react with anti-L-FABP or anti-I-FABP. The 14 kDa protein labeled with [3H]7,7-azo-TC was radioimmunoprecipitated from cytosol by rabbit antiserum raised against purified I-BABP. I-BABP was shown to have a blocked N-terminus; however, its mixed internal sequence generated from cyanogen bromide-cleaved protein and amino acid composition indicated that it was related to (although clearly distinct from) both I-FABP and L-FABP. These studies have isolated a 14 kDa bile acid-binding protein from rat ileal cytosol which is immunologically and biochemically distinct from I-FABP and L-FABP.     

Adipocyte differentiation of 3T3-L1 cells involves augmented expression of a 43-kDa plasma membrane fatty acid-binding protein.

A previously described 43-kDa plasma membrane fatty acid-binding protein (FABPPM) was not observed by immunohistochemical methods in proliferating 3T3-L1 fibroblasts. However, it was detectable in plasma membranes by the second day of confluent growth, prior to accumulation of visible lipid droplets, and was strongly expressed in 8-day differentiated adipocytes. These observations were confirmed by extraction of plasma membrane proteins and subsequent immunoblotting. Kinetics of initial [3H]oleate uptake by both fibroblasts and adipocytes consisted of the sum of a saturable and a non-saturable component. During differentiation the saturable component increased progressively. Vmax increased from 3 to 25 to 110 pmol.s-1.mg cell protein-1 between the fibroblast, the 4-day, and 8 day adipocyte stages; Km was 24 nM in fibroblasts and approximately 55 nM in both 4- and 8-day differentiated adipocytes. By contrast, the rate constant for nonsaturable oleate influx decreased progressively from 0.026 to 0.010 ml.s-1.mg protein-1 between the fibroblast and 8 day adipocyte stages. In 8-day adipocytes saturable oleate uptake was inhibited by up to 55% by antibodies against rat liver FABPPM; these antibodies had no effect on uptake of 2-deoxyglucose or the medium chain fatty acid octanoate. They also had no effect on oleate uptake by fibroblasts. These studies support the hypothesis that FABPPM is a component of a saturable transport mechanism for long chain fatty acids.     

How costly is clutch formation in the Audouin's Gull Larus audouinii?

During the Audouin's Gull's breeding season at the Ebro Delta in 1993, 24 fresh eggs from eight three-egg clutches (modal clutch-size) were collected at the peak of the laying period. Eggs were processed to obtain formalin-fixed yolks, which were halved and stained using the potassium dichromate method. Digitized images of the yolks were examined to assess the daily rates of yolk deposition. We used these data in combination with egg compositional analysis to build a model of energy demands during the formation of an average clutch in Audouin's Gull. To show how the different parameters of clutch formation affect the daily energy investment peak, we performed a simulation analysis in which the rapid yolk development (RYD) period, the follicle triggering interval (FTI), the laying interval (LI) and the albumen synthesis period (ASP) were allowed to vary simultaneously. In our sample, the mean RYD period was seven days with a range from six to eight days. There were no significant differences in yolk volume among eggs in a clutch, but albumen volume was significantly smaller in third eggs. According to our model the albumen synthesis of the a-egg coincides with the energy demand peak for clutch formation. This peak represents an increase by ca. 42% in female energy requirements. Values obtained from the simulation analysis showed that only the ASP of the a-egg and the RYD durations of the second and third follicles produced noticeable reductions in peak energy investment. We predict that in gulls, whose laying intervals seem to be kept constant, significant increases of the durations of the RYD periods of second and third eggs, or even significant reductions of yolk size of these eggs, may operate simultaneously to match the energy demands during clutch formation to the prevailing food conditions.