Protein extraction by reverse micelles: a study of the factors affecting the forward and backward transfer of alpha-chymotrypsin and its activity

alpha-chymotrypsin is taken as a model protein to investigate three aspects of the protein extraction by reverse micelles: (1) the comparison between the two forward transfer techniques, i.e., the liquid-liquid and the solid state-liquid transfer; (2)the back-transfer, i.e., the capability of the protein to be recovered from the micellar solution; and (3) the maintainance of the enzyme activity at the end of the extraction cycle. Concerning the forward transfer from the liquid phase, we study first the effect of salt initially present in the aqueous phase on the equilibrium concentration of the extracted species; further, we study the forward protein extraction from the solid state, and the effect of pH, salt, and protein concentration on the transfer efficiency. Concerning the back transfer, we find the somewhat surprising result, that the percentage of protein back-extraction depends on the type and concentration of salt used for the forward transfer. Preliminary data concerning an alternative method for the back-transfer using silica gel to liberate the protein from the micellar environment, are presented. Finally, it is found that the enzyme activity depends again on the type and concentration of salt used for the forward transfer.     

Extracellular Concentrations of Dopamine and Metabolites in the Rat Caudate After Oral Administration of a Novel Catechol-O-Methyltransferase Inhibitor Ro 40–7592

The effect of the systemic administration of a novel, orally active, catechol-O-methyltransferase (COMT) inhibitor, Ro 40-7592, on the in vivo extracellular concentrations of dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), was studied by transcerebral microdialysis in the dorsal caudate of freely moving rats. Ro 40-7592 (at doses of 3.0, 7.5, and 30 mg/kg p.o.) elicited a marked and long-lasting reduction of HVA, and at doses of 7.5 and 30 mg/kg, an increase of DOPAC output, but it failed to increase DA output. The administration of L-beta-3,4-dihydroxyphenylalanine (L-DOPA, 20 and 50 mg/kg p.o.) with a DOPA decarboxylase inhibitor (benserazide) increased both HVA and DOPAC output, but failed to modify significantly extracellular DA concentrations in dialysates; in contrast, combined administration of L-DOPA+benserazide with Ro 40-7592 (30 mg/kg p.o.) resulted in a significant increase in DA output. Ro 40-7592 prevented the L-DOPA-induced increase in HVA output and markedly potentiated the increase in DOPAC output. To investigate to what extent the increase in extracellular DA concentrations was related to an exocitotic release, tetrodotoxin (TTX) sensitivity was tested. Addition of TTX to Ringer, although abolishing DA output in the absence of L-DOPA, partially reduced it in the presence of L-DOPA+Ro 40-7592 and even more so after L-DOPA without the COMT inhibitor. The results of the present study suggest that metabolism through COMT regulates extracellular concentrations of DA formed from exogenously administered L-DOPA but not of endogenous DA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gating kinetics of batrachotoxin-modified Na+ channels in the squid giant axon. Voltage and temperature effects.

The gating kinetics of batrachotoxin-modified Na+ channels were studied in outside-out patches of axolemma from the squid giant axon by means of the cut-open axon technique. Single channel kinetics were characterized at different membrane voltages and temperatures. The probability of channel opening (Po) as a function of voltage was well described by a Boltzmann distribution with an equivalent number of gating particles of 3.58. The voltage at which the channel was open 50% of the time was a function of [Na+] and temperature. A decrease in the internal [Na+] induced a shift to the right of the Po vs. V curve, suggesting the presence of an integral negative fixed charge near the activation gate. An increase in temperature decreased Po, indicating a stabilization of the closed configuration of the channel and also a decrease in entropy upon channel opening. Probability density analysis of dwell times in the closed and open states of the channel at 0 degrees C revealed the presence of three closed and three open states. The slowest open kinetic component constituted only a small fraction of the total number of transitions and became negligible at voltages greater than -65 mV. Adjacent interval analysis showed that there is no correlation in the duration of successive open and closed events. Consistent with this analysis, maximum likelihood estimation of the rate constants for nine different single-channel models produced a preferred model (model 1) having a linear sequence of closed states and two open states emerging from the last closed state. The effect of temperature on the rate constants of model 1 was studied. An increase in temperature increased all rate constants; the shift in Po would be the result of an increase in the closing rates predominant over the change in the opening rates. The temperature study also provided the basis for building an energy diagram for the transitions between channel states.     

Immunoaffinity purification and partial amino acid sequence analysis of catechol-O-methyltransferase from pig liver

Monoclonal antibodies (mAbs) against the soluble form (S-COMT) of catechol-O-methyltransferase (COMT, EC 2.1.1.6) were produced using a purified preparation of the enzyme from pig liver as antigen. The selected monoclonal antibodies recognized the enzyme with different capacities. One of them (Co60-1B/7) showed a significant cross reaction with S-COMT from rat and human liver. A protein band of 23 kDa was recognized by the mAbs on Western blots of the soluble fraction of pig liver. The mAbs were also able to recognize the membrane-bound form of the enzyme, which was found to be mainly localized in the microsomal fraction of pig and rat liver as well as of the human hepatoma cell line Hep G2. The protein bands detected in microsomes had a molecular mass of 26 kDa in pig and rat liver and displayed a slightly higher molecular mass (29 kDa) in the Hep G2 cell line. A single step method for the immunoaffinity purification of pig liver S-COMT was developed by using a Sepharose 4B column to which the mAb Co54-5F/8 was covalently coupled. Acid elution conditions were optimized to obtain the enzyme in active form with a good yield. SDS-PAGE analysis of the purified preparation revealed a single protein band with a molecular mass of 23 kDa with 154-fold enrichment in enzyme activity over the starting material. Since the N-terminus was blocked, purified enzyme preparations were cleaved with trypsin. Two fragments of 22 and 33 amino acids in length could be sequenced by Edman degradation.     

Activity feedback to the mammalian circadian pacemaker: influence on observed measures of rhythm period length.

In the mouse, activity is precisely timed by the circadian clock and is normally most intense in the early subjective night. Since vigorous activity (e.g., wheel running) is thought to induce phase shifts in rodents, the temporal placement of daily exercise/activity could be a determinant of observed circadian rhythm period. The relationship between spontaneous running-wheel activity and the circadian period of free-running rhythms was studied to assess this possibility. With ad libitum access to a running wheel, mice exhibited a free-running period (tau) of 23.43 +/- 0.08 hr (mean +/- SEM). When running wheels were locked, tau increased (23.88 +/- 0.04 hr, p less than 0.03), and restoration of ad libitum wheel running again produced a shorter period (tau = 23.56 +/- 0.06 hr, p less than 0.05). A survey of free-running activity patterns in a population of 100 mice revealed a significant correlation between the observed circadian period and the time of day in which spontaneous wheel running occurred (r = 0.7314, p less than 0.0001). Significantly shorter periods were observed when running was concentrated at the beginning of the subjective night (tau = 23.23 +/- 0.04), and longer periods were observed if mice ran late in the subjective night (tau = 23.89 +/- 0.04), F (1, 99) = 34.96, p less than 0.0001. It was previously believed that the period of the circadian clock was primarily responsive to externally imposed tonic or phasic events. Systematic influences of spontaneous exercise on tau demonstrate that physiological and/or behavioral determinants of circadian timekeeping exist as well.     

Purification and characterization of a microbial, NADP-dependent bile acid 7 alpha-hydroxysteroid dehydrogenase

A constitutively expressed 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) has been purified over 1200-fold, to apparent homogeneity, from an intestinal anaerobic bacterium. The purified protein had a subunit molecular mass of 32 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sepharose CL-6B gel filtration gave a native molecular mass estimate of 124 kDa, suggesting that this enzyme existed as a tetramer of identical subunits. Sulfhydryl reactive compounds were potent inhibitors of 7 alpha-HSDH activity, however, metal ion chelators had no effect upon catalytic activity. The purified enzyme was highly NADP-dependent. Bile acid substrate utilization studies revealed that the enzyme was specific for the oxidation of an unhindered 7 alpha-hydroxyl group. A wide variety of bile acids and analogs were used as substrates including glycine and taurine conjugates, and methyl esters, amines, and bile alcohols. The purified 7 alpha-HSDH obeyed Michaelis-Menten kinetics. Hanes plots of substrate saturation kinetics revealed that most bile acid substrates had Km values ranging from 4 to 20 microM, while Vmax was 601 and 674 mumol/min/mg in the direction of bile acid oxidation and reduction, respectively. Primary kinetic plots and product inhibition patterns were consistent with an ordered sequential mechanism, with NADP(H) binding first. The N-terminal amino acid sequence analysis of the purified enzyme revealed a striking homology to several short, non-zinc alcohol/polyol dehydrogenases and a putative, cholate-inducible, hydroxysteroid dehydrogenase from the same organism. The high specific activity together with the stability, substrate range, and ease of purification, make this enzyme an excellent candidate for use in quantitating primary bile acids both in laboratory and clinical samples. Spectrofluorometry allowed for the quantitation of as little as 10 nM of both free and conjugated primary bile acids.     

Pyrrolizidine alkaloids sequestered by Solomon Island Danaine butterflies. The feeding preferences of the Danainae and Ithomiinae

The plant sources and identity of pyrrolizidine alkaloids sequestered by six species of Solomon Island Danainae have been investigated. The secretions of the pheromone-disseminating hairpencils of the males have also been analysed and the componenets, including pyrrolizidine metabolites, have been identified. The first confirmed example of Danaine larvae, rather than adults, acquiring pyrrolizidine alkaloids is reported and its relevance to hypotheses concerning the origin of the requirement of Danaine and Ithomiine butterflies for pyrrolizidine alkaloids is discussed.     

Balbiani ring RNA in the cytoplasm of Chironomus thummi salivary gland cells

In this paper experimental results on the size, transport and stability of cytoplasmic Balbiani ring RNA and on its appearance in polysomes are presented. Cytoplasmic RNA of salivary gland cells from Chironomus thummi contains two large RNA fractions of about 20×10⁶ dalton and 10×10⁶ dalton in size. These RNA fractions correspond both to Balbiani ring BR 1 RNA and BR 2 RNA and are apparently transported from nucleus into cytoplasm without a significant size reduction. Chase experiments illustrate a great stability of giant cytoplasmic Balbiani ring RNA molecules and exclude the possibility of a precursor-product relationship between these and smaller BR RNA molecules also found in cytoplasm. A part of giant cytoplasmic Balbiani ring RNA molecules is bound to poly(U)-sepharose columns and should, therefore, contain poly(A)-sequences. — Polysomes of salivary gland cells extracted by a gentle lysis procedure and centrifuged through sucrose gradients are characterized by a rather broad sedimentation profile. Polysome sizes up to about 800 S have been detected, but in no case a distinct polysome fraction corresponding in size to Balbiani ring RNA has been observed. Hybridization of polysomal RNA with salivary gland chromosomes in situ resulted in labelling of both Balbiani rings BR 1 and BR 2.     

Baclofen and γ-hydroxybutyrate: similar effects on cerebral dopamine neurones

Baclofen (20 mg/kg) caused an increase in the content of homovanillic acid (HVA) and dopamine (DA) in rat brain 2–3 h after drug injection without appreciable changes in the level of other monoamines and their main metabolites. Six and eight hours after baclofen, the content of HVA but not that of DA was reduced. Moreover, baclofen initially (20 min after injection) reduced, but later (105 min post drug) enhanced the accumulation of HVA induced by probenecid. The shortlasting (20 min) initial reduction of HVA elevation in probenecid-pretreated animals as well as the longlasting (6–8 h) decrease of HVA levels in rats injected with baclofen alone are interpreted to be due to a decreased release and metabolism of DA, probably as a consequence of the blockade of impulse flow in mesolimbic and nigro-striatal DA neurones. The increase in HVA and DA seen during the first few hours is thought to result from enhanced DA synthesis similar to that known for γ-hydroxybutyrate (GHB). This initial rise in HVA due to synthesis stimulation probably masked a reduction of HVA to be expected immediately after baclofen injection. The similarity between baclofen and GHB is stressed by the finding that baclofen counteracted the increase of HVA occuring after chlorpromazine and D-amphetamine but not that induced by the benzoquinolizine derivative, Ro 4-1284.