Calorimetric and Fourier transform infrared spectroscopic studies on the interaction of glycophorin with phosphatidylserine/dipalmitoylphosphatidylcholine-d62 mixtures

Glycophorin has been isolated in pure form from human erythrocyte membranes and reconstituted into lipid vesicles composed of binary mixtures of bovine brain phosphatidylserine (PS) and acyl-chain perdeuterated dipalmitoylphosphatidylcholine (DPPC-d62). The effect of protein on lipid melting behavior and order has been monitored with differential scanning calorimetry and Fourier transform infrared spectroscopy (FT-IR). The phase diagram for PS/DPPC-d62 is consistent with that previously reported for PS/DPPC (Stewart et al. (1979) Biochim. Biophys. Acta 556, 1-16) and indicates that acyl chain perdeuteration does not greatly alter the lipid mixing characteristics. The use of deuterated lipid allows the examination of lipid order by FT-IR of each lipid component in the binary mixtures as well as in the ternary (lipid/lipid/protein) systems. Addition of glycophorin to a 30:70 PS/DPPC-d62 binary lipid mixture results in a preferential glycophorin/PS interaction leading to bulk lipid enriched in DPPC-d62. This is revealed in two ways: first, through cooperative calorimetric transitions increased in temperature from the binary lipid system and second, through FT-IR melting curves of the DPPC-d62 component which shows transitions increased in both onset and completion temperatures in the presence of protein. In addition, non-cooperative melting events are observed at temperatures below the onset of phase separation. The FT-IR data are used to assign these non-cooperative events to the melting of the PS component. For the 50:50 lipid mixture with protein, two transitions are observed in the DSC experiments. The IR results indicate that both lipid components are involved with the lower temperature event.     

Comparison of physicochemical properties of purified mucus glycoproteins isolated from respiratory secretions of cystic fibrosis and asthmatic patients

The major nonreduced mucus glycoproteins (mucins) from sputa of cystic fibrosis (CF) and asthmatic patients have been purified to electrophoretic homogeneity and subjected to physical and chemical characterization. The sputum specimens were solubilized in buffer containing 0.22 M KSCN and fractionated on Bio-Gel A-5m, followed by digestion with DNase, rechromatography on the same column, and chromatography on hydroxylapatite. Sodium dodecyl sulfate gel electrophoresis of purified mucins gave a single band. Carbohydrate analyses of the purified mucins showed no significant differences in the sugar components from the two mucins. However, the CF mucin contained substantially higher (11%) sulfate content than that observed for the asthmatic mucin (5.9%). Amino acid analyses indicated that the CF mucin had higher levels of serine plus threonine (35%) as compared to the asthmatic mucin (29%). In contrast, CF mucin contained a lower content of aspartate, glutamate, and glycine than that observed for the asthmatic mucin. Molecular weights of 3.8 X 10(6) and 3.5 X 10(6) were obtained for CF and asthmatic mucins, respectively, from light-scattering studies of mucins in the presence of 6 M guanidine hydrochloride. Reduction of the disulfide bonds of the two mucins did not alter their molecular weights. Liquid chromatographic studies on Sepharose CL2B showed that CF mucin forms aggregates sufficiently large to be excluded from the gel. As compared to the CF mucin, the asthmatic mucin formed fewer of these large aggregates under identical experimental conditions. Reduction and alkylation of the mucins resulted in their inability to form aggregates. The higher state of aggregation of CF mucin may influence the viscoelastic properties of the CF lung's mucus secretions.     

Radioiodinated surface proteins of rabbit trophectoderm cells

Summary We have previously used surface iodination to discriminate between the protein patterns of epithelial cell surfaces in uteri of rabbits receptive (Day 6.5) or nonreceptive (Day 4) to nidation (Ricketts et al. 1984). In this paper, we describe application of the same technique to the trophoblastic surface of rabbit blastocysts collected on the same days of pregnancy. Analysis of labelled proteins by polyacrylamide-gel electrophoresis under denaturing conditions did not reveal qualitative differences between the two days of pregnancy. Scanning densitometry was used to quantitate the area under each protein peak on an autoradiogram; these areas were used as variables in statistical analysis of the protein pattern of individual animals. Quantitative differences between the protein patterns of the two surfaces were detected by canonical variate analysis of the pattern of relative areas of labelled protein peaks. In proteins separated on 7.5% gels, this statistical analysis correctly assigned blastocysts from 8 out of 10 animals to one of two groups according to day of pregnancy. The discrimination was not statistically significant, however, in protein patterns on 12.5% gels, used to give better separation in the lower range of molecular weights. The same analysis in the uterus unequivocally separated the surface iodination patterns from these same days of pregnancy. Thus the changes detected by surface iodination appear to be less pronounced on the trophectoderm than on the uterine epithelium in relation to the time of ovoimplantation.     

The analysis of recombinant interleukin-2 by thermospray liquid chromatography-mass spectrometry

The complete sequence of recombinant human interleukin-2 expressed in Escherichia coli has been confirmed by thermospray liquid chromatography-mass spectrometry (TS-LC-MS) of a tryptic digest derived from 100 micrograms (7 nmol) of reduced carboxymethylated interleukin-2. The preparation was shown by this method to contain predominantly unprocessed N-terminal initiator Met, with some authentic N-terminal Ala; the rest of the protein was as predicted from the DNA sequence, though some deamidated material was noted. TS-LC-MS proved to be a rapid and efficient method for surveying the protein tryptic peptide products allowing all the data to be collected in one chromatographic run; all tryptic fragments were identified by their molecular ions including those for the larger peptides (Mr 1500-3500) which, due to the presence of doubly and triply charged molecular ions, were brought within the mass range of the instrument (1800 Da). It is proposed that TS-LC-MS is a good general method for analyzing recombinant protein digests with respect to sequence confirmation, processing, and post-translational modification, and since each chromatographic peak is identified allows for subsequent monitoring of the protein by LC using uv detection. The method suffers from the disadvantages that all the sample is consumed during the experiment and that no fragment (sequence) ions are generally observed.     

Phytochrome control of oscillating levels of phenylalanine ammonia lyase in Hordeum vulgare shoots

Five-day-old etiolated barley shoots respond to brief illumination with red light by increasing their level of PAL ca 50% within 5 hr. When assayed s     

The effect of microsomal enzyme inducing drugs on reproductive function in the ewe.

A number of drugs cause marked increases in the steroid hydroxylase activity of hepatic microsomes. Beginning 2 days after estrus, 117 mature ewes were each given 14 injections over a 27-day period of phenobarbital sodium, diphenylhydantoin, chlorcyclizine HCl or phenylbutazone. Blood samples for luteinizing hormone (LH) and progesterone determination by radioimmunoassay (RIA) were taken on day 10 of the first estrous cycle (day 18 if no heat was observed) and on days 5 and 10 of the second cycle. On day 10 of the second cycle, the ewes were given an intravenous injection of 1 ml of 6% solution of pentobarbitol sodium anesthetic per 4.5 kg body weight, and the length of anesthetic sleep time was measured. The ewes were then killed and corpora lutea and liver were weighed.In 33 ewes treated with either phenobarbitol sodium or phenylbutazone, sleep time was shortened (18 min $\text{vs}$ 29 min in untreated controls, P<.01), indicating that enzyme induction had occurred. For 41 ewes treated with either chlorcyclizine HCl or diphenylhydantoin, sleep time was lengthened to 93 min (P<.01 $\text{vs}$ controls), indicating impaired liver function. Electron micrographs of liver cells verified that enzyme induction or hepatic degeneration had occurred.     

Muscarinic acetylcholine receptor activation causes inhibition of cyclic AMP accumulation, prolactin and growth hormone secretion in GH3 rat anterior pituitary tumour cells

Acetylcholine, oxotremorine and carbachol, compounds that exhibit muscarinic agonist activity, maximally inhibited basal prolactin secretion from GH3 cells by approx. 50% and intracellular cyclic AMP levels by approx. 20%. Both parameters were inhibited with similar potencies by each agonist. These inhibitory effects were blocked by a muscarinic but not by a nicotinic receptor antagonist. In the presence of VIP or IBMX, which raise intracellular cyclic AMP levels and stimulate hormone release, the degree of muscarinic inhibition was increased, but the potency remained unchanged. Similar changes in the secretory rate of prolactin and growth hormone occurred in these and in cell perifusion experiments. These results suggest that the inhibition of hormone secretion from GH3 cells by muscarinic agonists is mediated by a decrease in intracellular cyclic AMP levels.     

Morphometry and growth of sea pen species from dense habitats in the Gulf of St. Lawrence,eastern Canada

We examined four species of sea pen (Anthoptilum grandiflorum, Halipteris finmarchica, Pennatula aculeata and Pennatula grandis) collected from the Gulf of St. Lawrence and mouth of the Laurentian Channel, eastern Canada. An exponential length–weight relationship was found for all four species, where growth in weight was progressively greater than growth in length with increasing colony size. Halipteris finmarchica, P. grandis and P. aculeata presented the better allometric fits, explaining over 80% of the variance. In addition, a count of growth increments visible in transverse sections in 86 A. grandiflorum and 80 P. aculeata samples was made. Presumed ages ranged between 5 and 28 years for A. grandiflorum and 2 and 21 years for P. aculeata. Radiocarbon assays were inconclusive and could not be used to confirm these ages; further age validation is required. Radial growth of the rod is slow during the first years, increasing at intermediate sizes of the colony and slowing down again for large colonies. Similar results were obtained from the relationship between colony length and number of growth increments where a logistic model was the best fit to the data. On average Spearman’s rank correlations showed 11% of shared variance between sea pen length or weight and environmental variables. Bottom temperature and salinity, depth and summer primary production were significantly correlated to sea pen size for most species.     

The binding of 1,N6-ethenoNAD to bovine liver glutamate dehydrogenase: studies using the time-correlated single photon counting fluorescence technique

The time-correlated single photon counting (TCPC) fluorescence technique has been used as a novel approach to investigate ligand-protein interaction, for the case of the binding of the fluorescent coenzyme analogue 1,N6-ethenoNAD (epsilon NAD) to bovine liver glutamate dehydrogenase in the presence of glutarate, a substrate analogue which stabilizes the complex. System calibration was performed using solutions of epsilon ADP and carefully purified epsilon NAD mixed at variable molar ratios (pH 7.0, 0.05 M sodium phosphate buffer, 20 degrees C). The fluorescence lifetimes obtained after deconvolution were 2.4 ns (for epsilon NAD) and 23 ns (for epsilon ADP), in good agreement with literature values obtained under similar conditions. epsilon NAD binds to glutamate dehydrogenase in the presence of 50 mM glutarate, with a fluorescence quantum yield enhancement factor, Q, of about 17-fold, as previously reported (Favilla, R. and Mazzini, A. (1984) Biochim. Biophys. Acta 48-57). For this system, fluorescence lifetime values were obtained after deconvolution as 2.4 ns for free epsilon NAD and 21 ns for bound epsilon NAD. These values did not vary appreciably with enzyme concentration nor with degree of saturation, thus reflecting the existence of only one spectroscopically relevant type of complex. Addition of either GTP or ADP did not affect the lifetime of epsilon NAD bound to the enzyme, but only its affinity, thus allowing calculations of binding strengths. In the case of a simple binding (i.e., in the absence of GTP) the dissociation constant of the complex could be derived from a simple relationship, in which only the ratio between the pre-exponential factors and the parameter gamma, which represents the molar fraction of epsilon NAD molecules free in solution in the open conformation, are to be taken into account. The results are in good agreement with those reported by some of us (reference above) using a steady-state fluorescence technique, which by itself is, however, unable to resolve the number of relevant species present in the system.