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WALP peptides consist of repeating alanine-leucine sequences of different lengths, flanked with tryptophan “anchors” at each end. They form membrane-spanning α-helices in lipid membranes, and mimic protein transmembrane domains. WALP peptides of increasing length, from 19 to 31 amino acids, were incorporated into N-monomethylated dioleoylphosphatidylethanolamine (DOPE-Me) at concentrations up to 0.5mol% peptide. When pure DOPE-Me is heated slowly, the lamellar liquid crystalline (Lα) phase first forms an inverted cubic (QII) phase, and the inverted hexagonal (HII) phase at higher temperatures. Using time-resolved x-ray diffraction and slow temperature scans (1.5°C/h), WALP peptides were shown to decrease the temperatures of QII and HII phase formation (TQ and TH, respectively) as a function of peptide concentration. The shortest and longest peptides reduced TQ the most, whereas intermediate lengths had weaker effects. These findings are relevant to membrane fusion because the first step in the Lα/QII phase transition is believed to be the formation of fusion pores between pure lipid membranes. These results imply that physiologically relevant concentrations of these peptides could increase the susceptibility of biomembrane lipids to fusion through an effect on lipid phase behavior, and may explain one role of the membrane-spanning domains in the proteins that mediate membrane fusion.

The in meso method for membrane protein crystallization uses a lipidic cubic phase as the hosting medium. The cubic phase provides a lipid bilayer into which the protein presumably reconstitutes and from which protein crystals nucleate and grow. The solutions used to spontaneously form the protein-enriched cubic phase often contain significant amounts of detergents that were employed initially to purify and to solubilize the membrane protein. By virtue of their surface activity, detergents have the potential to impact on the phase properties of the in meso system and, by extension, the outcome of the crystallization process. The purpose of this study was to quantify the effects that a popular series of nonionic detergents, the n-alkyl-β-d-glucopyranosides, have on the phase behavior of hydrated monoolein, the lipid upon which the in meso method is based. Phase identity and phase microstructure were characterized by small-angle x-ray diffraction on samples prepared to mimic in meso crystallization conditions. Measurements were made in the 0–40°C range. Samples prepared in the cooling direction allow for the expression of metastability, a feature of liquid crystalline phases that might be exploited in low-temperature crystallization. The results show that the cubic phase is relatively insensitive to small amounts of alkyl glucosides. However, at higher levels the detergents trigger a transition to the lamellar phase in a temperature- and salt concentration-dependent manner. These effects have important implications for in meso crystallization. A diffraction-based method for assaying detergents is presented.

The phase properties of lipids have far-reaching consequences in membrane biology. Their influence ranges from domain formation in intact biomembranes to membrane protein reconstitution and crystallization. To exploit phase behavior in the spirit of rational design, it is imperative that the rules relating lipid molecular structure and liquid crystal or mesophase behavior be established. Phase behavior is quantitatively and concisely represented in the form of temperature-composition phase diagrams. A somewhat limited number of phase diagrams exists for the monoacylglycerols. The objective of the current study was to determine the quality of phase behavior prediction for a specific monoacylglycerol based on an analysis of the existing phase diagrams for related chain homologs. To this end, a phase diagram for the monononadecenoin (19:1c10)/water system was predicted in the temperature range from −15°C to 120°C and from 0% to 80% (w/w) water. The prediction was tested by constructing the corresponding phase diagram using low- and wide-angle x-ray diffraction, differential scanning calorimetry, and polarized light microscopy. The results show that the predicted and experimental phase diagrams agree remarkably well. They also highlight the need for additional phase studies of the type described to enlarge the data bank of phase diagrams and to strengthen the foundations of the rational design approach.

Thermodynamic reversibility of ion binding at a membrane/aqueous interface has been demonstrated using variable period X-ray standing waves generated above a gold mirror. The interface consists of a negatively charged and polymerized phospholipid monolayer bathed in a dilute zinc chloride solution. Zinc ion distribution in the diffuse double layer was monitored as the pH of the aqueous medium was titrated from a value of 5.8 to 2.0 and back again to pH 5.8. The ion distribution was modeled by an exponentially decaying function. As H⁺ concentration increased upon changing pH from 5.8 to 2.0, the amount of zinc in the diffuse double layer (integrated as a two-dimensional projection along the surface normal) decreased from 1.4 × 10¹³ to 1.0 × 10¹² ions/cm² and the concentration of bound zinc at the surface dropped by a factor of 6.4 from an initial value of 122 mM to a low of ca. 19 mM. Recovery of the pH 5.8 distribution profile was essentially complete upon back-titration from pH 2.0. These results demonstrate clearly that within the experimental error of the measurement ion distribution at the charged membrane interface is reversible upon adjusting the pH of the electrolyte solution which corresponds to altering the degree of protonation and, thus, the net charge on the exposed phosphate headgroups.

The “cubic phase method” for growing crystals of membrane proteins uses a complex mixture of water, lipid, protein, and other components. The current view is that the cubic phase is integral to the process. Thus additives from whatever source introduce the possibility of destabilizing the phase, thereby compromising the crystallization process. Detergents are used to solubilize membrane proteins and are likely to be ported into the cubic medium with the target protein. Depending on the identity and concentration of the detergent, the cubic phase, which itself is membranous, may be solubilized or destabilized in such a way as to render it unsuitable as a crystal growing system. The nonionic detergent n-dodecyl-β-d-maltopyranoside is commonly used in membrane protein work. In this study, we evaluate its effect on the cubic mesophase of hydrated monoolein. X-ray diffraction was used for phase identification and mesophase microstructure characterization. The results show that while low levels of the detergent are tolerated, increasing concentrations trigger a cubic-to-lamellar phase transition in a temperature-dependent manner. This finding is rationalized in the context of complementary molecular shapes of the lipid and the detergent and has implications for the mechanism of crystallization in lipidic mesophases as discussed.

This work is part of a long-term research effort to establish the relationship between lipid molecular structure and lyotropic and thermotropic mesophase propensity by comparing the equilibrium temperature−composition phase diagrams of a homologous series of monoacylglycerols. Here we report the temperature−composition phase diagram of monovaccenin (a C18:1c11 monoacylglycerol) in water, constructed using small-angle X-ray scattering in the temperature range of ca. 0−110 °C and the composition range of ca. 0−60% (w/w) water in the heating direction. The interpreted equilibrium phase diagram is based on several hundred discrete X-ray diffraction measurements in temperature−composition space recorded as a function of temperature in 5 °C increments and of composition in 4% (w/w) water increments on average. The phases identified and characterized structurally in this system include the lamellar crystal phase, the lamellar liquid crystal phase, two inverted cubic phases (Q²³⁰, Ia3d; Q²²⁴, Pn3m), the inverted hexagonal phase, and the fluid isotropic phase. The monovaccenin/water phase diagram is very similar to that of the monoolein (a C18:1c9 monoacylglycerol)/water system. However, there are important differences in transition temperatures and phase boundary positions between the two that we attribute to differences in the effective length and shape of the corresponding amphiphiles. The sensitive response to temperature and lipid identity of the average water channel radius in the fully hydrated, bicontinuous cubic phase makes monoacylglycerol systems important potential candidates for controlled drug release and membrane protein crystallization.

A bimodal change in yield and microsomal protein content of rabbit mammary gland was observed with the progress of pregnancy and lactation. The initial stimulus took place on day 22 of pregnancy and the second during early lactation. Palmitoyl-CoA:monopalmitoyl-sn-glycerol 3-phosphate palmitoyltransferase activity was monitored concurrently. This enzyme in rabbit mammary microsomes is composed of two isoenzymic species that differ with respect to the physical nature of the substrates with which each interacts. The activities of the two isoenzymes were recorded at progressive stages of pregnancy, lactation, and involution to determine if a regulatory role could be assigned to either or both species. Although the patterns were indefinite, total transacylase activity did increase over this period, i.e., the specific activity of LPAT-α was 12 and 24 nmoles/mg protein per min in pregnancy and lactation, respectively, while that of LPAT-β rose from zero to 90 nmoles/mg protein per min over the same period. The time of harvesting in relation to the interval between nursing periods is discussed in the light of these results.

The pituitary perfusion technique was used to investigate the possible interaction between the neurohypophyseal hormones and gonadotropin releasing hormone (GnRH). The perifusion of rat anterior pituitaries with 0.2 mU/ml arginine vasopressin (AVP) resulted in a significant (P< 0.05) increase in LH release over baseline, while higher doses had no effect. When combined with GnRH, this and higher concentrations of AVP did not alter the GnRH-induced release of LH. Three concentrations of oxytocin (OT): 0.02, 0.2 and 20 mU/ml, increased baseline secretion of LH (P< 0.05) while 2 mU/ml OT did not. When added to GnRH-containing perifusion media, 0.02 mU/ml OT caused significant (P< 0.05) enhancement and prolongation of the LH response to GnRH. All higher concentrations of OT and one concentration that was tenfold lower, did not exhibit potentiating effects. When the pituitary tissue was pretreated with AVP or OT prior to stimulation with GnRH, only the same concentration of OT (0.02 mU/ml) was effective (P< 0.01) while two concentrations of AVP (2 and 20 mU/ml) which had been ineffective previously, then enhanced the LH release due to GnRH (P< 0.05 and P< 0.01, respectively). It is proposed that the data from these experiments support the hypothesis that AVP and OT may have a role in reproduction via an interaction with GnRH at the level of the anterior pituitary.

The relative reactivities toward reduction by free flavin semiquinones of cytochromes (c-type cytochromes, cytochrome b5, c'-type cytochromes) iron-sulfur proteins (high-redox-potential ferredoxins, rubredoxins, low-redox-potential ferredoxins), and blue copper proteins (plastocyanin, azurins) are shown to correlate with calculations of the solvent exposure of the various prosthetic groups. In the case of the c-type cytochromes, one of the major centers of exposure is the sulfur atom of the thioether bridge that covalently links heme ring C to the protein. Charge-iterative extended Hückel calculations on a heme c model indicate that both porphyrin pi and Fe(III)d pi orbitals can delocalize onto the bridging sulfur atom. Unpaired spin densities are comparable to those obtained for individual aromatic porphyrin ring carbon atoms. Thus, the exposed sulfur of ring C may act to facilitate electron transfer.

For the structure determination of symmetric protein dimers it is necessary to distinguish between intra‐ and inter‐subunit NOEs. A method is presented to measure selectively the inter‐subunit NOEs using uniform 15N and 13C isotope labelling. This is accomplished by doubly filtered 2D NOE experiments on mixtures of native protein with isotope‐labeled protein. The method has been applied to the Arc represser and allows the characterization of virtually all proton‐proton NOEs in terms of their intra‐ or inter‐subunit nature.

1. Electrodecantation has been applied to the initial fractionation from crude material of pig kidney β-d-glucosidase and sheep testicular N-acetylglucosaminidase. 2. The isoelectric points determined by isoelectric focusing were pH4.9 for the β-d-glucosidase and pH6.3 for the N-acetylglucosaminidase. 3. Electrodecantation of pig kidney extract and sheep testicular extract was carried out at pH4.9 and 6.3 respectively. 4. A six- to ten-fold increase in specific activity could be obtained with good recoveries after a single cycle of electrodecantation. 5. The technique has also been used to purify further an extracellular Bacillus subtilis protease preparation. 6. Attempts to use electrodecantation for the concentration of very dilute enzyme solutions resulted in considerable loss of activity. 7. The limitations and potential use of the technique in laboratory-scale enzyme preparation are discussed.