Effect of cryoprotectants on release of various enzymes from buck spermatozoa during freezing

Semen samples from 12 bucks Were extended with 10 different extenders containing glycerol, DMSO, glycerol + DMSO, and glycerol + lactose in varying concentrations as cryoprotective agents. The activities of acrosin, hyaluronidase, alkaline phosphatase (AKP), aspartate aminotransferase (AST), alanine amino transferase (ALT) and lactic dehydrogenase (LDH) were assayed in equilibrated (Prefreeze) and frozen thawed (Postfreeze) semen samples. Significantly (P < 0.01) higher intracellular activity of acrosin was recorded in semen samples extended with lactose than with the other extenders, with the maximum being with Tris yolk glycerol lactose (TYGL(180)). Effects of extenders on acrosin activity were significant (P < 0.01) at both of the pre-and postfreeze stages. However, extracellular activities of hyaluronidase, alkaline phosphatase, transaminases (AST and ALT), and lactic dehydrogenase were significantly higher in extenders containing DMSO than lactose. Leakage of these enzymes was found to increase from the prefreeze to the post freeze stage.     

Study of interaction of export initiation domain of Escherichia coli mature alkaline phosphatase with membrane phospholipids during secretion

The efficiency of secretion of alkaline phosphatase from Escherichia coli depending on the primary structure of its N-terminal region and the content of zwitterionic phospholipid phosphatidylethanolamine and anionic phospholipids in membranes has been studied in this work to establish the peculiarities of interaction of mature protein during its secretion with membrane phospholipids. It has been shown that the effect of phosphatidylethanolamine but not anionic phospholipids on the efficiency of alkaline phosphatase secretion is determined by the primary structure of its N-terminal region. The absence of phosphatidylethanolamine appreciably reduces the efficiency of secretion of wild type alkaline phosphatase and its mutant forms with amino acid substitutions in positions +5+6 and +13+14. In contrast, secretion of the protein with amino acid substitutions in positions +2+3, significantly decreased as a result of such mutation, in the presence of phosphatidylethanolamine, reaches the level of wild type protein secretion in the absence of phosphatidylethanolamine. The results suggest an interaction of the N-terminal region of the mature protein under its translocation across the membrane with phosphatidylethanolamine.     

Mechanisms of interaction of amino acids with phospholipid bilayers during freezing

In this study we compare the ability of various amino acids to protect small unilamellar vesicles against damage during freeze/thaw. Liposomes were composed of 75% palmitoyloleoyl phosphatidylcholine and 25% phosphatidylserine. Damage to liposomes frozen in liquid nitrogen and thawed at 20 degrees C was assessed by resonance energy transfer. Cryoprotection by numerous amino acids was compared in the presence and absence of 350 mM NaCl. The majority of amino acids with hydrocarbon side chains increased membrane damage during freeze/thaw regardless of the presence of salt. However, amino acids with hydrocarbon side chains of less than three carbons long, e.g. glycine, alanine, and 2-aminobutyric acid, were cryoprotective only in the presence of salt. We suggest that NaCl selectively increases the solubility of such amino acids, allowing them to act as cryoprotectants. In contrast, amino acids with side chains containing charged amine groups were cryoprotective regardless of the presence of salt. The degree of charge on the second amine group is shown to be important for cryoprotection by these molecules. We present evidence that suggests an interaction between the positively charged, second amine group of the amino acid, and the negatively charged phospholipid headgroup.     

Membrane stabilization during freezing: the role of two natural cryoprotectants, trehalose and proline

The relative effectiveness of two natural cryoprotectants, proline and trehalose, in preserving membrane structure and function during freezing was studied. Isolated vesicles of sarcoplasmic reticulum (SR) from lobster muscle (Homarus americanus) were employed to study changes in structure and function during rapid freeze-thaw conditions. Both proline and trehalose were shown to effectively preserve the structure (assessed with freeze fracture) and function (assessed by the ability of the membranes to transport calcium) in the frozen vesicles. As a first step toward determining the mechanism of cryoprotection by these compounds, we have investigated their effectiveness in inhibiting freezing induced fusion between phospholipid vesicles. Pamiltoyloleoyl-phosphatidylcholine: phosphatidylserine (85:15 mole ratio) small unilamellar vesicles (SUVs) were made incorporating one of the following fluorescent probes, and energy donor, cholesteryl anthracene-9-carboxylate, or an energy acceptor, nitrobenzo-2-oxa-1,3-diazole phosphatidylethanolamine to investigate the amount of membrane mixing during rapid freeze-thaw cycles, and storage at -20 degrees C. Membrane mixing was measured as an energy transfer from donor to acceptor when donor vesicles and acceptor vesicles were mixed before a particular freezing treatment. Membrane mixing was correlated with structural changes in these membranes by freeze-fracture analysis. Both trehalose and proline were found to be more effective in preventing membrane mixing between SUVs than the standard protectants, glycerol and dimethylsulfoxide.     

The role of membrane phospholipids in the interaction of ribosomes with endoplasmic-reticulum membrane (Short Communication)

It is shown that the ionic head groups of the membrane phospholipids cannot be solely responsible for the attachment of the ribosome and that other membrane components must also be involved in the binding process.     

The interaction of insulin with phospholipids

1. A simple two-phase chloroform–aqueous buffer system was used to investigate the interaction of insulin with phospholipids and other amphipathic substances. 2. The distribution of ¹²⁵I-labelled insulin in this system was determined after incubation at 37°C. Phosphatidic acid, dicetylphosphoric acid and, to a lesser extent, phosphatidylcholine and cetyltrimethylammonium bromide solubilized ¹²⁵I-labelled insulin in the chloroform phase, indicating the formation of chloroform-soluble insulin–phospholipid or insulin–amphipath complexes. Phosphatidylethanolamine, sphingomyelin, cholesterol, stearylamine and Triton X-100 were without effect. 3. Formation of insulin–phospholipid complex was confirmed by paper chromatography. 4. The two-phase system was adapted to act as a simple functional system with which to investigate possible effects of insulin on the structural and functional properties of phospholipid micelles in chloroform, by using the distribution of [¹⁴C]glucose between the two phases as a monitor of phospholipid–insulin interactions. The ability of phospholipids to solubilize [¹⁴C]glucose in chloroform increased in the order phosphatidylcholine<sphingomyelin<phosphatidylethanolamine<phosphatidic acid. Insulin decreased the [¹⁴C]glucose solubilized by phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid, but not by sphingomyelin. 5. The significance of these results and the molecular requirements for the formation of insulin–phospholipid complexes in chloroform are discussed.     

Yeast mitochondrial inner membrane 30 kd hydrophobic protein: properties and interaction with phospholipids

A 30 kd hydrophobic protein is extracted from yeast mitochondrial inner membrane. It is present in wild yeast strains but absent in mitochondrial DNA lacking mutants. The isoelectric point of the protein and its solubility in various organic solvents are determined. The fluorescence of a tryptophan residue near the surface of the 30 kd protein dissolved in butanol-1, can be quenched by phospholipids containing unsaturated fatty acids. Results are in accordance with the 30 kd protein being an integral protein of the yeast mitochondrial inner membrane.     

Synaptotagmin activates membrane fusion through a Ca2+-dependent trans interaction with phospholipids

Synaptotagmin-1 is the calcium sensor for neuronal exocytosis, but the mechanism by which it triggers membrane fusion is not fully understood. Here we show that synaptotagmin accelerates SNARE-dependent fusion of liposomes by interacting with neuronal Q-SNARES in a Ca2+-independent manner. Ca2+-dependent binding of synaptotagmin to its own membrane impedes the activation. Preventing this cis interaction allows Ca2+ to trigger synaptotagmin binding in trans, accelerating fusion. However, when an activated SNARE acceptor complex is used, synaptotagmin has no effect on fusion kinetics, suggesting that synaptotagmin operates upstream of SNARE assembly in this system. Our results resolve major discrepancies concerning the effects of full-length synaptotagmin and its C2AB fragment on liposome fusion and shed new light on the interactions of synaptotagmin with SNAREs and membranes. However, our findings also show that the action of synaptotagmin on the fusion-arrested state of docked vesicles in vivo is not fully reproduced in vitro.     

Perturbation of membrane phospholipids alters the interaction between epidermal growth factor and its membrane receptors

This report presents electron microscopic evidence of statistically significant changes in the microtubule number concentration and length distribution after the attainment of monomer-polymer equilibrium (“steady state”). We also extend previous theoretical work on polymer redistribution (F. Oosawa, 1970, J. Theor. Biol.27, 69–86).     

Reconstitution of the glycosylphosphatidylinositol-anchored protein Thy-1: interaction with membrane phospholipids and galactosylceramide.

Glycosylphosphatidylinositol (GPI)-anchored membrane proteins are proposed to interact preferentially with glycosphingolipids and cholesterol to form microdomains, which may play an important role in apical targeting and signal transduction. The objective of the present study was to investigate the interaction of the GPI-anchored protein Thy-1 with phospholipids and a glycosphingolipid. Purified Thy-1 was reconstituted into lipid bilayer vesicles of dimyristoyl-phosphatidylcholine (DMPC) alone or in combination with galactosylceramide (GC). The ability of Thy-1 to perturb the gel to a liquid-crystalline phase transition of DMPC was examined by differential scanning calorimetry. As the mole fraction of Thy-1 increased, the phase transition enthalpy, deltaH, declined. Analysis indicated that each molecule of Thy-1 perturbed over 50 phospholipids, suggesting that, in addition to the anchor insertion into the bilayer, the protein itself may interact with the membrane surface. Inclusion of 5% w/w GC in the bilayer resulted in a striking change in the interaction of Thy-1 with phospholipids. At low Thy-1 content, there was a reduction in the phase transition temperature and an increase in phospholipid cooperativity, suggesting the formation of Thy-1/GC-enriched domains. DeltaH initially decreased with increasing Thy-1 content of the bilayer; however, at higher Thy-1 mole ratios, deltaH rose again. These results are interpreted in terms of a model whereby, at low protein:lipid mole ratios, Thy-1 preferentially sequesters GC to form enriched microdomains. At high protein:lipid mole ratios, Thy-1 may alter its conformation in response to steric crowding within these domains such that its interaction with the bilayer surface is reduced.     

Amides as cryoprotectants for freezing stallion semen: a review

Stallion semen cryopreservation, despite its impact on the horse industry, is not an established technology. During the last years, a number of modifications have been proposed to the freezing process, however, a large population of stallions still have poor semen quality and fertility after frozen-thawed. Glycerol toxicity could be a reason for the variation on stallion sperm freezability. There are limited publications concerning the use of alternative cryoprotectants for equine sperm. Glycerol is contraceptive for some species and other cryoprotectors, such as amides, have been show to be a good option for freezing semen of these species. Recent reports have shown encouraging data respecting the use of amides as cryoprotectants for stallions, with more remarkable improvements for semen from stallions that freeze poorly when glycerol is used.     

Interactions of annexins with membrane phospholipids.

The annexins are proteins that bind to membranes and can aggregate vesicles and modulate fusion rates in a Ca2(+)-dependent manner. In this study, experiments are presented that utilize a pyrene derivative of phosphatidylcholine to examine the Ca2(+)-dependent membrane binding of soluble human annexin V and other annexins. When annexin V and other annexins were bound to liposomes containing 5 mol % acyl chain labeled 3-palmitoyl-2-(1-pyrenedecanoyl)-L-alpha-phosphatidylcholine, a decrease in the excimer-to-monomer fluorescence ratio was observed, indicating that annexin binding may decrease the lateral mobility of membrane phospholipids without inducing phase separation. The observed increases of monomer fluorescence occurred only with annexins and not with other proteins such as parvalbumin or bovine serum albumin. The extent of the increase of monomer fluorescence was dependent on the protein concentration and was completely and rapidly reversible by EDTA. Annexin V binding to phosphatidylserine liposomes was consistent with a binding surface area of 59 phospholipid molecules per protein. Binding required Ca2+ concentrations ranging between approximately 10 and 100 microM, where there was no significant aggregation or fusion of liposomes on the time scale of the experiments. The polycation spermine also displaced bound annexins, suggesting that binding is largely ionic in nature under these conditions.     

Deep freezing of mouse one-cell embryos and oocytes using different cryoprotectants

The objective of this study was to compare iso-osmolar concentrations (1.5 M) of 1,2-propanediol, glycerol, dimethylsulphoxide and a combination of 1 M propanediol + 0.5M glycerol (PDGLY) as cryoprotectants for murine ovulated oocytes and one-cell embryos. A higher (P < 0.01) percentage of one-cell embryos developed to the two-cell stage when frozen-thawed with 1,2-propanediol (83%) as compared with glycerol (43%), dimethylsulfoxide (51%) or PDGLY (7%). Data recalculated on the basis of two-cell embryos/number of normal one-cell embryos after thawing indicated no differences among single cryoprotectant groups. More (P < 0.01) frozen-thawed, in-vitro fertilized oocytes developed to the two-cell stage when 1,2-propanediol (35%) was used as cryoprotectant as compared with glycerol (15%). Freezing-thawing resulted in a reduced number of two-cell embryos after oocytes were fertilized in-vitro as compared with fresh oocytes. 1,2-propanediol was a better cryoprotectant than glycerol, dimethylsulphoxide or PDGLY for deep freezing of murine oocytes or one-cell embryos.     

Incorporation of 32P into membrane phospholipids during chloroplast biogenesis

The composition of membrane phospholipids during chloroplast biogenesis was studied. The maximal level of phosphatidic acid was observed in the membrane fraction of proplastids. Phosphatidylglycerol was found to be the most abundant phospholipid component of grana thylakoids. The evidence from the in vivo experiments indicates that phosphatidic acid and phosphatidylglycerol incorporate the 32P label at a high rate at all stages of the chloroplast biogenesis. It is concluded that plastids are the site of the phosphatidylglycerol biosynthesis in the plant cell.