Protein Released Research Articles

Factors Affecting the in Vitro Release of Recombinant Human Interferon-γ (rhIFN-γ) from PLGA Microspheres

A long-acting depot formulation of recombinant human interferon-γ (rhIFN-γ) was achieved by microencapsulation of rhIFN-γ in polylactic-coglycolic acid (PLGA) microspheres by a water-in-oil-in-water technique. The release of protein was assessed with different release devices and buffer systems. The quality of the released protein was quantitated by sodium dodecyl sulfate-size exclusion chromatography, ELISA, and bioactivity assays. The microencapsulation process resulted in an encapsulation efficiency of 100% and the initial release of bioactive, native protein with no subsequent release. Further investigation suggested that the protein did not bind to the PLGA, but a constant and small amount of protein adsorbed to the filter device used for the release studies. The composition of the release media (pH, buffer species, salt concentration, ionic strength, and type and concentration of surfactants) had a profound effect on the in vitro release rate. The effect was mainly due to the differential solubility, stability, and aggregation of rhIFN-γ in the various systems for protein inside the microspheres or released into the bulk solution. The quality of the protein released from the microspheres was also affected by the buffer media upon storage at 5°C, which, in turn, affected the quantification of released protein. The bicinchoninic acid method typically used to quantitate protein release underestimated protein release because of aggregation. Protein released after several days was less active than the starting material and had lost activity as a result of the inherent instability of rhIFN-γ at 37°C. The release device, buffer species, pH, and excipients must be assessed in release studies of proteins from polymer matrices because the protein stability and release is dependent on these variables. These studies also indicated that rhIFN-γ was encapsulated and released from PLGA in a bioactive form, but its stability at 37°C, which was greatly affected by the release conditions, limits the duration of release of native, bioactive protein to 7days or less.

Heat-mediated activation of affinity-immobilized Taq DNA polymerase.

A novel strategy for heat-mediated activation of recombinant Taq DNA polymerase is described. A serum albumin binding protein tag is used to affinity-immobilize an E. coli-expressed Taq DNA polymerase fusion protein onto a solid support coated with human serum albumin (HSA). Analysis of heat-mediated elution showed that elevated temperatures (> 70 degrees C) were required to significantly release the fusion protein from the solid support. A primer-extension assay showed that immobilization of the fusion protein resulted in little or no extension product. In contrast, fusion protein released from the HSA ligand by heat showed high polymerase activity. Thus, a heat-mediated release and reactivation of the Taq DNA polymerase fusion protein from the solid support can be obtained to allow for hot-start PCR with improved amplification performance.

In vitro nasal transport across rabbit mucosa: Effect of oxygen bubbling, pH and hypertonic pressure on permeability of lucifer yellow, diazepam and 17 β-estradiol

The effect of pH, oxygen bubbling and hypertonic osmolarity in excised rabbit nasal mucosa on permeation of drugs was examined. For preincubation we used:(i) pH 5.2 saline, pH 7.4 saline and Krebs-Ringer solution (KR); (ii) the absence and presence of oxygen bubbling; and (iii) hypertonic KR. The control was put in KR with oxygen bubbling without preincubation. The permeation of lucifer yellow (LY; MW 521) as a hydrophilic drug and of diazepam (DP; MW 285) as a lipophilic drug and the amount of protein released from tissue were measured after preincubation. Also the enzyme activity in the nasal mucosa was examined by measuring the permeation of 17 β-estradiol (E2; MW 272) and estrone (El; MW 270) that was produced from E2 by 17 β-hydroxy-steroid dehydrogenase (17 β-HSD). The low pH increased the permeation of drugs, especially LY. The activity of 17 β-HSD appears to decrease by a low pH more than by bubbling. The protein from the mucosa after preincubation was released in the donor and receiver cells without bubbling irrespective of pH. High osmotic pressure in donor KR solution with bubbling also released more protein in the donor cell compared with a change in pH to 5.2 and 7.4 without bubbling. The results of the released protein amount are in agreement with those of the permeability study, suggesting a morphological change. The low pH increased the permeation of drug, and decreased the enzyme activity in the nasal mucosa without increasing the release of protein from the nasal mucosa compared with the release at pH 7.4. This finding can be applied to nasal absorption of drugs.

Influence of Formulation Variables on the In-Vitro Release of Albumin from Biodegradable Microparticulate Systems

Poly(D, L-lactide-co-glycolide) microspheres containing BSA were prepared by a modified solvent evaporation method using a double emulsion. These microspheres were characterized for size, morphology, surface adsorbed protein, encapsulation efficiency and release kinetics. The influence of two formulation variables (the procedure to obtain the first emulsion and the lyophilization of the microspheres once obtained) on the physical characteristics and release behaviour of the microspheres was also investigated. Sonicated microspheres were smooth and spherical, with a mean particle size of 20 μm and an encapsulation efficiency of 81%. When the first emulsion was prepared by vortex mixing the particles were irregular and porous, with a mean size of 31 μm and a lower encapsulation efficiency (56%). The sonication allows a more homogeneous emulsion as well as a lower percentage of albumin adsorbed on the surface. The in vitro release profile was described as a biexponential process with an initial burst effect due to the release of the protein adsorbed on the microsphere surface and a second sustained release phase due to protein diffusion through the channels or pores formed in the polymer coat. The release of BSA was dependent on the preparation method. The greatest burst release was found for microspheres formulated using the vortex mixer, 58% of the encapsulated protein was released during the first 24 h, whereas sonicated microspheres released 32.2%. This burst effect could be reduced by lyophilizing the micro-spheres following their preparation. The amount of protein released decreased to 28.3% and 51.6% in sonicated and non-sonicated microspheres respectively, when they were lyophilized.

Pressure-Induced Release of Basic 7S Globulin from Cotyledon Dermal Tissue of Soybean Seeds

Protein release by high-pressure treatment of soybean seeds was investigated. When soybean seeds were immersed in distilled water and pressurized at 100−700 MPa, a large amount of proteins, accounting for 0.5−2.5% of the total seed protein, were released from the seeds. By sodium dodecyl sulfate−polyacrylamide gel electrophoresis, immunoblot, and amino acid sequencing analyses, the major protein released was identified as basic 7S globulin (Bg), consisting of 16 and 27 kDa subunits. Bg was shown immunohistochemically to be localized in the dermal tissue of seed cotyledons, presumably in epidermal cells. There were almost no structural changes in seed inner cells, as determined by scanning electron microscopy, but considerably large changes in structures of cotyledon surface and epidermal cells were observed in the pressurized seeds. These results suggest that Bg localized in seed cotyledon dermal tissues is released from soybean seeds as a result of structural changes in cotyledon surface and epidermal cells that are caused by high-pressure treatment. Keywords: High pressure; basic 7S globulin; soybean seed

Fluid and protein secretion from ferret submandibular and parotid glands in response to sympathetic nerve stimulation or administration of sympathomimetics.

Electrical stimulation of the sympathetic innervation evoked secretion of submandibular and parotid saliva. By changing the mode of stimulation from a continuous to an intermittent one the fluid response increased and glandular blood flow improved. The volumes from the submandibular glands were larger than those from the parotid glands and further, the protein concentration of submandibular saliva was higher than that of parotid saliva. Adrenaline, isoprenaline and phenylephrine evoked larger fluid responses from submandibular than from parotid glands. However, the fluid response was small compared to the parasympathetic one. Substance P-evoked saliva was used as carrier for protein released by sympathetic nerve stimulation or administration of adrenaline and isoprenaline. In vitro tissues of submandibular and parotid glands responded to adrenaline with a dose-dependent release of protein. Taken together, the analytical pharmacology performed in vivo and in vitro, and including the antagonists phentolamine, dihydroergotamine, propranolol and metoprolol, showed that in submandibular glands, alpha(alpha 1)adrenoceptors were predominantly involved in fluid secretion and beta(beta 1)-adrenoceptors predominantly involved in protein secretion. In parotid glands, fluid secretion seemed solely to depend on alpha(alpha 1)-adrenoceptors, while beta(beta 1)-adrenoceptors seemed almost solely involved in protein secretion.

Is the primary cause of thermal inactivation of oxygen evolution in spinach PS II membranes release of the extrinsic 33 kDa protein or of Mn?

Incubation of PS II membranes at 45–50°C for several min resulted in strong inactivation of oxygen evolution, concomitant with release of Mn and the extrinsic proteins of 33, 23 and 17 kDa. No correlation was found between loss of the activity and release of the 33 kDa protein or Mn. However, involvement of the protein release in the mechanism of heat-inactivation was suggested by stabilization of the activity against heat-treatment by immobilization of the 33 kDa protein with a water-soluble carbodiimide. Furthermore, a linear correlation was found between extents of heat-inactivation and amounts of the 33 kDa protein released in the presence of 50 mM CaCl 2, which greatly accelerated inactivation of oxygen evolution, release of the 33 kDa protein and aggregation of PS II membranes at high temperatures. Evidence was obtained indicating that the 33 kDa protein released at high temperatures rebinds to its functional site when temperature is lowered but CaCl 2 suppresses rebinding of the protein by promoting intensive aggregation of the membranes. Thus, the activity survived in the presence of CaCl 2 is proportional to the amounts of the protein remained attached to the membranes during heat-treatment. By contrast, release of Mn was not affected by addition of CaCl 2 so that enhanced inactivation of oxygen evolution was not accompanied by corresponding increase in the amount of Mn released. It is concluded, therefore, that the primary cause of heat-inactivation of oxygen evolution is dissociation of the 33 kDa protein but not that of Mn.

Release of fatty acid binding protein and lactate dehydrogenase from isolated rat heart during normoxia, low-flow ischemia, and reperfusion.

The present study was performed to monitor the effect of low-flow ischemia and reperfusion on changes in the protein permeability of the cardiomyocyte cell membrane and the endothelial cell layer for two cytoplasmic proteins, i.e., fatty acid binding protein (FABP) and lactate dehydrogenase (LDH), which differ appreciably with respect to physicochemical characteristics. To accomplish this, isolated rat hearts were Langendorff perfused with separate collection of vascular and interstitial effluents. Control hearts were perfused normoxically for 300 min, whereas experimental hearts were subjected to 60 min normoxia (N), 180 min low-flow ischemia (I), and finally 60 min normoxic reperfusion (R). Protein release was measured in 15 min interval fractions. During the first 240 min of perfusion 0.2% of total tissue FABP and 1.1% of total tissue LDH were detected in the effluents in both groups. Moreover, in each case 80% of released FABP and LDH was found in the interstitial effluent. During R, following I in the experimental group, appreciable amounts of both proteins were released (2.2 and 5.1% of total tissue contents for FABP and LDH, respectively). During this period the percentage of protein released in the vascular effluent increased significantly for both proteins. It is concluded that the combination of low-flow ischemia and reperfusion increases the protein permeability of both the cardiomyocyte cell membrane and the endothelial barrier. Since the release patterns of FABP and LDH with respect to time were similar during the entire perfusion protocol, it is tempting to state that protein release from tissue is a nonspecific effect of a noxious intervention.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretory pathways in animal cells: With emphasis on pancreatic acinar cells

Studies over the past three decades have clearly established the existence of at least two distinct pathways for the intracellular transport and release of secretory proteins by animal cells. These have been identified as the regulated and constitutive pathways. Many observations have indicated that in certain cells, such as those of the exocrine pancreas and parotid glands at least, these pathways coexist in the same cells. Although the general scheme of protein transport within these pathways is well established, many fundamental aspects of intracellular transport remain to be unraveled. How are proteins transported through the endoplasmic reticulum? How are the transitional vesicles formed and what are the underlying mechanisms involved in their fusion with the cis-Golgi cisterna? Even the general mode of transfer through the Golgi stack is debated: Is there a diffusion through the stack by flow through intercisternal tubules and openings or is there a vesicle transfer system where membrane quanta hop from one cisterna to the other? What is the fate of secretory proteins in the trans-Golgi area and by what mechanisms is a fraction of newly synthesized molecules of a given secretory protein released spontaneously while the majority of such nascent molecules are diverted into a secretory granule compartment? In this review, we have examined these and other aspects of intracellular transport of secretory proteins using pancreatic acinar cells as our reference model and we present some evidence to support the existence of a paragranular pathway of secretion associated with secretory granule maturation.

Identification of an endogenous membrane anchor-cleaving enzyme for group A streptococcal M protein. Its implication for the attachment of surface proteins in gram-positive bacteria.

How streptococcal M protein or other surface proteins of gram-positive bacteria are anchored to the cell is poorly understood. Previously, we reported that M protein released after cell wall removal with a muralytic enzyme lacked the COOH terminal hydrophobic amino acids and charged tail predicted from DNA sequence. An endogenous membrane anchor-cleaving enzyme has now been identified with the ability to release M protein from isolated streptococcal protoplasts. At pH 5.5 in the presence of 30% raffinose, the streptococcal cell wall may be removed with a muralytic enzyme without releasing M protein from the resulting protoplasts indicating that the M molecule is attached through the bacterial cytoplasmic membrane. Release of M molecules occurs when the M protein-charged protoplasts are placed in raffinose buffer at pH 7.4. Although Zn2+, Cd2+, Ca2+, PHMB, and pHMPS inhibit the activity of the releasing enzyme, the blocking activity of Zn2+, Cd2+, and Ca2+ are reversible while PHMB and pHMPS are irreversible. PHMB-treated protoplasts are unable to release M protein at pH 7.4. However, M protein is liberated from these protoplasts when mixed with those prepared from M- streptococci serving as an enzyme source. The supernatant from M- protoplasts is unable to release M protein from PHMB-inactivated M+ protoplasts, confirming that the anchor-cleaving enzyme is membrane bound. Thus, the M protein releasing activity appears to be the result of a thiol-dependent anchor-cleaving enzyme. Streptococcal membranes treated with sodium carbonate and Triton X-114 still retain the M protein verifying that it is an integral membrane molecule. Evidence also is presented indicating significant sequence similarity between M protein and certain GPI-anchored proteins in the region responsible for protein anchoring.

A Mechanism for Anti-Asialo GM 1 Antibody-Induced Anaphylactoid Response in Mice Infected with Trichinella pseudospiralis

Intravenous injection of anti-asialo GM 1 antibody into mice infected with Trichinella pseudospiralis resulted in rapid acute illness or death accompanied by a dramatic rise in hematocrit values in these animals. The described antibody-induced changes were reversible by intravenous infusion of Hanks' balanced salt solution (HBSS). These effects were not seen in uninfected mice or in Trichinella spiralis-infected mice injected with anti-asialo GM 1 antibody. Viability of T. spiralis or T. pseudospiralis infective L1 larvae, both isolated worms and those housed in muscle, was unaffected by exposure to anti-asialo GM 1 antibody and complement. Infectivity of larvae of T. pseudospiralis decreased significantly following exposure to anti-asialo GM 1 antibody. Release of protein by T. pseudospiralis infective L1 larvae during incubation in the presence of anti-asialo GM 1 antibody was significantly greater than that released by worms incubated in normal rabbit serum or HBSS. Protein released by infective L1 larvae of T. pseudospiralis was identified as Trichinella excretory/secretory antigens by immunoblot. Intravenous injection of T. pseudospiralis excretory/secretory products resulted in anaphylaxis in T. pseudospiralis-infected mice but not in uninfected or T. spiralis-infected mice. Excretory/secretory product-induced anaphylactoid response also was reversible by the intravenous injection of HBSS or by injection of an antihistamine. Significantly higher levels of total IgE were observed in sera from mice infected with T. pseudospiralis compared to uninfected or T. spiralis-infected mice. Binding of anti-asialo GM 1 antibody to the surface of T. pseudospiralis muscle larvae induced release of excretory/secretory antigen by the parasite.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor (IGF)-binding protein release by human fetal fibroblasts: dependency on cell density and IGF peptides

Insulin-like growth factors (IGFs) are bound to specific binding proteins in extracellular fluids in vivo and when released by cells in vitro. One class of binding protein (IGF-BP), a peptide of 26 kDa purified from amniotic fluid, has been shown to modulate IGF bioactivity on isolated human fibroblasts. We have determined the factors that control release of IGF-BP from monolayers of human fetal fibroblasts using a radioimmunoassay, and have compared this with the effects of these factors on the release of IGF-I and -II. Separation of cell-conditioned cultured medium on SDS-PAGE, and subsequent immunoblotting with antibody against IGF-BP showed that fibroblasts released a single species of immunoreactive protein of an estimated molecular weight of 30 kDa. This was not the predominant binding protein released by cells since major bands of approximately 42 kDa and 39 kDa were visualized following separation by SDS-PAGE and ligand blotting with 125I-labelled IGF-I. The 30 kDa IGF-BP was released in parallel with radioimmunoassayable IGF-I and -II over 48 h. However, a significant inverse correlation was found between the release of IGF-BP, IGF-I, IGF-II and cell density. The exposure of fibroblasts to 1.3 nmol/l or greater of IGF-I or -II caused a significant release of IGF-BP. Maximum release was seen in sparse cultures with little or no release from confluent cultures. IGF-I and -II were approximately equipotent with a fourfold increase in IGF-BP release at 19.7 nmol/l. Insulin caused a release of IGF-BP and IGF-I and -II from fibroblasts at supraphysiological concentrations (16.7 nmol/l) which again was maximal on sparse cell cultures. Increases in IGF-BP, IGF-I and -II release were also found in the presence of human placental lactogen (23.3 nmol/l), but human GH, epidermal growth factor, fibroblast growth factor and platelet-derived growth factor were without effect. The results show that human fetal fibroblasts released an IGF-BP immunologically similar to that seen in amniotic fluid together with IGF-I and -II, that IGF-BP release was enhanced by exogenous addition of IGF peptides, and that the release of all three peptides was a property of sparsely plated, rapidly growing cells. These findings strengthen the hypothesis that the cellular expression of IGF-binding proteins may represent an important level of control in IGF physiology.

Heparin modulates the secretion of a major excreted protein-like molecule by vascular smooth muscle cells

Previous work from our laboratory has shown that heparin specifically induces the release of a pair of proteins of approximately 35 000 and 37 000 Da into the culture medium of vascular smooth muscle cells (SMC). In this report, we demonstrate that the previously identified 37 000-Da smooth muscle protein is composed of two protein species with very similar molecular weights based on migration pattern in SDS-polyacrylamide gels. The larger molecular weight species in this doublet has a similar molecular weight and shares antigenic determinants with major excreted protein (MEP), a lysosomal proteinase previously shown to be secreted by normal and transformed fibroblasts and epidermal cells. Antisera to MEP precipitated the higher molecular weight band from the doublet; preimmune serum was not reactive with the smooth muscle protein. Exposure of smooth muscle cells to heparin resulted in decreased amounts of immunoprecipitable protein released into the medium. Thus, it now appears that three proteins in the 35 000–38 000 molecular weight range are modulated by heparin, and that the largest of the heparin-modulated vascular SMC proteins has a similar molecular weight and is immunologically related to MEP. The release of MEP-like protein from SMC is decreased by heparin, while the remaining two heparin-modulated proteins are increased in the presence of heparin.

Defensive behaviour and the division of labour in the African honeybee (Apis mellifera scutettata)

1. Venom protein release and alarm pheromone production by African worker honey-bees (Apis mellifera scutellata) were documented with respect to adult age. Behavioural assays were used to explore relationships among venom protein release, the alarm pheromone bouquets and the recruitability of bees to colony defence as a function of age. 2. The concentration of venom protein released by young worker honeybees is low (10–30 μg/bee), gradually increasing with age, reaching a plateau of 60 μg/bee at 17 days and remaining at this value in older bees. 3. Nine alarm pheromones from the sting were identified and analysed gas chromatographically. Of these, only isopentyl acetate, isopentyl alcohol, benzyl alcohol and n-hexyl acetate showed concentration profiles that clearly differed with age. The mandibular gland alarm pheromone 2-heptanone, had a reasonably well-defined concentration profile as related to age. 4. Behavioural assays showed: (1) more bees of all ages responded to an isolated sting (sting alarm pheromones) than to (2) an isolated pair of mandibular glands (2-heptanone). A response equivalent to (1) occurred when both a sting and a pair of mandibular glands were presented together. Older bees (15–25 days old) responded more readily to the donor stings, mandibular glands and stings and mandibular glands together of 15–25 day old bees than did younger bees. The responses of older bees (25 days old) were more rapid and of longer duration than of younger bees. 5. Our combined data on the production of venom protein, alarm signals and their efficacy in recruitment; and the latency and duration of worker responses can be correlated with age. The defensive capacity of African worker bees is one which develops in relation to age and is consistent with defence being yet another element associated with the division of labour in honeybees.

Biosynthesis and release of proteins by isolated pulmonary Clara cells.

The major proteins synthesized and released by Clara cells were identified and compared with those synthesized and released by mixed lung cells. Highly purified Clara cells (85.9 +/- 2.4%) and mixed lung cells (Clara cells 4%, Type II cells 33%, granulocytes 18%, macrophages 2.7%, ciliated cells 1.2%) were isolated from rabbit lungs, incubated with Ham's F12 medium in collagen/fibronectin-coated plastic culture dishes in the presence of 35S-methionine for periods of 4 and 18 hrs. Radiolabelled proteins were isolated from the cells and from the culture medium, electrophoresed on polyacrylamide gels in the presence of SDS under reducing conditions, and then autoradiographed. After 4 and 18 hr of incubation of the Clara cells the major radiolabelled cell-associated proteins were those with molecular weights of 6, 48, and 180 Kd. The major radiolabelled proteins released by Clara cells into the medium after 4 hrs of incubation had molecular weights of 6, 48, and 180 Kd, accounting for 42, 16, and 10%, respectively, of the total extracellular protein-associated radioactivity. After 18 hr of incubation the 6 and 48 Kd proteins represented 30 and 18% of the total released radioactivity, and the relative amount of the 180 Kd protein had decreased to 3%. With the mixed lung cells, the major proteins released into the medium had molecular weights of 6 and 48 Kd. Under nonreducing conditions the 6 Kd protein released by Clara cells had an apparent molecular weight of 12 Kd. Labelling isolated Clara cells with a mixture of 14C-amino acids also identified this low molecular weight protein as the major secretory product of the Clara cell. The 6 Kd protein did not label when the cells were incubated with 14C-glucosamine indicating that it was not a glycoprotein. These data demonstrate the release of several proteins from isolated Clara cells but the major protein had a molecular weight of 6 Kd.

Platelet-derived collagenase inhibitor: characterization and subcellular localization.

Purified human platelets were found to contain a collagenase inhibitor that is immunologically, functionally, and chromatographically identical to that produced by human skin fibroblasts. None of the other formed elements of the blood (erythrocytes, granulocytes, mononuclear cells) possessed detectable quantities of this protein. Virtually all the collagenase inhibitor contained within platelets was released following platelet activation with thrombin. Similarly, platelet activation accompanying blood clotting also resulted in the release of this protein, the ratio of plasma to serum inhibitor levels being approximately equal to 0.5. When platelets were subjected to subcellular fractionation, essentially all of the platelet-associated collagenase inhibitor was found to be located in the alpha-granule. Studies with radiolabeled inhibitor failed to detect uptake of inhibitor by platelets. Furthermore, immunologically reactive protein of similar quantity to that found in platelets was identified in human megakaryocyte lysates. Thus, the data suggest that the collagenase inhibitor is endogenously produced and stored within platelet alpha-granules. The platelet-derived collagenase inhibitor was antigenically identical to the collagenase inhibitor from human skin fibroblasts in double immunodiffusion and, like its fibroblast counterpart, inhibited collagenase on a 1:1 stoichiometric basis. When subjected to several of the chromatographic procedures utilized to purify the fibroblast protein, the platelet inhibitor behaved in an indistinguishable manner. Platelet factor 4, previously reported to be a collagenase inhibitor, was found to be immunologically unrelated to the platelet-derived collagenase inhibitor. Furthermore, platelet factor 4 displayed no collagenase inhibitory activity. Although the function of platelet-derived collagenase inhibitor is unknown, such a protein released by activated platelets may serve to regulate collagen turnover during the early stages of the inflammatory process.

Mechanical and chemical injury to thylakoid membranes during freezing in vitro

The relative contributions of membrane rupture due to osmotic stress and of chemical membrane damage due to the accumulation of cryotoxic solutes to cryoinjury was investigated using thylakoid membranes as a model system. When thylakoid suspensions were subjected to a freeze-thaw cycle in the presence of different molar ratios of NaCl as the cryotoxic solute and sucrose as the cryoprotective solute, membrane survival first increased linearly with the osmolality of the solutions used to suspend the membranes, regardless of the molar ratio of salt to sucrose. It subsequently decreased when the ratio of sucrose to salt was not sufficiently high for complete cryopreservation by sucrose. There was an optimum of cryopreservation at intermediate osmolalities (approx. 0.1 osmol/kg). This optimum of cryopreservation at a given sucrose concentration could be shifted to lower solute concentration, if mixtures of NaCl and NaBr were used instead of NaCl alone. At suboptimal initial osmolalities, damage is attributed mainly to membrane rupture. Under these conditions, cryopreservation is not influenced by the chaotropicity of the suspending medium. At supraoptimal initial solute concentrations, solute (i.e., chemical) effects determine membrane survival. Under these conditions, increased ratios of sugar to salt increased cryoprotection. In mixtures of NaCl and NaBr at constant molar ratios of salt to sucrose, chemical membrane damage was quantitatively related to the lyotropic properties of the ions used. The degree of chemical damage becomes more pronounced with rising osmolalities of the suspending media. With NaF as the cryotoxic solute, damage was more severe than should be expected from its lyotropic properties. This may reflect a specific interaction of fluoride with the membranes. Protein release from the membranes during freezing in the presence of different anions was qualitatively comparable at identical ratios of sugar to salt. However, the total amount of protein released was correlated linearly with membrane inactivation, even when different anions acted on the membranes. Gel electrophoretic analysis of proteins released from thylakoid membranes during freezing revealed discrete bands indicative of mechanical and chemical damage, respectively.

Loss of membrane proteins from thylakoids during freezing

Slow freezing of thylakoids (about 0.5 K/min) in media containing an excess of cryotoxic solutes results in the release of membrane proteins and in the rapid inactivation of photophosphorylation and other processes dependent on structural intactness of membranes. Although loss of photophosphorylation was observed before protein release became extensive, both processes were dependent on the ratio of cryotoxic to cryoprotective solutes in the membrane suspensions. Moreover, inactivation of photophosphorylation and protein release during freezing were similarly influenced by different cryotoxic solutes. Cryotoxicity of different anions increased with increasing Stoke's law hydrated anion radius ( I > Br > Cl > F ). Divalent cations were more cryotoxic than monovalent alkali cations. Cryotoxicity thus followed a Hofmeister lyotropic power series. Two opposing effects of low temperature influenced protein release. Lowering the temperature increased accumulation of cryotoxic solutes in the membrane vicinity which promoted protein dissociation. However, rates of protein dissociation increased with increasing temperature. While the freezing temperature unequivocally determined the concentration of solutes in a solution coexisting with ice, both the extent of membrane inactivation and protein release depended on the initial concentration of solutes and on membrane concentration. Apparently, the volumes of the unfrozen hydrophilic phase and of the membranes are also important factors in freezing injury. The polypeptide pattern of released proteins differed drastically from that of thylakoids, but the same main proteins were released during freezing of membranes in the presence of different cryotoxic solutes. More than 35 polypeptide bands were observed in SDS-polyacrylamide gel electrophoretograms of released proteins. While the amount of protein released differed depending on freezing conditions and the composition of the suspending medium, more than 10% of the total membrane protein could be solubilized during freezing in the presence of sodium bromide or sodium iodide. Protein release during freezing is thought to be caused primarily by a suppression of intramembrane electrostatic interactions and ion competition although solute effects on water structure may also play a role.

Rapidly transported proteins released from nerve and isolated with myelin

An in vitro preparation was used to determine if rapidly transported radiolabeled protein which is isolated with the myelin fraction of frog sciatic nerves is similar to rapidly transported radiolabeled protein released from sciatic nerves. Sciatic nerves along with their eighth and ninth dorsal root ganglia were placed in modified Warburg flasks. The center well of the flasks contained 100 μCi [ 3H]leucine. The preparation was incubated for 24 h, the sciatic nerve removed and the myelin isolated. The solution in which the sciatic nerve portion of the preparation incubated was removed, dialyzed and lyophilized. The radiolabeled proteins isolated from myelin and those released from the nerve were separated on 10% SDS polyacrylamide gels. Results of this separation revealed that a radiolabeled protein of 100,000 dalton was isolated with myelin and released from sciatic nerves. Incubating isolated myelin in 10 mM EDTA for 14–16 h broughtabout the release of a protein of 100,000 daltons.

Release of Soluble Protein and Nitrogen in Alfalfa. II. Infleunce of Potassium and Nitrogen Fertilizer on Three Cultivars1

Reduced incidence of ruminant bloat was reported on legume pastures fertilized with high levels of potassium (K) or K plus nitrogen (N). Amount of soluble protein released from alfalfa (Medicago sativa L.) into sodium maleate buffer (pH 6.9) was proposed as possible estimate of ruminant bloat potential in comparative studies. Our objective was to determine whether K and N fertilization would influence concentration of several N fractions released from alfalfa.‘Ramsey,’ ‘Saranac,’ and ‘520’ alfalfa cultivars were fertillzed in the fall with 0 (control), 350, and 700 kg as K2SO4/ha and in the following spring with these same levels of N as NH4NO(34 3 × 3 factorial). The upper 10 to 15 cm of told‐bud stage alfalfa in first and second crops was analyzed for several N fractions.As N applied increased, mean concentrations of total N (TN), total soluble N (TSN), soluble nonprotein (SNPN), and soluble protein N (SPN) increasedm first‐harvest herbage. Mean concentrations of SPN increased 10.8% at 300 kg N/ha and 13.3% at 700 kg over that in alfalfa not fertilized with N. As the K level increased, mean concentrations of TN, TSN, and SNPN decreased in first‐harvest herbage. Fertilization with K did not affect concentration of SPN. Mean concentrations of TN, TSN, and SNPN in Saranac and TN, TSN, and SPN in 520 first‐harvest herbage were slightly but significantly greater than those in Ramsey. Cultivars did not affect concentrations of N fractions in secondharvest herbage.We found no evidence that fertilization of alfalfa with K or N reduced its apparent bloat potential, as estimated by SPN release. Instead, we showed that alfalfa fertilized with high levels of N had a slightly increased apparent bloat potential.