Structural relationship between the two small hydrophobic apoproteins in bovine pulmonary surfactant

Lipid extracts of bovine pulmonary surfactant contain two very hydrophobic surfactant-associated proteins (SP) designated SP-B (15 kDa nonreduced) and SP-C (3.5 kDa). These two low molecular weight apoproteins were delipidated and purified on silica SEP-PAK cartridges using various reagents. Dansylation studies revealed that the 15 kDa apoprotein has three N-termini: Phe, Leu and Ile, while the 3.5 kDa apoprotein has two N-termini: Leu and Ile. In either protein, only a very small amount of N-Ile is present. Quantitative N-terminal dansylation analysis of the 15 kDa protein indicated that Phe and Leu (plus Ile) are present in a 1:1 ratio. Carboxy-terminal analysis showed that the 15 kDa protein contains C-terminal Gly, and the 3.5 kDa protein contains C-terminal Leu. Gas-phase amino terminal sequencing of the 15 kDa protein revealed almost exclusively the Phe-polypeptide (SP-B). These results suggest that the 15 kDa apoprotein is not an oligomer of SP-B and SP-C. The reason that analysis of SP-B reveals N-terminal Leu and Ile by dansylation which cannot be confirmed by amino acid sequencing is not known.     

Analysis of pulmonary surfactant apoproteins by isoelectric focusing

Apoproteins of Mr 38 000, 32 000 and 26 000 are found in surfactant isolated from rat lungs. The surfactant isolated from monkey lungs, on the other hand, contains the 38 kDa apoprotein and not the 32 and 26 kDa apoproteins. These preparations of pulmonary surfactant contain, in addition, several serum proteins. We have used a combination of salt- and sucrose-density gradient centrifugations to isolate and further purify surfactant from the washings of rat lungs. Thus, a preparation of pulmonary surfactant was obtained which contained exclusively the 38, 32, 26 and 10-12 kDa apoproteins, and which was rich in phosphatidylcholine and phosphatidylglycerol. Using an immunoassay and an immunoblotting technique, it was established that the 38, 32 and 26 kDa apoproteins are not serum proteins. The surfactant apoproteins of rat and monkey were further subjected to the high-resolution of isoelectric focusing. Thus, rat surfactant apoproteins resolved into 11 bands in the pH range 4.64-5.53. A second-dimensional electrophoresis in a sodium dodecyl sulfate system led to the migration of the 11 bands, separated by first-dimensional isoelectric focusing, into three distinct groups with apparent molecular weights of 38 000, 32 000 and 26 000, respectively. Upon isoelectric focusing, the apoproteins from monkey lung surfactant also separated into several bands in the pH range 5.18-5.82. After electrophoresis in the second dimension as above, these bands migrated as a single group with an apparent molecular weight of 38 000. Neuraminidase treatment of rat surfactant apoproteins, and subsequent IEF, led to the disappearance of several low-pI variants with a concomitant increase in the amounts of higher-pI variants. Thus, the sialic acid content of surfactant apoproteins accounts for, in large part, the observed charge heterogeneity.     

Comparative studies on the biophysical activities of the low-molecular-weight hydrophobic proteins purified from bovine pulmonary surfactant

Two low-molecular-weight hydrophobic proteins with nominal molecular weights Mr = 15,000 and Mr = 3,500 have been isolated from the lipid extracts of bovine pulmonary surfactant by several methods, including (a) dialysis plus silicic acid chromatography, (b) elution from Waters SEP-PAK silica cartridges with a variety of solvent mixtures, and (c) ultrafiltration. As detailed in the text, these proteins have been designated surfactant-associated protein-BC (SP-BC) (15 kDa: nonreduced), and SP-C (3.5 kDa). The biophysical activities of reconstituted surfactant containing these proteins and the phospholipids present in lung surfactant have been compared with the biophysical activities of bovine lipid extract surfactant on a pulsating bubble surfactometer using a phospholipid concentration of 10 mg/ml. At this concentration, unmodified lipid extract surfactant reduces the surface tension of the pulsating bubble to near 0 within 10 pulsations at 20 cycles per min. Similar biophysical properties were observed with modified lipid extract surfactant in which the relative concentration of hydrophobic protein had been reduced from 1 to 0.4% (W/W) of the phospholipids by addition of dipalmitoylphosphatidylcholine (DPPC) or DPPC plus phosphatidylglycerol. Reconstituted surfactants, which contained partially delipidated SP-BC (15 kDa: nonreduced) obtained by method (a) at a relative concentration of 0.1%, were also capable of reducing the surface tension to near 0 mN/m. Preparations of SP-BC (15 kDa: nonreduced) obtained by method (b), which had been subjected to very low pH levels during isolation and were extensively delipidated, exhibited full biophysical activity only at higher protein concentrations and with prolonged pulsation. Extensively delipidated samples of SP-BC obtained by method (c) exhibited impaired biophysical activities, even when prepared with neutral organic solvents. Reconstituted surfactant samples containing SP-C (3.5 kDa) obtained by any of the methods listed above were only able to reduce the surface tension at minimum bubble radius to approx. 20 mN/m. The biophysical activity of SP-C (3.5 kDa) was not significantly affected by low pH or extensive delipidation. Reconstituted samples containing mixtures of SP-BC (15 kDa: nonreduced) and SP-C (3.5 kDa) were more effective than samples containing either protein alone. Furthermore, with samples containing both hydrophobic proteins the final surface tensions at maximum bubble radius were attained within a few bubble pulsations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reconstitution of surfactant activity by using the 6 kDa apoprotein associated with pulmonary surfactant.

Lipid extracts of bovine pulmonary surfactant containing the 6 kDa apoprotein, but lacking the 35 kDa apoprotein, can mimic the essential characteristics of pulmonary surfactant on a pulsating-bubble surfactometer. Reconstituted surfactant can be produced by combining silicic acid fractions containing 6 kDa apoprotein and phosphatidylglycerol with phosphatidylcholine. Treatment of the protein-containing fraction with proteolytic enzymes abolishes its efficacy. These results indicate that the presence of the 6 kDa apoprotein can account for some of the essential physical and biological characteristics of pulmonary surfactant. Immunodiffusion studies indicate that, contrary to earlier suggestions, the 6 kDa apoprotein is not structurally related to the major surfactant apoprotein that has a molecular mass of 35 kDa.     

Identification of surfactant proteolipid SP-B in human surfactant and fetal lung

Surfactant proteolipid (SP-B) is one of several hydrophobic peptides detected in organic extracts of pulmonary surfactant and associated with the dramatic surface-active properties of surfactant phospholipids. In the present study human SP-B was identified as a protein with a relative molecular weight (Mr) of 7,500-8,000 under reducing conditions; protein of Mr 18,000 was detected under nonreducing conditions by immunoblot analysis of organic extracts of bovine and human surfactant utilizing an antiserum directed against a 60-amino acid synthetic SP-B peptide. This peptide antiserum was subsequently used to identify SP-B in explant cultures of 18- to 23-wk gestation human fetal lung. Immunoprecipitation of explants labeled with [35S]methionine after 48 h of culture identified proteins of Mr 40,000-42,000, 25,000, and 18,000 after electrophoresis under nonreducing conditions. The Mr 18,000 form was reduced to Mr 7,500-8,000 in the presence of beta-mercaptoethanol. These molecular forms likely represent the SP-B precursor protein, a proteolytic intermediate, and the mature SP-B peptide, respectively. Immunocytochemistry with the peptide antiserum localized SPL(Phe) in granular inclusions in the apical region of type II-like epithelial cells, a pattern of staining similar to that observed for the major surfactant-associated protein of Mr 26,000-38,000 (SP-A). SP-B is a novel pulmonary surfactant-associated protein that is synthesized by the human alveolar type II epithelial cell as an Mr 40,000-42,000 precursor that is subsequently proteolytically processed to Mr 7,500-8,000.     

Effects of Hydrophobic Amino Acid Substitutions on Antimicrobial Peptide Behavior

Antimicrobial peptides (AMPs) are naturally occurring components of the immune system that act against bacteria in a variety of organisms throughout the evolutionary hierarchy. There have been many studies focused on the activity of AMPs using biophysical and microbiological techniques; however, a clear and predictive mechanism toward determining if a peptide will exhibit antimicrobial activity is still elusive, in addition to the fact that the mechanism of action of AMPs has been shown to vary between peptides, targets, and experimental conditions. Nonetheless, the majority of AMPs contain hydrophobic amino acids to facilitate partitioning into bacterial membranes and a net cationic charge to promote selective binding to the anionic surfaces of bacteria over the zwitterionic host cell surfaces. This study explores the role of hydrophobic amino acids using the peptide C18G as a model system. These changes were evaluated for the effects on antimicrobial activity, peptide-lipid interactions using Trp fluorescence spectroscopy, peptide secondary structure formation, and bacterial membrane permeabilization. The results show that while secondary structure formation was not significantly impacted by the substitutions, antibacterial activity and binding to model lipid membranes were well correlated. The variants containing Leu or Phe as the sole hydrophobic groups bound bilayers with highest affinity and were most effective at inhibiting bacterial growth. Peptides with Ile exhibited intermediate behavior while those with Val or α-aminoisobutyric acid (Aib) showed poor binding and activity. The Leu, Phe, and Ile peptides demonstrated a clear preference for anionic bilayers, exhibiting significant emission spectrum shifts upon binding. Similarly, the Leu, Phe, and Ile peptides demonstrated greater ability to disrupt lipid vesicles and bacterial membranes. In total, the data indicate that hydrophobic moieties in the AMP sequence play a significant role in the binding and ability of the peptide to exhibit antibacterial activity.     

Purification and characterization of honey bee vitellogenin

A protocol has been developed for the purification of vitellogenin from the honey bee, Apis mellifera. Purification allows for the first characterization of a vitellogenin from the large order Hymenoptera. Hymenopteran vitellogenins are unusual among insect vitellogenins in that they contain only one type of apoprotein. The honey bee vitellogenin was isolated from hemolymph of honey bee queens by a combination of density gradient ultracentrifugation, ion-exchange chromatography, and affinity chromatography. The native vitellogenin particle is a very high density glycolipoprotein containing approximately 91% protein, 7% lipid, and 2% carbohydrate. Phospholipid and diacylglycerol are the major lipid components. The equilibrium density (1.28 g/ml) is the same as that for Manduca sexta vitellogenin, which contains a much higher proportion of lipid. The covalently bound carbohydrate moiety of the particle is high in mannose. The amino acid composition of vitellogenin is similar to those of vitellogenins from other insect species. The N-terminal amino acid sequence of the apoprotein was determined, the first such sequence for any insect vitellogenin. When analyzed by sodium dodecyl sulfate (SDS)-gel electrophoresis, A. mellifera vitellogenin resolved into a single band with an apparent Mr of 180,000. Gel filtration under reducing and native conditions yielded estimated Mr values of about 300,000.     

Effect of surfactant-associated protein-A (SP-A) on the activity of lipid extract surfactant

The properties of natural bovine surfactant and its lipid extract have been examined with a pulsating bubble surfactometer which assesses the ability of surfactant lipids to adsorb to the air/liquid interface and reduce the surface tension to near 0 dynes/cm during dynamic compression. Studies conducted at 1 mg/ml phospholipid revealed that the surface activity (i.e., the ability to produce low surface tensions) of lipid extracts could be enhanced by incubating the sample at 37 degrees C for 120 min or by addition of CaCl2. In contrast, incubation at 37 degrees C only slightly improved the biophysical activity of natural surfactant and the addition of CaCl2 had a more modest effect than with lipid extracts. With 20 mM CaCl2, the surfactant activity of lipid extract surfactant was similar to that of natural surfactant. Incubation with EDTA reduced the biophysical activity of natural surfactant. Experiments in which increasing amounts of lipid extract were replaced by natural surfactant revealed that small amounts of natural surfactant enhanced the surfactant activity of lipid extract. The biophysical activity of lipid extract surfactant was also increased by the addition of soluble surfactant-associated protein-A (SP-A) (28-36 kDa) purified from natural bovine surfactant. These results indicate that SP-A (28-36 kDa) improves the surfactant activity of lipid extracts by enhancing the rate of adsorption and/or spreading of phospholipid at the air/liquid interface resulting in the formation of a stable lipid monolayer at lower bulk concentrations of either phospholipid or calcium.     

Substrate specificity of platypus venom L-to-D-peptide isomerase

The L-to-D-peptide isomerase from the venom of the platypus (Ornithorhyncus anatinus) is the first such enzyme to be reported for a mammal. In delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used N-terminal segments of defensin-like peptides DLP-2 and DLP-4 and natriuretic peptide OvCNP from the venom as substrates. The DLP analogues IMFsrs and ImFsrs (srs is a solubilizing chain; lowercase letters denote D-amino acid) were effective substrates for the isomerase; it appears to recognize the N-terminal tripeptide sequence Ile-Xaa-Phe-. A suite of 26 mutants of these hexapeptides was synthesized by replacing the second residue (Met) with another amino acid, viz. Ala, alpha-aminobutyric acid, Ile, Leu, Lys, norleucine, Phe, Tyr, and Val. It was shown that mutant peptides incorporating norleucine and Phe are substrates and exhibit L- or D-amino acid isomerization, but mutant peptides that contain residues with shorter, beta-branched or long side chains with polar terminal groups, viz. Ala, alpha-aminobutyric acid, Ile, Val, Leu, Lys, and Tyr, respectively, are not substrates. It was demonstrated that at least three N-terminal amino acid residues are absolutely essential for L-to-D-isomerization; furthermore, the third amino acid must be a Phe residue. None of the hexapeptides based on LLH, the first three residues of OvCNP, were substrates. A consistent 2-base mechanism is proposed for the isomerization; abstraction of a proton by 1 base is concomitant with delivery of a proton by the conjugate acid of a second base.     

藻红蓝蛋白裂合异构酶对几种脱辅基藻胆蛋白的催化作用

PecE/PecF是层理鞭枝藻藻红蓝蛋白α亚基(α-PEC)生物合成的裂合异构酶。以4种脱辅基藻胆蛋白为底物,初步研究了PecE/PecF对底物蛋白的催化专一性。结果表明,PecE/PecF可催化藻蓝胆素(PCB)与高度同源的层理鞭枝藻不同亚种的α-PEC脱辅基蛋白的体外重组,也可催化经128位Trp定点突变到Phe而得到的α-PEC脱辅基蛋白的体外重组,但PecE/PecF对PCB与藻蓝蛋白α亚基(α-CPC)脱辅基蛋白的体外重组无催化作用。A-PEC脱辅基蛋白的重组不受表面活性剂Triton X-100的影响,而Triton X-100可改进PCB与α-CPC脱辅基蛋白的重组。     

Cloning and expression of the bioluminescent photoprotein pholasin from the bivalve mollusc Pholas dactylus

Pholasin is the photoprotein responsible for luminescence in the bivalve Pholas dactylus and consists of a luciferin tightly bound to a glycosylated protein. It is a sensitive indicator of reactive oxygen species. A full-length clone encoding apopholasin was isolated from a P. dactylus light organ cDNA library. The unprocessed apoprotein contained 225 amino acids, starting with a signal peptide of 20 amino acids, 3 predicted N-linked glycosylation sites, 1 O-linked site, no histidines, and 7 cysteines. The recombinant apoprotein was expressed in cell extracts and insect cells. The size of the apoprotein expressed in cell extracts and the cytosol of insect cells was 26 kDa but that of the fully processed protein was 34 kDa, as was native pholasin. Both the processed and unprocessed recombinant apoproteins were recognized by a polyclonal antibody raised against native pholasin. Acid methanol extracts from Pholas added to recombinant apoprotein resulted in chemiluminescence triggered by sodium hypochlorite but not photoprotein formation. These results have important implications in understanding the molecular evolution of bioluminescence and will allow the development of recombinant pholasin as an intracellular indicator of reactive oxygen species.     

Purification of canine surfactant-associated glycoproteins A. Identification of a collagenase-resistant domain

A procedure for purification of surfactant-associated glycoproteins A from canine surfactant was established utilizing preparative isoelectric focusing as a major purification step in absence of detergents. The proteins migrated as charge trains, isoelectric points 4.2-5.0. Unglycosylated forms of surfactant-associated protein A1 (26 kDa) and glycoproteins A2 and A3 (32-36 kDa) were identified by silver-staining and immunoblot analysis. These forms were demonstrated to be identical polypeptides by fingerprint analysis of 125I-labeled peptides generated by tryptic-chymotryptic digests of the iodinated proteins. Size and charge heterogeneity were related to varying amounts of N-linked complex carbohydrates, including sialic acid, which were sensitive to endoglycosidase F and neuraminidase but resistant to endoglycosidase H. A collagenase-sensitive region was demonstrated which was required for sulfhydryl-dependent oligomerization of the molecule. Collagenase treatment resulted in removal of approx. 10 kDa from the glycoprotein molecule. Collagenase-resistant fragments of 21-23 kDa migrated with carbohydrate-dependent size and charge heterogeneity and were reduced to 16 kDa by endoglycosidase F. Amino acid composition of the surfactant glycoproteins demonstrated high glycine content which was diminished after digestion with collagenase. Several glycine-rich tryptic peptides were isolated by reverse-phase chromatography. Partial sequence information shows Gly-X-Y repeat sequences containing hydroxyproline residues. The major canine surfactant-associated proteins, glycoproteins A contain complex-type N-linked carbohydrate. In addition, a separate collagen-like peptide domain is present and is required for sulfhydryl-dependent oligomerization.     

Immunologically related multimeric forms of 30–40 kDa peptides associated with lung surfactant in various mammalian species

A comparative study of lung surfactant associated proteins was undertaken to determine which mammalian species would best serve as models for investigating alterations of the human lung surfactant system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified surfactants in the presence of dithiothreitol revealed that surfactant invariably contains at least one peptide with molecular weight of 30 000–40 000. In the absence of disulfide reducing agents, the above peptides were in the form of high molecular-weight proteins (> 400 kDa) in primates and cat, whereas in dog, rat and rabbit, the protein was a 72 kDa dimer. The 30–40 kDa peptide subunits were isolated from human, rat and dog surfactants and found to contain four or five residues of hydroxyproline. Antisera to either the human 34 kDa peptide or high-molecular-weight proteins reacted with the high-molecular-weight bands, the 34 kDa subunit and at least six intermediate disulfide-linked forms separated from purified human surfactant by electrophoresis under nonreducing conditions. Following electrophoresis in the presence of dithiothreitol, both antisera detected the 34 kDa peptide as well as other peptides ranging in molecular weight from 23 000 to 160 000. The isolated 34 kDa peptide readily reaggregated into disulfide-linked forms including 68 and 100 kDa complexes which were not reduced by 40 mM dithiothreitol. We conclude that the 34 kDa surfactant-associated peptide forms a complex system of monomeric and multimeric proteins, which varies among the species and could conceivably vary in distribution during lung development or disease.     

Proteolipid in bovine lung surfactant: its role in surfactant function

The chemical and biophysical properties of the proteins in the lipid extracts of lung surfactant have not clearly been determined. These proteins were isolated from lung surfactant lipids by Sephadex LH-20 chromatography and purified with silicic acid chromatography followed by dialysis against organic solvents. The proteolipid thus obtained had a protein to phospholipid ratio of 3 to 1 (w/w). The proteolipid apoprotein had a nominal molecular weight of ca. 5 kDa. We evaluated the functional role of this proteolipid by combining it with proteolipid-depleted surfactant lipids or synthetic dipalmitoylphosphatidylcholine (DPPC) and then measuring with a pulsating bubble surfactometer. The proteolipid and DPPC recombinant reproduced the surface activity of natural lung surfactant. We conclude that this 5 kDa proteolipid apoprotein is a functionally important constituent of lung surfactant.     

Monoclonal antibodies against human pulmonary surfactant apoproteins: specificity and application in immunoassay

Monoclonal antibodies were prepared against pulmonary surfactant apoproteins which were isolated from lung lavages of patients with alveolar proteinosis with the following steps: solubilization of the surface-active fraction by Triton X-100, delipidation with butanol-ethanol extraction followed by column chromatographies on Blue-Sepharose and DEAE-Toyopearl in the presence of dithiothreitol. The fraction including 62 and 36 kDa proteins, i.e., pulmonary surfactant apoproteins, was used for the immunization. Monoclonal antibodies against the pulmonary surfactant apoproteins were prepared using hybridoma technology. The monoclonal antibodies prepared, PC6 and PE10, recognized the same proteins, i.e., 62 and 36 kDa proteins, in the patients' lavages. They also recognized 37 and 34 kDa proteins in human lung lavage and amniotic fluid. Quantitation of the apoproteins by enzyme-immunoassay using the monoclonal antibodies has been developed. A combination of PC6 and PE10 was found to be useful for a two-site sandwich enzyme-linked immunosorbent assay (ELISA), where it gave a good dose response and was capable of measuring 10-1280 ng of the apoprotein/ml. The specificity of the monoclonal antibodies in animal species was tested by this sandwich ELISA. The results indicated that the monoclonal antibodies obtained in this study are specific for the human lung.     

No evidence for vitamin K-dependent carboxylation of canine surfactant apoproteins, 28-36 kDa.

Recent research has shown that rat surfactant apoproteins (26-38 kDa) are vitamin K-dependent [Rannels, Gallaher, Wallin & Rannels (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5952-5956]. We have investigated the effect of the vitamin K antagonist warfarin on this family of apoproteins in surfactant from dog lung. Our data suggest that warfarin does not interfere with synthesis and secretion of these proteins into dog lung surfactant. Abnormal surfactant apoproteins, produced in response to warfarin treatment of the dog, were also not found in lung surfactant. 4-Carboxyglutamic acid analysis of purified dog apoproteins also failed to detect the vitamin K-modification. When vitamin K-dependent 14C labelling of precursors of vitamin K-dependent proteins was carried out, fluorography of these precursors, when electrophoresed into SDS/polyacrylamide gels, revealed 14C-labelled proteins of apparent molecular mass 74, 46, 42, 34, 31 and 23 kDa. Antibodies produced against purified dog surfactant apoproteins recognized precursors of the surfactant apoproteins in lung microsomes but did not recognize any 14C-labelled carboxylase substrates. These precursors appeared on immunoblots with apparent molecular mass 29, 32, 33 and 50 kDa. Our data suggest that there are significant differences between this class of surfactant apoproteins in the rat and the dog.     

Effects of surfactant-associated protein SP-B synthetic analogs on the structure and surface activity of model membrane bilayers.

The effect of several synthetic peptides based on the sequence of human pulmonary surfactant-associated protein B (SPB) on the molecular packing of model membrane lipids (7:1 dipalmitoyl phosphatidylcholine (DPPC)/dipalmitoyl phosphatidylglycerol (DPPG)) was studied using fluorescence anisotropy. This information was then correlated with complementary biophysical data obtained on both a modified Wilhelmy-Langmuir balance and a pulsating bubble surfactometer. The SP-B peptides examined in these studies are synthetic human SP-B Phe1-Ser78 (SP-B 1-78, full-length sequence), synthetic human SP-B Phe1-Thr60 (SP-B 1-60), synthetic human SP-B Phe1-Ala20 (SP-B 1-20), synthetic human SP-B Ala20-Thr60 (SP-B 20-60), synthetic human SP-B Leu27-Ser78 (SP-B 27-78), synthetic human SP-B Leu40-Thr60 (SP-B 40-60) and synthetic human SP-B Tyr53-Ser78 (SP-B 53-78). trans-parinaric acid was utilized to detect changes in ordering of lipids within the interior upon incorporation of synthetic SP-B peptide, whereas 1-hexadecanoyl-2-[N-(7-nitro-2-benzoxa-1,3-diazol-4-yl)-a min ohexanoyl] phosphatidylcholine (6-NBD-PC) and 1-acyl-2-[N-(7-nitro-2-benzoxa-1,3-diazol-4-yl)aminohexanoyl ] phosphatidylglycerol (6-NBD-PG) were utilized to determine alterations in lipid order at the surface of the model membrane bilayer. With the exception of SP-B 40-60, which corresponds to the most hydrophobic segment of the full-length SP-B, none of the other peptide significantly perturbed the interior bilayer as determined by fluorescence anisotropy of trans-parinaric acid. Incorporation of any of the peptides with the exception of SP-B 40-60, resulted in an increase in anisotropy of NBD-PC. The most significant enhancements resulted from the addition of SP-B 1-78, SP-B 1-20, SP-B 27-78 or SP-B 53-78. The magnitude of anisotropy increase with these peptides is similar to that observed with an equivalent molar ratio of native SP-B isolated from a bovine source. These observations suggest that these four synthetic peptides have the structural and compositional characteristics required for surface ordering of the membrane bilayer in a manner similar to that observed with native SP-B, thereby facilitating the surfactant-like properties of phospholipid mixtures.     

Immunologically related multimeric forms of 30-40 kDa peptides associated with lung surfactant in various mammalian species

A comparative study of lung surfactant associated proteins was undertaken to determine which mammalian species would best serve as models for investigating alterations of the human lung surfactant system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified surfactants in the presence of dithiothreitol revealed that surfactant invariably contains at least one peptide with molecular weight of 30 000-40 000. In the absence of disulfide reducing agents, the above peptides were in the form of high-molecular-weight proteins (greater than 400 kDa) in primates and cat, whereas in dog, rat and rabbit, the protein was a 72 kDa dimer. The 30-40 kDa peptide subunits were isolated from human, rat and dog surfactants and found to contain four or five residues of hydroxyproline. Antisera to either the human 34 kDa peptide or high-molecular-weight proteins reacted with the high-molecular-weight bands, the 34 kDa subunit and at least six intermediate disulfide-linked forms separated from purified human surfactant by electrophoresis under nonreducing conditions. Following electrophoresis in the presence of dithiothreitol, both antisera detected the 34 kDa peptide as well as other peptides ranging in molecular weight from 23 000 to 160 000. The isolated 34 kDa peptide readily reaggregated into disulfide-linked forms including 68 and 100 kDa complexes which were not reduced by 40 mM dithiothreitol. We conclude that the 34 kDa surfactant-associated peptide forms a complex system of monomeric and multimeric proteins, which varies among the species and could conceivably vary in distribution during lung development or disease.     

Purification and properties of cathepsin D from the mammary glands of lactating rabbits

Cathepsin D was purified from the lactating rabbit mammary gland by a rapid procedure, which included fractionation with (NH4)2SO4, acid precipitation, double affinity chromatography on pepstatin-Sepharose 4B and gel filtration on Sephadex G-100, resulting in approximately 360-fold purification of the enzyme over the homogenate and approximately 16% recovery. After isoelectric focusing, the enzyme dissociated into four (pI 5.8, 6.3, 6.5 and 7.2) multiple forms, but appeared homogeneous on polyacrylamide gel electrophoresis. Cathepsin D has a Mr of 45 kDa as determined by Sephadex G-100 column chromatography. On sodium dodecylsulfate/polyacrylamide gel electrophoresis the enzyme gave a single protein band, corresponding to Mr of 45 kDa. The amino acid composition of the enzyme is similar to that of cathepsins D from other tissues. A single N-terminal amino acid was glycine. Cathepsin D contains 6.4% carbohydrates consisting of mannose, galactose, fucose and glucosamine at a ratio of 3:9:2:2. Cathepsin D is inhibited by pepstatin with Ki of 2.5 X 10(-9) M and irreversibly by N-diazoacetyl-N'-2.4-dinitrophenyl-ethylene diamine. The enzyme hydrolyzes bovine hemoglobin with the maximal activity at pH 3.0 with Km = 10(-5) M and HLeu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe with Km = 4 X 10(-5) M and Rcat = 0.95 s-1. The major cleavage sites were Leu15-Tyr16, Phe24-Phe25 and Phe25-Tyr26 during hydrolysis of the oxidized insulin B-chain by cathepsin D.     

Self‐assembly of short peptides composed of only aliphatic amino acids and a combination of aromatic and aliphatic amino acids

The morphology of structures formed by the self‐assembly of short N‐terminal t‐butyloxycarbonyl (Boc) and C‐terminal methyl ester (OMe) protected and Boc‐deprotected hydrophobic peptide esters was investigated. We have observed that Boc‐protected peptide esters composed of either only aliphatic hydrophobic amino acids or aliphatic hydrophobic amino acids in combination with aromatic amino acids, formed highly organized structures, when dried from methanol solutions. Transmission and scanning electron microscopic images of the peptides Boc‐Ile‐Ile‐OMe, Boc‐Phe‐Phe‐Phe‐Ile‐Ile‐OMe and Boc‐Trp‐Ile‐Ile‐OMe showed nanotubular structures. Removal of the Boc group resulted in disruption of the ability to form tubular structures though spherical aggregates were formed. Both Boc‐Leu‐Ile‐Ile‐OMe and H‐Leu‐Ile‐Ile‐OMe formed only spherical nanostructures. Dynamic light scattering studies showed that aggregates of varying dimensions were present in solution suggesting that self‐assembly into ordered structures is facilitated by aggregation in solution. Fourier transform infrared spectroscopy and circular dichroism spectroscopy data show that although all four of the protected peptides adopt well‐defined tertiary structures, upon removal of the Boc group, only H‐Phe‐Phe‐Phe‐Ile‐Ile‐OMe had the ability to adopt β‐structure. Our results indicate that hydrophobic interaction is a very important determinant for self‐assembly and presence of charged and aromatic amino acids in a peptide is not necessary for self‐assembly. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.