Protein constituents of the mouse spermatozoon: I. An electrophoretic characterization

Total protein constituents of the mouse spermatozoon have been fractionated and characterized by polyacrylamide gel electrophoresis. Three spermatozoan fractions were obtained following homogenization with 1% sodium dodecylsulfate (SDS) and sucrose gradient centrifugation: SDS-soluble proteins, SDS-insoluble tail components, and SDS-insoluble head components. Purities of these fractions were assessed at greater than 95% using Nomarksi differential interference microscopy. Subsequently, the SDS-insoluble sperm heads were further fractionated into five protein subclasses by ultracentrifugation and ion-exchange chromatography. SDS-Polyacrylamide gel electrophoresis indicates that each of these spermatozoan fractions contains distinct protein species. Furthermore, the electrophoretic profiles are highly reproducible and show no evidence of cross-contamination or proteolysis. The SDS-soluble fraction, which includes proteins from the plasma membrane, acrosome, axoneme, matrix and cristae of the mitochondria, contains one major 39,000-molecular weight band and numerous minor bands with molecular weights ranging from ～30,000 to greater than 100,000. In contrast, electrophoresis of the SDS-insoluble tail proteins reveals the presence of at least nine prominent bands with apparent molecular weights between 21,000 and 89,000. Ultrastructural analysis suggests that this fraction contains proteins from the outer dense fibers, fibrous sheath, outer mitochondrial membranes, and structural elements of the neck region of the sperm tail. Two subfractions of the SDS-insoluble sperm heads each contain one of the two mouse protamines. In addition, the acidic and moderately basic head fractions each contain a limited number of distinct protein bands with molecular weights ranging from 14,000 to 76,000. These proteins are apparently derived from either the spermatozoan nucleus or the associated perinuclear material, since all other sperm head structures are solubilized during SDS treatment. One- and two-dimensional electrophoresis on acetic acid-urea polyacrylamide gels indicates that the moderately basic fraction may contain minor components that resemble certain histones and/or spermatidal basic nuclear proteins.     

用单克隆抗体和蛋白水解酶研究人绒毛膜促性腺激素的抗原决定簇

 本文报道,在不同酶解条件下,用嗜热菌蛋白酶、糜蛋白酶、胰蛋白酶,酶解人绒毛膜促性腺激素(hCG)得四个片段,而用胃蛋白酶则得三个片段;这些酶解片段在聚丙烯酰胺凝胶电泳中的条带位置不同,说明为非均一的酶解产物。应用蛋白质转移电泳技术和在硝酸纤维素膜上进行免疫酶标染色,检测出抗hCG的单克隆抗体9C_2和8B_5可与完整hCG及所有hCG酶解片段反应;而7F_3、BAH和10E_8只与完整hCG反应。表明hCG分子中可能存在序列型和结构型的两种不同的抗原决定簇。     

Relationship between phosphate content and serological activities of the mannans of Candida albicans strains NIH A-207, NIH B-792, and J-1012.

The mannans from Candida albicans strains NIH A-207 (serotype A), NIH B-792 (serotype B), and J-1012 (serotype C) were fractionated on a column of diethylaminoethyl-Sephadex into five subfractions containing different amounts of phosphate. Antibody-precipitating activities of the mannan subfractions of strains NIH A-207 and NIH B-792 were proportional to their phosphate content, while those of strain J-1012 did not show regularly proportional precipitin activity. A similar tendency was also observed in the cross-reaction between the mannan su,fractions of strains NIH A-207 and J-1012 and their heterologous antisera. The mannans of strain NIH B-792 showed lower cross-reactivities against antisera of strains NIH A-207 and NIH B-792, i.e., only two subfractions containing larger amounts of phosphate were able to react with these antisera.     

Chymotrypsinogen: an activating enzyme in the mitochondrial membrane of rat submaxillary gland.

A peripheral membrane protease was purified from mitochondria of rat submaxillary gland. On non-denaturing PAGE the purified enzyme showed a single protein band with the enzyme activity. It yielded two protein bands with molecular weights of 39 KDa and 20 KDa on SDS-PAGE, indicating that the enzyme is composed of two protein components. The enzyme activity was strongly inhibited by SBTI, aprotinin and benzamidine. PMSF, TLCK and EDTA did not produce inhibition. The enzyme could hydrolyze different synthetic substrates having arginine at the P1 position with highest affinity for the substrate Bz-Phe-Val-Arg-p-nitroanilide was noted. The enzyme showed significant activation of chymotrypsinogen A.     

雌核发育银鲫和两性生殖彩鲫精子蛋白组份的比较研究

对雌核发育银鲫和两性融合彩鲫精子蛋白组份进行了比较分析。通过分级抽提得到精子的不同组份精浆、精头的膜、鞭毛和脱膜精头等,然后经不同的凝胶电泳系统,比较分析了银鲫精子和其两性亲缘种彩鲫精子相应组份可溶性蛋白成份的差异。研究表明,经分级抽提的银鲫精子和彩鲫精子的各个组份都含有其特定的蛋白谱带。精浆蛋白在两种鱼之间和两种鱼的不同个体之间都存在一定差异。精头膜、鞭毛和脱膜精头的可溶性蛋白在同种鱼不同个体间高度一致,但在两种鱼之间表现出差异。两种鱼精头膜的可溶性蛋白在SDS－PAGE电泳图谱上基本一致,而在非变性聚丙烯酰胺凝胶电泳图谱上则具有各自的特征性谱带。鞭毛可溶性蛋白的SDS－PAGE分析在雌核发育银鲫中揭示出一条特异的蛋白带。脱膜精头的可溶性蛋白在SDS－PAGE电泳图谱上差异明显,存在几条特征性蛋白带,并经Acid-Urea PAGE系统分析,证实这些特征性蛋白为碱性蛋白。这些发现为进一步鉴定雌核发育银鲫雄鱼精子的特异性蛋白和揭示其分子机制打下了基础。     

Saturating Irradiance-Induced Photoinhibition without Monomerisation of Photosystem 2 Dimer in Soybean Leaves

The oligomeric state of photosystem 2 (PS2) complex in soybean leaves treated with saturating irradiance was studied by non-denaturing polyacrylamide gel electrophoresis (PAGE) and gel filtration chromatography. PS2 dimers resolved by non-denaturing PAGE accounted for about 75 % of total PS2 complex and there was no significant difference in the ratio of PS2 dimer to monomer between samples from saturating irradiance-treated and fully dark-adapted leaves. Furthermore, BBY particles were resolved into four chlorophyll-enriched fractions by gel filtration chromatography. From their molecular masses and protein components, these fractions were deduced to be PS2 dimer, PS2 monomer, oligomeric light-harvesting complex 2 (LHC2), and monomeric LHC2. Also, no change in the proportion of PS2 dimer in total PS2 was observed in the granal region of thylakoid membranes from soybean leaves after saturating irradiation. Hence the dimer is the predominant natural form of PS2 in vivo and no monomerisation of PS2 dimer occurs during saturating irradiance-induced photoinhibition in soybean leaves.     

Molecular Weight Determination of Gliadin Fractions in Gel Filtration by SDS-Polyacrylamide Gel Electrophoresis and Sedimentation Equilibrium

Gliadin was fractionated into three fractions; ω-gliadin, Fraction III (γ-gliadin) and Fraction IV (α- and β-gliadin). The determination of the molecular weights (MW) of the three fractions was performed by both SDS-polyacrylamide gel electrophoresis (SDS–PAGE) and sedimentation equilibrium. In SDS–PAGE, ω-gliadin gave three bands (MW 50,000, 54,000 and 64,000), Fraction III two bands (MW 38,000 and 46,000) and Fraction IV two bands (MW 33,000 and 38,000), The sedimentation analysis showed that each fraction was fairly homogeneous relative to molecular weight. The molecular weights obtained by sedimentation were 28,000 for Fraction III and 27,000 for both Fraction IV and ω-gliadin. The disagreement in molecular weight between sedimentation and gel electrophoresis was discussed.     

Identification of bacterial periplasmic glycine betaine-binding protein after electrophoresis and affinity labeling

Antibodies were elicited in rabbits against periplasmic proteins obtained by cold osmotic shock from the Gram-negative eubacterium Rhizobium meliloti. When analyzed by crossed immunoelectrophoresis (CIE), the periplasmic proteins gave rise to 20 distinct immunoprecipitates corresponding to the same number of bands in polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions and in SDS-PAGE. The periplasmic glycine betaine-binding protein (GB-BP) was identified by autoradiography after affinity labeling with [14C]glycine betaine in PAGE and in CIE gels. The binding proved to be quite specific to glycine betaine, since the GB-BP was not labeled by choline (a metabolic precursor of glycine betaine in Escherichia coli and Rhizobium meliloti) and 15 distinct L-amino acids, including L-proline which, like glycine betaine is also an osmoprotectant. Affinity labeling of the GB-BP with [14C]glycine betaine after protein separation by PAGE or CIE is a simple and sensitive technique permitting the GB-BP to the unambiguously detected and identified in samples of complex protein mixtures containing down to 2 micrograms of GB-BP in PAGE and only 0.2 micrograms in CIE.     

Detection of DNA-protein binding in western blots by phosphorus-labeled and biotinylated DNA probes

Eukaryotic DNA-binding proteins can be detected by a filter binding assay combining protein blotting on nitrocellulose, incubation with DNA by filtration, and the application of radioactively or nonradioactively labeled DNA probes. Basic nuclear and non-nuclear standard proteins are assayed in dot blots as well as in Western blots from sodium dodecyl sulfate gels. The DNA-binding ability of fractionated proteins is compared employing two different blotting techniques, conventional electro-transfer and protein-renaturating capillary transfer. Biotinylated DNA probes exhibit high sensitivity and a distinct discrimination of detection signals corresponding only to defined DNA-binding proteins. In contrast, phosphorus-labeled DNA probes show higher sensitivity, but less effective resolving power, especially for bands localized close to each other. Using the DNA-incubation procedure described, biotinylated DNA probes are preferable to radioactively-labeled probes for screening DNA-binding proteins in complex protein fractions.     

Multiple antigens in the rat visceral yolk sac induce teratogenic antisera

Preparative isoelectric focusing was used to fractionate the supernatant from a homogenate of day 19 rat visceral yolk sac. Three fractions, of pI ranges 3.5-5.0, 5.0-7.0, and 7.0-9.0, were isolated and used to immunize rabbits, by four or six weekly injections, each containing 5 mg protein. The resulting antisera were all teratogenic when injected into rats on day 9 of gestation, but widely differing potencies were observed. The most potent antiserum was that against yolk sac components focusing in the pI 7.0-9.0 range: An optimum teratogenic dose of 50 mg protein per kg body weight was observed, and a dose of 100 mg/kg was shown to cause 100% embryonic resorption. Antiserum against the fraction focusing in the pI 3.5-5.0 range was the least teratogenic: A significant incidence of embryonic malformation and death was seen only at doses of 600 mg/kg and above. The two fractions that yielded the more teratogenic antisera were refocused over narrower pH ranges, yielding four subfractions in the pI 5.0-7.0 range and eight subfractions in the pI 7.0-9.0 range. Antisera against each of these 12 fractions were raised in rabbits; most of these antisera were shown to be teratogenic, although of differing potencies. It is concluded that the yolk sac contains many antigens that can elicit antibodies with teratogenic and yolk sac-localizing properties.     

Heterogeneity of Escherichia coli-expressed human muscle creatine kinase

Creatine kinase (CK) plays an important role in maintaining a constant ATP:ADP ratio during periods of high energy usage. Elevated levels of CK give an early indication of myocardial infarction. The enzyme has four major isozymes with heterogeneity being observed for each of them. In many cases the source of the heterogeneity is unclear. However, some of the isoforms are known to result from exposure to serum proteases, and analysis of the plasma isoforms provides an estimate of the time of onset of the infarction. Somewhat surprisingly, isoelectric focusing (IEF) experiments provided evidence of heterogeneity in human muscle CK (HMCK) expressed in E. coli. To investigate this further, HMCK was purified to apparent homogeneity utilizing Blue Sepharose affinity chromatography and HiPrep Q anion exchange chromatography. Additional purification on a PBE 94 chromatofocusing column resulted in four fractions, three of which, HMCK I - III, were characterized. The three isoforms are all active and have similar kinetic parameters. They exhibited identical bands on SDS PAGE but different anodal mobility on non-denaturing gels. Modification of C-terminal and/or cysteine residues has been ruled out, and deamidation of asparagine or glutamine residue(s) is proposed to be the cause of isoform formation. In addition each of these isoforms showed a similar four-band pattern on a carrier ampholytes-based IEF gel. Two-dimensional IEF analysis showed that an equilibrium was established between the four bands, suggesting that the four components were unstable and generated only when the protein was subjected to IEF.     

Induction of lesions in deoxyribonucleic acid by low molecular weight substances from normal and colicin E2-treated cells of Escherichia coli.

A fraction containing a variety of low molecular weight substances was extracted into 80% aqueous acetone from both a colicin E2-treated cell culture of Escherichia coli and an untreated one. The extract was divided into five fractions by Sephadex G15 chromatography. One of them, Fraction B, was separated into three subfractions by Sephadex G10 chromatography. Two subfractions, Fraction BI and Fraction BII, were further fractionated by several chromatographic systems. DNA was incubated with an aliquot from each of these fractions and was then analyzed by sedimentation in an alkaline sucrose density gradient. The activity which caused a decrease in the sedimentation coefficient of the DNA was found in some of these fractions. The activity from colicin E2-treated cells was compared with that from untreated ones. It was revealed that colicin E2 induces some increases in the activity toward DNA in one of the subfractions, Fraction BI, and also causes the appearance of a new species in another fraction, Fraction BII, which potentiates the activity in Fraction BI. These colicin E2-induced changes appeared at 5 min after the addition of colicin E2. The possible significance of such reactions for the action of colicin E2 are discussed.     

Characterization of a soluble carnitine acetyltransferase from Candida tropicalis

Carnitine acetyltransferase was purified from the cytoplasmic fraction of Candida tropicalis grown on alkanes in continuous culture. By ion-exchange chromatography the enzyme was resolved in two fractions with the same specific activity of 80 U/mg. The molecular mass of both enzyme forms, determined by non-denaturing gradient gel electrophoresis, was 540 kDa. After SDS electrophoresis only one band of 64 kDa was detected indicating that both enzymes are oligomers each containing eight subunits. Isoelectric focusing in agarose under non-denaturing conditions demonstrated the presence of at least four different charged species in the pH range between 5.6 and 6.7. After isoelectric focusing in 9 M urea/1% Nonidet P-40 gels, both enzyme forms were resolved into four bands. Peptide mapping, performed by cyanogen bromide cleavage of polypeptides separated by denaturing isoelectric focusing followed by second-dimension SDS electrophoresis, revealed a very high degree of homology between these polypeptides. The presence of the octameric form of carnitine acetyltransferase already in the starting material was demonstrated by non-denaturing gradient gel electrophoresis and immunoblotting. Antibodies against carnitine acetyltransferase from C. tropicalis ATCC 32113 formed precipitation lines with extracts from several Candida species but not with extracts of Candida utilis, Candida ethanothermophilum and an another strain of C. tropicalis.     

Changing patterns of vitellin-related peptides during development of the cattle tick Boophilus microplus

The major components of protein extracts from the cattle tick Boophilus microplus eggs and larvae of various ages were characterized by molecular sieving chromatography, ion exchange chromatography and SDS-PAGE. The fractions analysed showed a changing chromatographic pattern development. A serum raised against the components of a fraction showing characteristics of vitellin strongly reacted in Western blots with the major peptides of extracts from eggs, larvae, gut and ovary. Comparison of patterns obtained by electrophoresis in non-denaturing PAGE, stained with Coomassie blue or with benzidine/hydrogen peroxide, revealed that the major proteins of these extracts are haemoproteins, possibly in different aggregation states or heterogeneous in composition.     

Specific and azurophilic granules from rabbit polymorphonuclear leukocytes. I. Isolation and characterization of membrane and content subfractions

The specific and azurophilic granules of rabbit polymorphonuclear heterophils (PMNs) have been isolated and fractionated into membrane and extractable subfractions. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) revealed several features of the protein composition of the two granules: (a) Whereas each type of granule had 40-60 proteins separable on one-dimensional gradient gels, few of the proteins were common to both granules. (b) The proteins of the extractable fractions (which comprised approximately 98% of the total granule protein) of each granule were distinct from the proteins of the membrane fractions (which comprised approximately 2% of the total granule protein). (c) The extractable proteins co- migrated with those collected from the medium of ionophore-treated, degranulating PMNs and therefore were defined as content proteins. These results were confirmed by radiolabeling studies. Lactoperoxidase- catalyzed iodination of intact granules did not label the content proteins but did label proteins that co-migrated with major granule membrane proteins. Moreover, disruption of the granules before iodination led to labeling of both content and membrane proteins. We conclude that the membranes of specific and azurophilic granules, which arise from different faces of the Golgi complex, are composed of unique sets of membrane proteins some of which are exposed on the cytoplasmic face of the granules.     

Isolation and Characterization of Acidic Peptides in Soy Sauce

A soy sauce sample was fractionated by gel filtration on a Sephadex G–15 column, then the fractions were subfractionated on the basis of acidity by ion exchange chromatography on a QAE-Sephadex A–25 column. The acidic subfractions with various acidities were further fractionated, using a preparative amino acid analyzer and by paper chromatography to separate the acidic peptide components.

Four dipeptides and sugar derivatives of ten dipeptides and two tripeptides were isolated and characterized as the major acidic peptides in soy sauce. However, it was difficult to anticipate any direct contribution of these peptides to the flavor construction in soy sauce on the basis of their contents and taste intensities.     

Isolation and properties of individual components of cattle transferrin: The role of sialic acid

Homozygous cattle transferrin has been fractionated into six main peaks by DEAE-Sephadex chromatography. These correspond to the six transferrin components seen in starch gel electrophoresis of normal serum. In addition, six more minor components were isolated from DEAE-Sephadex, making 12 in all, and these could be divided into six pairs. Treatment of whole transferrin with neuraminidase yielded only two bands. Treatment of the individual fractionated bands showed that the slower band of each pair had the same mobility as the slow band from treated whole transferrin, while the faster band from each pair corresponded with the fast band of treated whole transferrin. These observations, and the results of sialic acid assays, showed that the difference between the pairs of bands was caused by differing numbers of sialic acid residues (0–5) per molecule of protein, but that sialic acid was not responsible for the difference between the bands within a pair. The mode of genetic control is discussed and probably involves three loci. Other physicochemical properties of cattle transferrins, namely, molecular weight, effect of iron addition, and behavior in isoelectric focusing, were also studied.     

Separation of oligo(adenosine diphosphate ribose) fractions with various chain lengths and terminal structures.

Oligo(adenosine diphosphate ribose) preparations with chain lengths of 3 to 10 adenosine diphosphate ribose units were fractionated according to their chain lengths and their terminal structures by hydroxyapatite column chromatography and then polyacrylamide gel electrophoresis. The peak fractions from the hydroxyapatite column were each separated into two distinct subfractions by gel electrophoresis. The two subfractions were found to differ in chain length and terminal structure. A linear correlation was observed between the mobility and the logarithm of the chain length of oligo(adenosine diphosphate ribose) on gel electrophoresis, irrespective of the terminal structure.     

The effect of Echis carinatus crude venom and purified protein fractions on carbohydrate metabolism in rats

Echis carinatus crude venom was fractionated into 11 protein fractions by preparative native polyacrylamide gel electrophoresis (PAGE). All fractions except fractions 5 and 10 appeared as a single band on analytical native PAGE. Purified venom fractions 1, 4, 8, 10 and 11 appeared as single bands on SDS-PAGE whereas fractions 2, 3 and 7 contained two bands and fraction 6 contained three bands. Fractions 1 and 3 exhibited basic pI (7.3 and 7.6) respectively, while fractions 2, 4, 6, 8, 10 and 11 showed an acidic pI. Amino acid analysis also showed that crude venom is rich in acidic amino acids. A significant hyperglycaemia was produced by i.p. injection of E. carinatus crude venom, after 15 min of envenomation which persisted even after 24 h. Along with hyperglycaemia there was a significant decrease of liver glycogen at 15 min and 1, 12 and 24 h. A significant decrease of plasma [pyr + lac] levels was found from 15 min to 24 h. The liver [pyr + lac] levels increased significantly after 24 h. Skeletal muscle [pyr + lac] level was significantly decreased after 24 h of envenomation. Fractions 2 and 6 produced the highest increase in plasma glucose after 12 h and fraction 7 after 24 h. The plasma insulin level was significantly decreased by these three fractions (2, 6 and 7). So it can be hypothesized that the hyperglycaemia may result from a direct effect of a venom component on plasma insulin. Fractions 7, 8 and 11 caused the highest decrease in plasma [pyr + lac] while fractions 1, 2, 3, 4 and 8 produced the most significant decrease in liver [pyr + lac]. The most significant increase in lactate dehydrogenase level was also produced by fractions 1, 2, 3, 4 and 8.