Purification and partial peptide sequence analysis of the boar 32 kDa sperminogen

Boar 32 kDa sperminogen was purified from acid extracts of washed epididymal spermatozoa, and partial peptide sequence was determined. Boar sperminogen was purified from the acid extracts of boar spermatozoa by gel filtration through Sephadex G-75 column, followed by preparative SDS-PAGE. Gelatin zymographic analysis of the gel-filtered fractions showed that sperminogen was composed of three separate proteolytic bands. Among the three proteolytic bands, the 32 kDa sperminogen band which showed the strongest proteolytic activities upon activation was sliced out and eluted from the gel fragments. The eluted 32 kDa sperminogen was then subjected to peptide sequencing. Since the N-terminus of the 32 kDa sperminogen was blocked for peptide sequencing by Edman degradation method, the internal amino acid sequence of the sperminogen was obtained from the CNBr-digested peptides of sperminogen. The amino acid sequence of the analyzed peptide of the 32 kDa sperminogen showed 100% identity with that of proacrosin.     

Structure of human erythrocyte spectrin. I. Isolation of the alpha-I domain and its cyanogen bromide peptides

The alpha-I domain of human erythrocyte spectrin was produced by a mild tryptic digestion of the intact molecule and purified by a single step affinity chromatography procedure using a monoclonal antibody. A tryptic peptide representing the alpha-I domain, which migrated on polyacrylamide gels as an 80,000-dalton peptide, was subjected to automated Edman-Begg degradation. Products from automated sequencing were identified by reverse-phase high performance liquid chromatography. Two smaller proteolytic products of the alpha-I domain (T74 and T50) were also subjected to automated sequence analysis. CNBr cleavage of the alpha-I domain produced nine unique peptides which were separated by gel filtration on a high performance liquid chromatograph. Peptides were further purified by reverse-phase chromatography and characterized by amino acid analysis. Partial sequences were determined by automated NH2-terminal sequence analysis. A single aspartate-proline bond, which was partially hydrolyzed during the cyanogen bromide cleavage reaction, was also identified. These sequence data include the first 86 residues of the alpha-I domain, and the spectrin oligomer binding site has been tentatively localized within the first 39 residues. The sequence of 293 residues of a total 633 residues in the alpha-I domain is presented and represents the first structural information for this protein.     

河蚌C反应蛋白一级结构分析

经亲和层析纯化的河蚌 C反应蛋白 ( CRP)具有 SDS- PAGE纯度 ,用经改进的双偶联 Ed-man方法测得其 N端残基为谷氨酸 ,而不是高等动物 (人与家兔 ) C反应蛋白 N端的焦谷氨酸 .河蚌 C反应蛋白 N端的一级结构由固相 Edman方法测得 ,依次为 H2 N- E- T- A- Y- S- C- I- T- A- V- ;C端的一级结构由羧肽酶 A降解法测得 ,依次为 - L/V- S- S- T- Y- COOH,也不同于人和家兔的 C反应蛋白 .在河蚌 CRP的胰蛋白酶酶解肽段中 ,其 N端及 C端的结构也得到了证实 .河蚌 C反应蛋白经 V8蛋白内切酶酶解 ,溴化氰裂解 ,肽段经 HPLC反相柱分离 ,共得到 35个肽段 ,所有肽段的氨基酸序列均由气相氨基酸自动分析仪测得 .结合河蚌 C反应蛋白的胰蛋白酶酶解肽段的分析结果 ,其一级结构已初步拼接完成 .在其一级结构中发现有类似于其它 CRP的 Ca2 + 结合部位和磷酸胆碱结合部位 .河蚌 C反应蛋白的分子结构中存在微观不均一性 .从已知河蚌 C反应蛋白的分子特点 ,包括分子量 ,糖基化比例 ,一级结构不均一等特点 ,可以推测它与高等动物的免疫蛋白有许多相关之处 .对于河蚌 C反应蛋白分子结构的分析 ,将有助于免疫系统蛋白的发生 ,进化等方面的研究     

Acanthamoeba actin and profilin can be cross-linked between glutamic acid 364 of actin and lysine 115 of profilin

Acanthamoeba profilin was cross-linked to actin via a zero-length isopeptide bond using carbodiimide. The covalently linked 1:1 complex was purified and treated with cyanogen bromide. This cleaves actin into small cyanogen bromide (CNBr) peptides and leaves the profilin intact owing to its lack of methionine. Profilin with one covalently attached actin CNBr peptide was purified by gel filtration followed by gel electrophoresis and electroblotting on polybase-coated glass-fiber membranes. Since the NH2 terminus of profilin is blocked, Edman degradation gave only the sequence of the conjugated actin CNBr fragment beginning with Trp-356. The profilin-actin CNBr peptide conjugate was digested further with trypsin and the cross-linked peptide identified by comparison with the tryptic peptide pattern obtained from carbodiimide-treated profilin. Amino-acid sequence analysis of the cross-linked tryptic peptides produced two residues at each cycle. Their order corresponds to actin starting at Trp-356 and profilin starting at Ala-94. From the absence of the phenylthiohydantoin-amino acid residues in specific cycles, we conclude that actin Glu-364 is linked to Lys-115 in profilin. Experiments with the isoforms of profilin I and profilin II gave identical results. The cross-linked region in profilin is homologous with sequences in the larger actin filament capping proteins fragmin and gelsolin.     

Rabbit testis proacrosin: Immunological similarities between testis,sperm proacrosins and the initially formed testis acrosin

Anti-rabbit proacrosin IgG was prepared from goat serum following immunization with a homogeneous preparation of rabbit testis proacrosin. The “auto-activation” products of purified testis proacrosin were separated into 68,000 and 34,000 molecular weight (mol wt) acrosins by Sephadex G-100 column chromatography. Immunodiffusion analysis of testis and epididymal sperm proacrosins and acrosins on agarose gel against goat anti-rabbit testis proacrosin showed immunological identity between rabbit testis and sperm proacrosins and the initial testis acrosin (mol wt 68,000). However, the 34,000 mol wt form of testis acrosin showed weaker reaction with the antibody and only partial identity with the proacrosin and the 68,000 mol wt form of acrosin. These results suggest that there is no major structural difference between testis and sperm proacrosins and between proacrosin and the 68,000 mol wt acrosin, but such a structual change occurs when the 34,000 mol wt acrosin is formed.     

Purification, characterization, and partial amino acid sequence of nerve growth factor from cobra venom.

The nerve growth factor (NGF) from Naja naja (cobra) venom has been purified and its structure compared to the NGF from mouse submaxillary gland. A two-step purification procedure has been devised, consisting of a gel filtration step in 1 M acetic acid followed by chromatography of the active pool on carboxymethylcellulose at pH 5. The molecular weight of the native protein was found to be 28000, and this value was reduced by approximately one-half under denaturing conditions. These values are comparable to those obtained for mouse 2.5S or betaNGF. Tryptic peptide maps of S-[14C]carboxymethyl NGF gave the number of labeled peptides expected for a structure composed of two identical or very similar subunits. Thus, the quaternary structures of mouse and cobra NGF are the same. Cyanogen bromide (CNBr) treatment of Naja naja NGF produced three fragments, of which two were purified to homogeneity. These fragments and the whole protein were analyzed in the automated protein Sequencer. The amino-terminal CNBr fragment of the protein was also subjected to digestion by thermolysin and the resultant peptides were purified and characterized. These data plus those from the characterization of the tryptic peptides provided the basis of the construction of a tentative primary structure of Naja naja NGF which is approximately 60% identical with mouse NGF.     

Identification of amino acid residues at the interface of a bacteriophage T4 regA protein-nucleic acid complex.

The bacteriophage T4 regA protein (M(r) = 14,6000) is a translational repressor of a group of T4 early mRNAs. To identify a domain of regA protein that is involved in nucleic acid binding, ultraviolet light was used to photochemically cross-link regA protein to [32P]p(dT)16. The cross-linked complex was subsequently digested with trypsin, and peptides were purified using anion exchange high performance liquid chromatography. Two tryptic peptides cross-linked to [32P]p(dT)16 were isolated. Gas-phase sequencing of the major cross-linked peptide yielded the following sequence: VISXKQKHEWK, which corresponds to residues 103-113 of regA protein. Phenylalanine 106 was identified as the site of cross-linking, thus placing this residue at the interface of the regA protein-p(dT)16 complex. The minor cross-linked peptide corresponded to residues 31-41, and the site of cross-linking in the peptide was tentatively assigned to Cys-36. The nucleic acid binding domain of regA protein was further examined by chemical cleavage of regA protein into six peptides using CNBr. Peptide CN6, which extends from residue 95 to 122, retains both the ability to be cross-linked to [32P]p(dT)16 and 70% of the nonspecific binding energy of the intact protein. However, peptide CN6 does not exhibit the binding specificity of the intact protein. Three of the other individual CNBr peptides have no measurable affinity for nucleic acid, as assayed by photo-cross-linking or gel mobility shifts.     

Identification of the peptide bond cleaved during activation of human Clr

CNBr cleavage of unreduced proenzyme Clr yielded fragment CP2b, isolated by gel filtration and highpressure gel permeation chromatography. This fragment (˜ M_τ 55000) comprised at least 4 disulphidelinked peptides, which were separated by gel filtration after reduction and alkylation. Peptide CP2bRA4, overlapping the A- and B-chain regions in proenzyme Clr was digested by V8 staphylococcal protease, and the digest separated by reversed-phase HPLC. N-terminal sequence analysis of peptide CP2bRA4SP9 established that Clr activation involves the cleavage of a single Arg-Ile bond, located in the sequence: Gln-Arg-Gln-Arg-Ile-Ile-Gly-Gly     

Bovine Peripheral Nervous System Myelin P2 Protein: Chemical and Immunological Characterization of the Cyanogen Bromide Peptides

Cleavage of bovine P2 protein by cyanogen bromide (CNBr) produced peptide fractions CN1, CN2, and CN3 which were isolated by gel filtration chromatography. CN2 was found to contain two NH2-terminals (lysine and valine) and accounted for both of the cysteine residues of P2. When reduced carboxymethylated P2 (RCM-P2) was digested with CNBr, peptides CN1 and CN3 were obtained as were (1) a peptide with NH2-terminal lysine (Lys) that contained no homoserine and only one cysteine residue and (2) a peptide with NH2-terminal valine (Val) that was co-eluted with CN3. These data and the chemical characterization of all the CNBr peptides obtained from P2 and RCM-P2 suggest that isolated P2 protein has a structure composed of the CNBr peptides in the order CN3-CN1-CN2(Val)-CN2(Lys) with an intrachain disulfide bond between the cysteine residues located in the two constituent peptides of CN2, CN2(Lys) and CN2(Val). To locate the neuritogenic region(s) within the P2 protein structure, CN1, CN2, and CN3 were tested for the ability to induced experimental allergic neuritis (EAN) in Lewis rats. The disease-inducing sites of P2 protein were found only in CN1; neither CN2 nor CN3 produced disease. EAN induced by CN1 was comparable to that induced with P2 protein as determined by disease onset, clinical symptoms, and histologic lesions.     

Isolation of a tripeptide showing weak acetylthiocholine hydrolysing activity from a soluble form of monkey basal ganglia acetylcholinesterase by limited trypsin digestion

Acetylcholinesterase was purified from the soluble supernatant of monkey (Macaca radiata) brain basal ganglia by a three-step affinity purification procedure. The purified enzyme showed two major protein bands corresponding to molecular weights of approximately 65 kDa and approximately 58 kDa which could be labelled by [3H]diisopropylfluorophosphate. When the purified enzyme was subjected to limited trypsin digestion followed by gel filtration on Sephadex G-75 or Sephadex G-25 column, a peptide fragment of molecular weight approximately 300 Da having a weak acetylthiocholine hydrolysing activity was isolated. The amino acid sequence analysis of this peptide showed a sequence of Gly-Pro-Ser. When the [3H]DFP labelled enzyme was subjected to limited trypsin digestion and Sephadex G-75 column chromatography, a labelled peptide corresponding to approximately 430 Da was isolated. The kinetics, inhibition characteristics and binding characteristics to lectins of this peptide were compared with the parent enzyme. A synthetic peptide of sequence Gly-Pro-Ser was also found to exhibit acetylthiocholine hydrolysing activity. The kinetics and inhibition characteristics of the synthetic peptide were similar to those of the peptide derived from the purified acetylcholinesterase, except that the synthetic peptide was more specific towards acetylthiocholine than butyrylthiocholine. The specific activity (units/mg) of the synthetic peptide was about 123700 times less than that of the purified AChE.     

Purification, Biological Activities, and Molecular Cloning of a Novel Mannose-Binding Lectin from Bulbs of Zephyranthes candida Herb （Amaryllidaceae）

A novel mannose-bindlng aggiutinln was purified from bulbs of Zephyranthes candida Herb by extraction, precipitation with 80% （NH4）2SO4, and ion-exchange chromatography on DEAE-Sepharose followed by gel flitration on Sephscryl S-100. The purified Z. candida agglutlnln （ZCA） migrated as a single band of 12 kDa on sodium dodecyi suifate-poiyecryiamide gel electrophoresis under reducing and non-reducing conditions. The apparent molecular mass of the iectln, as datermlned by gel filtration chromatography, was 48 kDa. The results Indicated that ZCA was composed of four Identical subunlts of 12 kDa each （homotetramerlc nature）. The ZCA agglutlhated rabbit erythrocytes, Escherichla coil and Saccharomyces cerevislae ceils at concentrations of 0.95, 1.90, and 31.30 μg/mL, respectively. Bloassays Indicated that ZCA has a significant effect on wheat aphid survival. Mortality after 7 d was 〉 90% at 0.26%. A degenerate primer was designed In accordance with the N-terminal partial sequence of purified ZCA. The full-length cDNA was cloned by 3＇- and 5＇-rapid amplification of cDNA ends. The full-length cDNA had 661 bp and the sequence encoded an open reading frame of 168 amino acids. The mature protein of ZCA Includes 109 amino acid residues and the molecular weight of the protein was 12.1 kDa. The result show that the zca gene encodes a protein precursor with a signal peptlde, a mature protein, and a C-terminal cleavage amino acids sequence. Molecular modeling of ZCA Indicated that Its three-dimensional atructure strongly resembies that of the snowdrop aggiutinin. Blocks＇ analysis revealed that the deduced amino acid sequence of ZCA has three functional domains specific for agglutination and three carbohydrate binding boxes （QDNY）.     

Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 5 from the dacA mutant strain of Escherichia coli (TMRL 1222)

The localization of the active site of penicillin-binding protein 5 from the dacA mutant of Escherichia coli strain TMRL 1222 has been determined. The protein was purified to homogeneity and labeled with [14C] penicillin G. The labeled protein was digested with trypsin, and the active site tryptic peptide was purified by a combination of gel filtration and high-pressure liquid chromatography. Sequencing of the purified [14C]penicilloyl peptide yielded the sequence Arg-Asp-Pro-Ala-Ser-Leu-Thr-Lys, which corresponds to residues 40-47 of the gene sequence (Broome-Smith, J., Edelman, A., and Spratt, B. G. (1983) in The Target of Penicillin (Hakenbeck, R., Holtje, J.-V., and Labischinski, H., eds) pp. 403-408, Walter de Gruyter, Berlin). The catalytic amino acid residue that forms a covalent bond with penicillin was identified by treating the purified [14C]penicilloyl peptide with a mixture of proteases and then separating the radioactive products using high-pressure liquid chromatography. Analysis of the radioactive peaks by amino acid analysis confirmed that it is the serine residue that reacts with the beta-lactam ring of penicillin.     

Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 1b of Escherichia coli

This paper reports the sequence of the active site peptide of penicillin-binding protein 1b from Escherichia coli. Purified penicillin-binding protein 1b was labeled with [14C]penicillin G, digested with trypsin, and partially purified by gel filtration. Upon further purification by high-pressure liquid chromatography, two radioactive peaks were observed, and the major peak, representing over 75% of the applied radioactivity, was submitted to amino acid analysis and sequencing. The sequence Ser-Ile-Gly-Ser-Leu-Ala-Lys was obtained. The active site nucleophile was identified by digesting the purified peptide with aminopeptidase M and separating the radioactive products on high-pressure liquid chromatography. Amino acid analysis confirmed that the serine residue in the middle of the sequence was covalently bonded to the [14C]penicilloyl moiety. A comparison of this sequence to active site sequences of other penicillin-binding proteins and beta-lactamases is presented.     

Amino acid sequence of a cyanogen bromide fragment containing the two tryptophanyl residues of lobster arginine kinase (Homarus vulgaris).

Lobster arginine kinase [EC 2.7.3.3] contains 2 tryptophanyl residues and 9 methionyl residues. The whole carboxymethylated protein was first subjected to CNBr cleavage and the resulting fragments were isolated by gel filtration and other experimental approaches. One fragment, CB5, which contains 60 residues including the two tryptophanyl residues and two of the five cysteinyl residues of the protein, was characterized and the results are reported inthis paper. The overall strategy for the establishment of the complete sequence of this fragment was based on the use of three types of peptides: (a) whole cyanogen bromide peptide CB5 which was partially characterized by automatic Edman degradation using a sequencer: 42 steps were performed out of 60 residues, (b) tryptic peptides of CB5, (c) peptides formed by cleavage of S-carboxymethylated arginine kinase (whole protein) at the two tryptophanyl residues with BNPS-skatole. The complete amino acid sequence of the CNBr polypeptide (CB5) which contains the two tryptophanyl residues of the whole protein was established.     

Molecular organization of 19 S calf thyroglobulin

Controlled freezing and thawing of 19 S calf thyroglobulin resulted in a specific and reproducible breakdown of the protein. Beside the elementary chain (300,000 Da), new, discrete bands are revealed by gel electrophoresis in sodium dodecyl sulfate under reducing conditions. These new species consist of a major peptide of 100,000 Da and several faster-migrating bands. Most of these polypeptides were purified by preparative gel electrophoresis and individually digested in formic acid with CNBr. A comparative gel electrophoresis under denaturating and reducing conditions of (i) the fragments obtained from the native protein, (ii) the electrophoretically purified elementary chain, (iii) the 100,000-Da peptide, and (iv) a smaller fragment (of about 50,000) was performed. It revealed a very close homology among the peptide maps of the intact 19 S, the elementary chain, and the 100,000-Da peptide. Furthermore, it was shown that the digestion products of the smaller fragment, present in the peptide map of the native protein, were absent in both the elementary chain and the 100,000-Da species. These results support the idea that calf thyroglobulin, even though it has an apparently complex molecular organization, contains structural motifs which are repeated in the elementary chain.     

Demonstration of a boar testicular protein band that is immunoreactive to proacrosin and proacrosin binding protein antibodies.

A testicular protein band has been identified and shown to be immunoreactive to both of the proacrosin (53-55 kd) and the proacrosin binding protein (28 kd) antibodies. pH 4.5 extracts of boar testis were prepared and subjected to Western blot analysis using polyclonal antibodies of the proacrosin and the proacrosin binding protein. In addition to their respective antigens, a distinct high molecular weight protein band of approximately 200 kd was detected by both of the antibodies. Gelatin SDS-PAGE analysis of the extracts showed that this protein band was proteinase active. These results suggest that the proacrosin molecule is present as a much higher molecular weight form in the boar testis than the currently known 53-55 kd forms that have been isolated from spermatozoa.     

Isolation and characterization of a soluble, immunoactive peptide of glial fibrillary acidic protein

A soluble immunoactive peptide with a molecular weight of 16 000 was isolated and purified from the cyanogen bromide digest of the insoluble 50 000 dalton glial fibrillary acidic protein by Sephacryl S-200 gel filtration followed by DEAE-Bio-gel A chromatography. The homogeneity of the peptide was established by SDS-polyacrylamide gel electrohporesis and isoelectric focusing. The peptide from several species showed immunocrossreaction with rabbit antibody to intact glial fibrillary acidic protein. The peptide has a pI value of 5.32. The amino acid sequence of 28 residues from the amino terminus of the calf peptide has been determined.     

Is sperminogen a modified proacrosin? Isolation, purification, and partial characterization of low-molecular-mass boar proacrosin

Low-molecular-mass zymogen was extracted from boar spermatozoa together with proacrosin using 10% acetic acid supplemented with 10% glycerol, and was purified by the sequential use of gel filtration on Sephadex G-75 and (FPLC) reversed-phase chromatography. LMM zymogen represented approximately 5% of the latent trypsin-like activity present in the sperm extract. SDS-PAGE indicated a molecular mass of 33 kDa. The zymogen reacted with both mouse monoclonal and rabbit polyclonal antibodies to boar acrosin. Determination of the N-terminal sequence of 34 amino-acid residues revealed its identity with the known N-terminal sequence of boar proacrosin.     

Difference of acrosomal serine protease system between mouse and other rodent sperm

A fraction of acrosomal proteins dispersed during calcium ionophore A23187‐induced acrosome reaction was prepared from cauda epididymal sperm of wild‐type and acrosin‐deficient mice, rat, and hamster. The acrosome‐reacted sperm were further extracted by Nonidet P‐40 to obtain the detergent‐soluble protein fraction. Activities of serine proteases in the two protein fractions were examined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the presence of gelatin. A mixture of 42‐ and 41‐kDa gelatin‐hydrolyzing proteases was found in both fractions of the wild‐type mouse sperm, whereas the acrosin‐deficient mouse sperm contained the active 42‐kDa protease and apparently lacked the activity of the 41‐kDa protease. However, exogenous bovine pancreatic trypsin compensated for the absence of acrosin in the protein fractions of the mutant mouse sperm; the gelatin‐hydrolyzing activity of the 41‐kDa protease appeared when the sperm proteins of the mutant mice were treated with pancreatic trypsin. Two‐dimensional polyacrylamide gel electrophoresis revealed that the 42‐ and 41‐kDa proteases were distinguished from acrosin by the isoelectric point and immunoreactivity with affinity‐purified antibody against an oligopeptide corresponding to the N‐terminal amino acid sequence of mouse proacrosin. Moreover, the gelatin‐hydrolyzing proteins corresponding to these two proteases were not detected in rat and hamster sperm, in spite of the treatment of the sperm extracts with pancreatic trypsin, and the total amount of gelatin‐hydrolyzing activities in mouse was much smaller than those in rat and hamster. These results may reflect the difference of the serine protease system for the sperm penetration through the egg zona pellucida between mouse and other rodent animals, possibly explaining why the acrosin‐deficient mouse sperm are capable of penetrating the zona pellucida. Dev. Genet. 25:115–122, 1999. © 1999 Wiley‐Liss, Inc.