Specific cleavage of the native type III collagen molecule with trypsin. Similarity of the cleavage products to collagenase-produced fragments and primary structure at the cleavage site.

Solutions of native Type III collagen (chain composition, [α1(III)]₃) exhibit a rapid and dramatic decrease in relative viscosity when incubated with trypsin. Cleavage products of the reaction were precipitated with ammonium sulfate and isolated in denatured form by molecular sieve chromatography. They were found to be comprised of: α1(III)-T1 (molecular weight, 71,000) derived from the NH₂-terminal portion of the Type III molecule; and α1(III)-T2 (molecular weight, 24,000) from the COOH-terminal portion of the molecule. Determination of the amino acid sequence at the NH₂-terminal portion of α1(III)-T2 as well as at the COOH-terminus of α(III)-T1 demonstrated that the products arose from specific cleavage of the type III molecule at an arginine-glycine bond corresponding to residues 780–781 in the repetitive triplet sequence of the α1(III) chain. The results suggest that the trypsin-susceptible bond in the native Type III collagen molecule resides in a region characterized by a relative lack of the normal collagen helicity.     

Identification of the milk fat globule membrane proteins: I. Isolation and partial characterization of glycoprotein B

The salt soluble proteins from the fat globule membrane of cow's milk were resolved into three fractions by Sephadex column chromatography in sodium dodecyl sulfate. One of the fractions, termed glycoprotein B, was purified by rechromatography to essentially one band on sodium dodecyl sulfate gel electrophoresis. It was found to contain 14% carbohydrate including sialic acid, mannose, galactose, glucose, glucosamine and galactosamine. The amino acid composition of glycoprotein B was determined; it has amino terminal serine and carboxyl terminal leucine. The molecular weight of this glycoprotein as estimated by sodium dodecyl sulfate gel electrophoresis is 49 500.     

Comparative chemical studies of the polyhedral proteins of the nuclear polyhedrosis viruses of Bombyx mori and Galleria mellonella

Amino and carboxyl terminal groups, amino acid composition, and peptide maps of polyhedral proteins of the nuclear polyhedrosis viruses (NPV) of Bombyx mori and Galleria mellonella were investigated. It is shown that both the proteins have a tyrosine residue as their carboxyl terminal group and no amino terminal group. Amino acid compositions of the proteins are similar. The proteins are found to have 242 residues. From the amino acid composition, a molecular weight of 28,000 was calculated. The tryptic peptide maps of both the proteins differed only in a few peptides.It is inferred that the polyhedral proteins of B. mori and G. mellonella NPV have a closely similar primary structure.     

Assembly and processing of procollagen type III in chick embryo blood vessels

The processing of [3H]proline-labeled procollagen III in excised chick embryo blood vessels was found to differ significantly from that of procollagen I in the same tissue. While first the amino propeptides and then the carboxyl propeptides were fairly rapidly cleaved from procollagen I, only the carboxyl propeptides were split off procollagen III, leaving pN-collagen III. This intermediate, which is only slowly converted to collagen III by loss of amino propeptides, was characterized by its sedimentation properties, isolation of the amino propeptide, and reaction with purified antibodies that are specific against bovine amino propeptide III. It is interchain disulfide-linked, both through the amino propeptide and the carboxyl ends of the collagen chains. The conversion of procollagen III to pN-collagen III either in blood vessels, or after isolation by a carboxyl procollagen peptidase obtained from chick tendon fibroblast cultures, is inhibited by 50 mM arginine. Underhydroxylated procollagen III was isolated from blood vessels treated with alpha, alpha'-dipyridyl. Its amino propeptides reacted with the above antibodies but were not linked to each other. In contrast, its carboxyl propeptides were interchain disulfide-bridged, supporting previous suggestions that the carboxyl propeptides play a role in the assembly of procollagen trimer.     

肠毒素C2蛋白C,N末端对其活性的影响

目的:分析金黄色葡萄球菌肠毒素C2(SEC2)中N,C末端对其超抗原活性和可溶性表达能力的影响。方法:应用基因工程技术对SEC2的N,C末端进行部分删除,获得三种突变蛋白,并对其进行体外超抗原活性和可溶性表达能力的比较。结果:对SEC2的N,C末端的删除都在一定程度上影响其超抗原活性和可溶性表达能力,其中,N末端的两个删除突变体的超抗原活性分别降低40%和48%,而删除C末端则使其可溶性表达水平下降到野生型的20%左右。结论:SEC2蛋白分子的N末端对其超抗原活性起主要作用,C末端对其可溶性表达具有显著影响,而完整的SEC2分子对于其发挥最大生物学活性是必要的。     

Grucuronic acid-containing glycopeptide from squid cartilage

A glycopeptide fraction containing glucuronic acid as a component sugar was extracted and purified from squid cartilage to give a single band migrating much slower than hyaluronic acid in cellulose acetate electrophoresis. The molecular weight of the glycopeptide was fairly large since its K_av value in Sephadex G-200 chromatography was 0.18; however, it was soluble in 66% ethanol. This glycopeptide contained glucuronic acid, glucosamine, galactosamine, galactose, and fucose. The total amino acid content was 1.87 μmol of amino acid per mg of the glycopeptide. Threonine, serine and proline represented 80% of the amino acids. Digestion with chondroitinase ABC or reaction with nitrous acid did not result in degradation of the glycopeptide; however, it was completely degraded by reaction with 0.5 M KOH at 37°C. Two hexasaccharides were separated from the alkaline degradation products, and they both contained glucuronic acid, fucose, galactosamine, and reducing terminal glucosamine in the molar ratio, 2:1:2:1. These results indicated that the glycopeptide contains glucuronic acid-containing sugar chains that are distinct from any known glycosaminoglycan.     

Isolation and characterization of polypeptides from human plasma enhancing the growth of human normal cells in culture

The isolation in pure form and the chemical characterization is first described here of four polypeptides from human plasma which stimulate [³H] thymidine uptake into glial cells in culture and increase the number of human embryonic lung fibroblasts. The polypeptides have a molecular weight of 5000. Although they differ in charge the amino acid compositions are essentially identical and each peptide has four disulphide bridges. The amino and carboxyl terminal residues are aspartic acid and threonine respectively. The peptides are tentively designated Somatomedin B.     

Characterization of lens capsule collagen: evidence for the presence of two unique chains in molecules derived from major basement membrane structures

A collagen fraction representing two-thirds of the collagenous sequences in bovine lens capsules has been isolated following limited pepsin digestion and purified by DEAE- and carboxymethyl-cellulose chromatography in native form. The denaturation products of this collagen contain two types of components. The more acidic components (C and 50K₁) are, respectively an α-chain-sized collagenous polypeptide and a mixture of smaller molecular weight proteolytic cleavage products of the C chain. The more basic components (80K and 50K₂) represent, respectively, a collagenous polypeptide with an apparent M_r = 80,000 and a mixture of smaller molecular weight components derived through proteolysis of the 80K component. The C chain and 80K components are unique with respect to chromatographic properties, amino acid composition, and cyanogen bromide cleavage products. These data are interpreted to indicate that lens capsule basement membrane collagen molecules collectively contain at least two genetically distinct collagen chains: the C chain representing the collagenous domain of one type of chain and the 80K component representing the major portion of the collagenous domain of a second type of chain, designated the D chain.     

The role of colicin receptors in the uptake of ferrienterochelin by Escherichia coli K-12.

The amino terminal sequences of the 4 caseins synthesized by translation of ovine mammary mRNAs in a wheat germ cell-free system have been investigated by automated Edman degradation. The 3 “calcium-sensitive” caseins (α_s1, α_s2 and β) and κ-casein were synthesized as precaseins with amino terminal hydrophobic extensions of 15 and 21 amino acid residues respectively, resembling “signal peptides” of other secretory proteins. The extra pieces of the 4 caseins, which start with a methionyl residue, end with an alanyl residue which may be one of the signals recognized by the mammary membrane-bound enzyme responsible for the specific cleavage of precaseins. The amino terminal extensions of α_s1, α_s2 and β-caseins show a high degree of homology suggesting that they have derived from a common ancestor.     

Crystal Structure of Penicillin-Binding Protein 3 (PBP3) from Escherichia coli

In Escherichia coli, penicillin-binding protein 3 (PBP3), also known as FtsI, is a central component of the divisome, catalyzing cross-linking of the cell wall peptidoglycan during cell division. PBP3 is mainly periplasmic, with a 23 residues cytoplasmic tail and a single transmembrane helix. We have solved the crystal structure of a soluble form of PBP3 (PBP3_57–577) at 2.5 Å revealing the two modules of high molecular weight class B PBPs, a carboxy terminal module exhibiting transpeptidase activity and an amino terminal module of unknown function. To gain additional insight, the PBP3 Val88-Ser165 subdomain (PBP3_88–165), for which the electron density is poorly defined in the PBP3 crystal, was produced and its structure solved by SAD phasing at 2.1 Å. The structure shows a three dimensional domain swapping with a β-strand of one molecule inserted between two strands of the paired molecule, suggesting a possible role in PBP3_57–577 dimerization.     

The characterization of type I and type III collagens from human peripheral nerve.

The normal chemical features of peripheral nerve collagens were determined on postmortem, histologically normal adult human femoral nerve. 1. Genetically distinct type I, [alpha1(I)2]alpha2, and type III, [alpha1(III)]3, were isolated by differential salt precipitation and the component subunit chains, alphal(I), alpha2 and alphal(III) were obtained by ion-exchange chromatography and gel filtration. 2. The molecular weight of alphal(I) and alpha2 of type I collagen was 95 000 and that for type III was 280 000. Reduction of type III with dithiothreitol yielded expected alpha1(III) chains of 95 000 molecular weight. 3. The amino acid composition of the three collagen chains, alpha1(I), alpha2, and alpha1(III), was the same as previously reported values for the corresponding chains from human skin except for slightly elevated hydroxylysine content. 4. Peripheral nerve collagen was found to contain 81% type I collagen and 19% type III. These results indicate that peripheral nerve collagen characteristics closely simulate that of human skin and differ from that of human aorta and other parenchymal organs. These data will permit a chemical analysis for possible abnormalities of peripheral nerve collagen in various neurogenic disorders.     

Identification of Bilirubin Binding Site in Type I Collagen

The interaction of bilirubin with collagen in the significance of jaundice incidence have been previously reported and investigated. The novel peptide sequences containing bilirubin binding domain was identified and located to develop a basis for further studies investigating the interactions of collagen with bilirubin in the present study. In this study an intricate interaction between bilirubin and collagen was characterized and their binding domain has been established using in-gel digestion and LC–MS/MS analysis based on the collagen sequencing and peptide mass fingerprinting. The biotinylated bilirubin derivatives bind to α₁(I) chain but not to α₂(I) chains which clearly designates that bilirubin shows greater affinity to α₁ chains of collagen. The intact proteins collected after analyzing the resulting complex mixture of peptides was used for peptide mapping. Using the electrospray method, among the other peptide sequence information obtained, the molecular weight of collagen alpha-2(I) chain was obtained by locating a 130 kDa weight peptide sequences with greater pi value (9.14) with 1,364 amino acid residues and collagen alpha-1(I) chain with 1,463 amino acid residues with 138.9 kDa molecular weight. This information leads to locate the exact sequence of these helices focussing on the domain identification. The total charge of the peptide domain sequences infers that the bilirubin participates in the electrostatic mode of interaction with collagen peptide. Moreover, other modes of interactions such as hydrogen bonding, covalent interactions and hydrophobic interactions are possible.     

Some newly characterized collagenases from procaryotes and lower eucaryotes

Summary Chemical and enzymatic properties of four collagenases newly isolated from anaerobic Clostridium histolyticum, aerobic Achromobacter iophagus, and from two lower eucaryotes, the fungus Entomophthora coronata and the insect Hypoderma lineatum are reviewed.The problems of their biosynthesis and precursors, namely the effect of induction of collagenase and neutral proteinase in Achromobacter by their macromolecular substrates are discussed.The two bacterial collagenases are Zn-metallo-enzymes; the highly purified Clostridium collagenase contains cyst(e)ine, serine phosphate and tryptophan additionally to amino acids reported previously. Achromobacter collagenase has the highest specific activity of all collagenases; it yields by autolysis enzymatically active degraded forms. The active dimer is composed of two identical subunits of molecular weight 35,000. Similarities between Achromobacter collagenase, thermolysin and Bacillus subtilis neutral proteinase in molecular weight, amino acid composition, and amino acids important for the active sites are discussed.The two collagenases from low eucaryotes are serine proteinases; Hypoderma collagenase is homologous to the trypsin family in the amino terminal sequence.The initial cleavage of native collagen by highly purified bacterial collagenases occurs in the central helical part of the a chains and not progressively from the amino terminal end. One of the two initial cleavages produced by Achromobacter collagenase is situated in the region cleaved specifically by vertebrate collagenases, but with different bond specificity. The same is true for the insect collagenase. Entomophthora collagenase is a proteinase of broad specificity which also cleaves collagen in its helical parts. All four collagenases also degrade other proteins according to their bond specificity.     

Developmental modulation of embryonic cardiac myocyte adhesion to cardiac collagens in vitro

The goal of this investigation is to identify molecules that mediate embryonic cardiac myocyte adhesion during chick cardiac morphogenesis. The assay used employs culturing embryonic myocytes on substrata containing embryonic heart proteins separated by molecular weight. This assay shows that embryonic myocytes from 10- to 14-day-old embryos will bind to 140,000 and 128,000 Da proteins present in embryonic hearts and do not require Mg²⁺ or Ca²⁺ for adhesion. Myocytes from embryos younger than 10 days or older than 14 days display little or no binding. Embryonic heart flbroblasts collected at these same ages do not bind to these proteins. The 140- and 128-kDa proteins were found to copurify in extraction procedures for procollagens. Amino acid analysis shows that both proteins contain high glycine and hydroxyproline, indicating that they are collagens. However, glycine and imino acid levels are low relative to other known collagens, indicating a nonhelical domain present in each molecule and most closely resembled levels present in procollagens. Immunoblots show that antisera to chick collagen type I recognizes the 128-kDa protein while anti-collagen type III recognizes the 140-kDa protein. Monoclonal antibodies to the amino terminal propeptide of collagen type I recognize the 128-kDa protein in immunoblotting procedures. Embryonic chick myocytes bind to 140/128 kDa proteins present in extracts of sympathetic trunk, although they do not bind to 140/128 kDa proteins in embryonic tendon. The findings thereby indicate that forms of type III and type I collagens in embryonic heart support direct adhesion of embryonic myocytes for a restricted period of cardiac myogenesis and that these proteins differ from collagen types I and III present in other tissues and from fully processed collagen types I and III.     

Active center and subunit structure of transaldolase from Candida utilis.

Crystalline transaldolase (type III) isolated from Candida utilis is composed of two identical subunits, as shown by the following lines of evidence. 1. Tryptic digestion of the performic acid oxidized enzyme yields the number of ninhydrin- and arginine-positive peptides expected for identical subunits. 2. All attempts to separate both subunits by molecular weight or charge differences have failed. 3. Cyanogen bromide cleavage and sodium dodecyl sulfate gel electrophoresis of S-carboxymethylated transaldolase revealed four distinct peptides designated C₂ to C₅ according to their decreasing molecular weight and one additional peak, C₁, in low yield, presumably an aggregate or partially degraded peptide.By chromatography on Sephadex G-100 the maleylated cyanogen bromide digest from ¹⁴C-labeled β-giyceryl-transaldolase could be separated into four peptide peaks which have been analyzed for their amino acid composition. The largest peptide C₂ with a molecular weight of 16,800 was identified as the active site containing fragment. The four fragments together account for all amino acid residues in the entire protein.From transaldolase (type I) containing four methionine residues three cyanogen bromide peptides could be identified. By addition of the individual peptides a molecular weight of 37,100 ± 3500 could be calculated, which is half the molecular weight of the native enzyme. From experimental data presented so far both isoenzymes of transaldolase can be regarded as “half-of-the-sites” enzymes.     

Complete amino acid sequence of 21-hydroxylase cytochrome P-450 from porcine adrenal microsomes

Adrenal microsomal 21-hydroxylase is essential for biosynthesis or metabolism of various steroid hormones. The complete amino acid sequence of this cytochrome P-450 from pig adrenal was determined by sequence analysis on several sets of proteolytic fragments. The mature protein consists of 492 amino acid residues, corresponding to a molecular weight of 55,484. Seven out of nine total cysteine residues are localized in the amino terminal half of the molecule. The carboxyl terminal half contains only two cysteines, one of which is located at the highly conserved heme-binding region proposed in all cytochromes P-450. A structural comparison between 21-hydroxylase and 17 alpha-hydroxylase reveals that there is a preponderance of sequence homology at the carboxyl terminal region. These studies indicate that a single gene product is expressed for steroid 21-hydroxylase in porcine adrenal glands.     

Human Brain βA4 Amyloid Protein Precursor of Alzheimer''s Disease: Purification and Partial Characterization

The major component of the amyloid deposition that characterizes Alzheimer's disease is the 4-kDa beta A4 protein, which is derived from a much larger amyloid protein precursor (APP). A procedure for the complete purification of APP from human brain is described. The same amino terminal sequence of APP was found in two patients with Alzheimer's disease and one control subject. Two major forms of APP were identified in human brain with apparent molecular masses of 100-110 kDa and 120-130 kDa. Soluble and membrane fractions of brain contained nearly equal amounts of APP in both humans and rats. Immunoprecipitation with carboxyl terminus-directed antibodies indicates that the soluble forms of APP are truncated. Carboxyl terminus truncation of membrane-associated forms of human brain APP was also found to occur during postmortem autolysis. The availability of purified human brain APP will facilitate the investigation of its normal function and the events that lead to its abnormal cleavage in patients with Alzheimer's disease.     

Cleavage of human fibroblast type I procollagen by mammalian collagenase: demonstration of amino- and carboxy-terminal extension peptides.

Human skin fibroblasts in monolayer culture synthesize and secrete precursor forms of collagen into the culture medium. The type I collagen precursor, the major precursor in the culture medium, was isolated on DEAE cellulose chromatography and subjected to mammalian collagenase cleavage. The amino terminal cleavage fragments had a higher molecular weight than α1^A and α2^A, but did not contain interchain disulfide bonds. The carboxy-terminal cleavage fragments formed high molecular weight aggregates which contained interchain disulfide bonds. These results indicate that human type I procollagen contains noncollagenous amino and carboxy-terminal extension peptides and that all of the interchain disulfide bonds are on the carboxy-terminal portion of the molecule.     

Procollagen and collagen produced by a teratocarcinoma-derived cell line,TSD4: Evidence for a new molecular form of collagen

Procollagen and collagen were isolated from the culture medium and cell layer of line TSD4 (obtained from mouse teratocarcinoma OTT6050). SDS-polyacrylamide gel electrophoresis of the highly purified procollagen fraction demonstrated that the fraction is composed of θ chains (150,000 daltons), pro α chains (130,000 daltons), and α chains (100,000 daltons). Limited pepsin digestion of this fraction yielded a single species of collagen molecules having a chain composition (α1)₃, as did collagen isolated from the cell layer. Each α1 chain appears to be slightly larger than α1 chains from calf or human type I and type III collagen. Amino acid analysis and cyanogen bromide peptide profiles of pepsin-treated TSD4 collagen demonstrated significant differences from those of other collagens (II, III, IV) of the type α1(X)₃, although similar to that of the α1 chain of type I collagen, [α1(I)]₂α2. Taken together, acrylamide gel electrophoresis, amino acid composition, electron microscopy, and cyanogen bromide peptide analysis indicate that this material represents a new molecular species of collagen not previously characterized, probably related to [α1(I)]₃.