Intact Monomer Research Articles

Evidence for covalent bonding of native hapten-protein complexes to smooth lipopolysaccharide ofBrucella abortus

Smooth lipopolysaccharide (S-LPS) of Brucella abortus was dissociated from noncovalently attached components by differential centrifugation and exhaustive treatment with guanidinium thiocyanate. Mild alkaline hydrolysis then released ester-linked fatty acids as well as native hapten (NH)-protein complexes. These complexes, comprising 15 to 20% by weight of the S-LPS, failed to dissociate in guanidinium thiocyanate or in boiling SDS, suggesting covalent attachment. Analyses for 2-keto-3-deoxyoctonate demonstrated that the released carbohydrate was NH, rather than degraded O-polysaccharide or intact S-LPS monomers. This provides strong evidence that NH-protein complexes are covalently linked to S-LPS, most likely through ester bonds.

Die Organisation der Pigmente im Lichtsammelnden Chlorophyll a/b-Protein-Komplex (LHC) aus Vicia faba I. Das Spektralformensystem der Chlorophylle im LHC

Summary The light-harvesting chlorophyll a/b-protein complex (LHC) from Vicia faba was isolated according to a modification of the method described by Shutilova et al. (1979) using Triton X-100 and followed by a removal of most of the detergent molecules. Continuing the investigations of the structure and function of chlorophyll forms in the LHC a method for shaping the profile curve to fit the optical spectra is proposed. All the spectra could best be fitted with synthetic functions consisting of Gaussian and Lorentzian portions in a fixed ratio for all the spectral forms of each pigment. In conformity with French et al. (1972) only four components of chlorophyll a in the longwavelength region are detectable in the spectra of LHC in different states of aggregation. Applying the exciton theory the numbers of individual pigment molecules and the molecular weight of an intact LHC monomer are estimated.

Heat-induced fragmentation of human plasma fibronectin

Human plasma fibronectin was found to undergo fragmentation during heat-denaturation, leading to artifacts in SDS-polyacrylamide gel electrophoretic analyses. Electrophoretic patterns of heated samples showed a progressive decrease in intact fibronectin chains (225 kDa) which coincided with the appearance of increasing amounts of numerous smaller components having molecular weights ranging from 10 000 to 200 000. The fragmentation was temperature-dependent, being undetectable after 2 h at 60°C, but detectable after 30 min at 70°C or as little as 2 min at 100°C. After 2 h at 100°C, the intact monomer was no longer visible. Neither mercaptoethanol nor SDS was required for fragmentation. Sterile filtration or pretreatment with inhibitors of proteolytic enzymes had no effect. Treatment with amines did not diminish the degradation, indicating that the process differs from heat-fragmentation of α 2-macroglobulin and complement proteins, which occurs at a reactive internal thiolester bond. Fibronectin fragmentation was highly pH-dependent, being markedly accelerated under acidic conditions, suggesting that autolytic cleavage of the peptide chain at acid-labile aspartyl bonds was responsible for this phenomenon.

13C nuclear magnetic resonance suggests a flexible proteoglycan core protein.

13C NMR was used to study the molecular dynamics of the chick limb bud proteoglycan core protein. Because only about 10% of the proteoglycan is protein, [2-13C]glycine and [3-13C]serine were incorporated into the core protein of the chick limb bud proteoglycan monomer using a chondrocyte culture system. The purified labeled monomer was studied in solution (50 mg/ml, 0.05 M sodium acetate/0.05 M sodium phosphate, pH 7.4) at 37 degrees C. Spin-lattice relaxation times, line widths, and nuclear Overhauser enhancements were measured for the labeled carbons in the protein and for the natural abundance carbons in the glycosaminoglycan chains. Analyses of these data show that correlation times for backbone reorientation of protein and polysaccharide chains in the intact monomer are predominantly in the range of 0.5-5.0 ns. These correlation times are 10(2)-10(3) times smaller than the minimum correlation time calculated for a rigid monomer, indicating that the core protein and polysaccharide backbones are segmentally flexible. Signal intensity data show that at least 80% of the protein backbone is flexible but do not exclude the possibility that 20% of the protein backbone has ordered structure. We observe both broad and narrow signal components in the spectrum of the intact monomer, showing that backbone motion is heterogeneous. The broad signal component is not observed after the monomer is digested with chondroitinase. This result and the strong concentration dependence of the 13C line width observed in solutions of chondroitin 4-sulfate suggest an assignment of the broad signal component to residues near the protein-polysaccharide linkage region. The difference in NMR parameters observed for free and substituted serine carbons confirms that motion of the substituted serine side chain is restricted.

Preliminary crystallographic data on the human λIII Bence Jones protein dimer Cle

A complete human λ Bence Jones protein dimer (Cle) has been isolated and crystallized. Protein Cle was characterized immunochemically and chemically as having a variable region amino acid sequence associated with light chains of the λ chain subgroup, λIII, and a constant region sequence characteristic of “non-Mcg” type λ chains. Bence Jones protein Cle contains two covalently bound intact monomers, each having a molecular weight of ~23,000. Crystals of Bence Jones protein Cle, obtained from ammonium sulfate solutions, diffract to 2.6 Å resolution and have the orthorhombic space group P2 12 12 1 with cell dimensions a = 113.0 A ̊ , b = 72.3 A ̊ , and c = 48.9 A ̊ . The asymmetric unit consists of a dimer with a molecular weight of ~ 46,000.

Nucleosome dissociation at physiological ionic strengths.

Monomer nucleosomes purified on isokinetic sucrose gradients are shown to dissociate into component DNA and histones at physiological ionic strength upon dilution to a DNA concentration below 20 microgram/ml. The starting material is 11S, contains 145-190 BP DNA, and equimolar amounts of the four core histones with slightly less H1. Dilution of monomers in the presence of 0.14 M NaCl results in the rapid conversion of 10-40% of the 3H thymidine labeled material from 11S to 5S (5S is coincident with the S value of monomer length DNA). The proportion of nucleosomes which dissociate increases with increasing NaCl concentration between 0.15 M and 0.35 M and decreases with increasing DNA concentration above 1 microgram/ml. Recycling 11S monomers, which remain after dissociation, through a second dilution in salt generates an equivalent proportion of 5S material as seen after the initial dilution. Thus, the dissociation does not result from special properties of a subset of nucleosomes. An equilibrium between intact monomer and free DNA and histones appears to be rapidly established under the conditions described and the dissociated DNA will reassociate with histones to form 11S monomers if conditions of high DNA concentration and low ionic strength are established.

Crystallographic data on a complete κ-type human Bence-Jones protein

A complete human κ-type Bence—Jones protein (Fin) has been isolated and crystallized. Immunochemical and physicochemical characterization of protein Fin indicates that it is of the κ-chain subgroup, κII, and that it consists of two non-covalently bound intact monomers having a molecular weight of ~23,000 Crystals of Bence—Jones protein Fin obtained from ammonium sulfate solutions have the orthorhombic space group P2 1 2 1 2 1 with cell dimensions a = 132.0 A ̊ , b = 93.3 A ̊ , and c = 42.3 A ̊ . The asymmetric unit consists of a dimer of molecular weight ~46,000.

Characterization and serology of the matrix protein from a nuclear-polyhedrosis virus of Trichoplusia ni before and after degradation by an endogenous proteinase.

The intact matrix protein from a nuclear-polyhedrosis virus of the cabbage looper (Trichoplusia ni), isolated after inhibition of an endogenous serine-type proteinase, was further purified by molecular-sieve chromatography. The matrix protein was associated with carbohydrate moieties, and the carbohydrate content was determined for the two major peptides isolated after proteolysis by the endogenous proteinase. The association-dissociation interactions of the intact and proteinase-hydrolysed monomer units were characterized at high and low pH. At pH1.9, proteinase-degraded matrix protein dissociated into two different peptide fractions, FI and FII. Fraction FII, a single peptide of 9400 daltons, comprised one-third of the monomer unit of 28 000 daltons. At pH9.5, the degraded peptides were tightly associated in units equivalent to the intact monomer. These monomer equivalents associated to form a series of interconverting aggregates. The predominant aggregate sedimented at 11S and had a mol.wt greater than or equal to 200 000. Two non-cross-reacting antigens were present in the aggregate mixture. The presence of these two antigens does not reflect the presence of two different matrix proteins; rather, the expression of the antigens correlates with the degree of aggregation of the matrix protein.

A Type k Bence Jones Protein Containing a Cysteinyl Residue in the Variable Region 1

The proteins precipitated with ammonium sulfate from the urine of a patient (Mat) with multiple myeloma were separated into three components by ion-exchange and gel chromatographies. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, amino acid analyses, immunochemical tests, and measurement of circular dichroism showed that these components were a dimer with a disulfide bond, a stable monomer, and a variable fragment, respectively. All three protein components reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) in Tris-HCl buffer at pH 8.0, indicating that they contained free sulhydryl groups. Partial reduction with dithiothreitol in the absence of denaturants yielded two SH groups per molecule from both the monomer and the dimer, and one SH group per molecule from the fragment. This indicates that the monomer of Mat protein contains a cysteinyl residue in the variable region in addition to a cysteinyl residue at the COOH terminus. The reactivities of the two SH groups of the partially reduced monomer toward iodoacetamide and iodoacetic acid were studied by polyacrylamide gel electrophoresis. The two SH groups had similar reactivities with iodacetamide, but the SH group at the COOH terminus was more reactive with iodoacetic acid than that in the variable region. The extrinsic Cotton effects of an azobenzene-2-sulfenyl group introduced into the SH group in the variable region were different from those of dye attached to the COOH terminal SH group, indicating that the two SH groups had different environments. The states of the SH groups of the intact monomer are discussed on the basis of these findings.

Specific cleavage of adenovirus-associated virus DNA by restriction endonuclease R· EcoRI—Characterization of cleavage products

The cleavage of adenovirus-associated virus type 2 (AAV2) DNA by the restriction endonuclease R· EcoRI was investigated using neutral and alkaline sucrose sedimentation, electrophoresis in composite agarose-acrylamide gels, and reassociation kinetics. Linear monomeric AAV2 DNA duplexes were cleaved at two specific sites to yield three fragments A, B and C, each of which is equivalent to a unique region of the genome of approximately 57.2, 38.2 and 4.6%, respectively. Fragment C may possibly consist of two smaller fragments, and in this case there would be three, rather than two, specific cleavage sites within a 4.6% region of the genome. Cleavage of oligomeric forms of AAV2 DNA (including linear and circular oligomers and network-like structures) yielded fragments A, B and C and two additional components, AB′ and AB . In alkaline sucrose, both AB′ and AB yielded only A and B strands. Component AB′ had a neutral sucrose sedimentation coefficient and electrophoretic mobility close to those of intact linear monomer AAV duplexes whereas AB sedimented more rapidly in neutral sucrose and was specifically trapped close to the electrophoretic origin in composite gels. In conditions expected to dissociate cohesive ends (80° in 1 × SSC), approximately 50% of both AB′ and AB released equimolar amounts of fragments A and B. Therefore, at least part of both AB′ and AB appear to consist of fragments A and B joined by cohesive ends. The remaining portion of both AB′ and AB apparently consists of more bizzare structures such as branched molecules (originating from DNA networks) or they may contain single-stranded regions. The probable physical order of fragments A, B and C in the AAV genome can be deduced.

Effects of chemical modification of f2 viral coat protein on its shell-forming activity

The coat protein of f2 bacteriophage has been modified in an attempt to determine the effects of changes in primary structure on the ability of this protein monomer to form shells in vitro. Cleavage by cyanogen bromide results in loss of ability to form shells; however, the fragments produced were active in inhibiting shell formation by unmodified protein. Reactions aimed at sulfhydryl and methionine residues resulted in precipitation of the protein monomer and attendant loss of ability to form shells. Arginine residues were reacted with phenylglyoxal hydrate: in the monomer, this prevented shell formation; although phage disintegrated upon treatment with reagent, the empty protein shells remained intact. Cleavage of the carboxyl-terminal tyrosine by carboxypeptidase produced a modified protein which was incapable of forming shells, but which prevented intact monomers from achieving the conformation necessary for shell formation. Shells and phage treated with carboxypeptidase did not release the carboxylterminal tyrosine residue.

The Occurrence and Significance of SLS CrystallitesIn Vivo

Electron microscopic studies of subcutaneous carrageenin granulomas induced in three different animal species revealed, in addition to several other ultrastructural changes in extracellular collagen fibers, the constant presence of normal SLS crystallites. This form of aggregation of tropocollagen molecules was observed throughout the entire evolution of the granuloma and is probably due to conditions created by the carrageenin in the tissue. It is suggested that SLS crystallites represent precipitation of intact tropocollagen monomers solubilized from previously constituted extracellular fibers, rather than aggregation of recently synthetized mononiers or precursors.The presence of SLS crystallites in one model of connective tissue degradation suggests that solubilization of established extracellular collagen fibers may be a regular feature of the general mechanism of collagen resorption.