Products Of Limited Digestion Research Articles

Traces of pFc’ in IVIG interact with human IgG Fc domains and counteract aggregation

To prevent multimer formation, intravenous immunoglobulin (IVIG) is often treated with traces of pepsin. So far, the mechanism behind this treatment has been unclear. Recently, we reported that human IgG4 binds other IgG molecules via Fc–Fc interactions. Here we show that IVIG treated with traces of pepsin (Nanogam) inhibits these interactions. We found that – besides IgG4 – peptides corresponding to IgG1 and IgG2 pFc’ (products of limited pepsin digestion) are responsible for the inhibitory action. Using radiolabeled pFc’, it was found that pFc’ binds directly to IgG1. Furthermore, recombinant CH3 fragments were found to also possess binding activity, and potencies of inhibition varied over 3 orders of magnitude amongst the subclasses, IgG4 being most potent. We propose that pFc’ formation explains how limited pepsin digestion diminishes adverse effects of IVIG. In particular, the presence of this fragment can enhance the stability of IgG products including IVIG and therapeutical monoclonal antibodies. Indeed, using a model system it was found that acid-induced aggregation of IgG is reduced in the presence of pFc’, suggesting a ‘chaperone-like’ activity of this fragment. Thus, pFc’ can modulate Fc interactions and may therefore reduce adverse effects of IVIG, in particular by preventing oligomerization.

Endopolyphosphatase in Saccharomyces cerevisiae undergoes post-translational activations to produce short-chain polyphosphates

Endopolyphosphatase (Ppn), responsible for cleavage of long chain inorganic polyphosphate (poly P) of several hundred residues to generate progressively shorter chains, has been identified in mammalian cells and purified from Saccharomyces cerevisiae. Disruption of the encoding gene, PHM5, in S. cerevisiae resulted in a mutant that showed limited growth and failure to survive in a minimal medium. The limited digestion products of the yeast enzyme Ppn1 judged to be P 3 and P 60 have now, with the homogeneous enzyme and improved separation methods, been demonstrated to be P i and P 3. Ppn1, a homotetramer of a 35-kDa subunit, is of vacuolar origin and requires protease activation of a 78 kDa (674-aa) precursor polypeptide (prePpn1). The protease-processed Ppn1 has been purified 3800-fold to homogeneity and the protease cleavage sites determined. Both termini of prePpn1 and the post-translational modification of N-glycosylations are essential for the protease-mediated maturation of Ppn1.

Thermodynamic studies of the core histones: stability of the octamer subunits is not altered by removal of their terminal domains.

We have investigated the role of the labile terminal domains of the core histones on the stability of the subunits of the protein core of the nucleosome by studying the thermodynamic behavior of the products of limited trypsin digestion of these subunits. The thermal stabilities of the truncated H2A-H2B dimer and the truncated (H3-H4)/(H3-H4)(2) system were studied by high-sensitivity differential scanning calorimetry and circular dichroism spectroscopy. The thermal denaturation of the truncated H2A-H2B dimer at pH 6.0 and low ionic strength is centered at 47.3 degrees C. The corresponding enthalpy change is 35 kcal/mol of 11.5 kDa monomer unit, and the heat capacity change upon unfolding is 1.2 kcal/(K mol of 11.5 kDa monomer unit). At pH 4.5 and low ionic strength, the truncated (H3-H4)/(H3-H4)(2) system, like its full-length counterpart, is quantitatively dissociated into two truncated H3-H4 dimers. The thermal denaturation of the truncated H3-H4 dimer is characterized by the presence of a single calorimetric peak centered at 60 degrees C. The enthalpy change is 25 kcal/mol of 10 kDa monomer unit, and the change in heat capacity upon unfolding is 0.5 kcal/(K mol of 10 kDa monomer unit). The thermal stabilities of both types of truncated dimers exhibit salt and pH dependencies similar to those of the full-length proteins. Finally, like their full-length counterparts, both truncated core histone dimers undergo thermal denaturation as highly cooperative units, without the involvement of any significant population of melting intermediates. Therefore, removal of the histone "tails" does not generally affect the thermodynamic behavior of the subunits of the core histone complex, indicating that the more centrally located regions of the histone fold and the extra-fold structured elements are primarily responsible for their stability and responses to parameters of their chemical microenvironment.

Ligand Interactions in the Adenosine Nucleotide-binding Domain of the Hsp90 Chaperone, GRP94: II. LIGAND-MEDIATED ACTIVATION OF GRP94 MOLECULAR CHAPERONE AND PEPTIDE BINDING ACTIVITY

The N-terminal domain of eukaryotic Hsp90 proteins contains a conserved adenosine nucleotide binding pocket that also serves as the binding site for the Hsp90 inhibitors geldanamycin and radicicol. Although this domain is essential for Hsp90 function, the molecular basis for adenosine nucleotide-dependent regulation of GRP94, the endoplasmic reticulum paralog of Hsp90, remains to be established. We report that bis-ANS (1,1'-bis(4-anilino-5-napthalenesulfonic acid), an environment sensitive fluorophore known to interact with nucleotide-binding domains, binds to the adenosine nucleotide-binding domain of GRP94 and thereby activates its molecular chaperone and peptide binding activities. bis-ANS was observed to elicit a tertiary conformational change in GRP94 similar to that occurring upon heat shock, which also activates GRP94 function. bis-ANS activation of GRP94 function was efficiently blocked by radicicol, an established inhibitory ligand for the adenosine nucleotide binding pocket. Confirmation of the N-terminal nucleotide binding pocket as the bis-ANS-binding site was obtained following covalent incorporation of bis-ANS into GRP94, trypsinolysis, and sequencing of bis-ANS-labeled limit digestion products. These data identify a ligand dependent regulation of GRP94 function and suggest a model whereby GRP94 function is regulated through a ligand-dependent conversion of GRP94 from an inactive to an active conformation.

Selective and asymmetric action of trypsin on the dimeric forms of seminal RNase.

Dimeric seminal RNase (BS-RNase) is an equilibrium mixture of conformationally different quaternary structures, one characterized by the interchange between subunits of their N-terminal ends (the MXM form); the other with no interchange (the M=M form). Controlled tryptic digestion of each isolated quaternary form generates, as limit digest products, folded and enzymatically active molecules, very resistant to further tryptic degradation. Electrospray mass spectrometric analyses and N-terminal sequence determinations indicate that trypsin can discriminate between the conformationally different quaternary structures of seminal RNase, and exerts a differential and asymmetric action on the two dimeric forms, depending on the original quaternary conformation of each form. The two digestion products from the MXM and the M=M dimeric forms have different structures, which are reminiscent of the original quaternary conformation of the dimers: one with interchange, the other with no interchange, of the N-terminal ends. The surprising resistance of these tryptic products to further tryptic action is explained by the persistence in each digestion product of the original intersubunit interface.

Isolation and partial characterisation of the non-cellulosic polysaccharides of flax fibre

The non-cellulosic polysaccharides of flax fibre cells were isolated using chemical extraction methods. Extraction of mature retted flax fibre with the calcium chelating agent trans-1,2-diaminocyclohexane- N, N, N′, N′-tetraacetic acid (CDTA) removed polysaccharides enriched in galacturonic acid. These pectic polysaccharides were fractionated by their solubility in water into two fractions that differed in their neutral sugar content. Extraction of the fibre with aqueous 24% (w/v) potassium hydroxide produced the first hemicellulosic fraction (Hc1), which was enriched in xylose and, to a lesser extent, glucose- and galactose-containing polysaccharides. Digestion of aliquots of this fraction using the fungal carbohydrase mixture, Driselase, yielded xylobiose and isoprimeverose, which are characteristic limit digestion products of xylans and xyloglucans, respectively. Digestion of Hc1 with a crude cellulase preparation from Aspergillus niger yielded oligosaccharide products, one of which was similar to a characteristic nonasaccharide subunit of xyloglucan. Further extraction of the fibre residue with aqueous 18% (w/v) potassium hydroxide supplemented with 4% (w/v) of boric acid removed a second hemicellulosic fraction (Hc2), which was enriched in mannose-containing polysaccharides. Digestion of this fraction with a highly purified endo-(1 → 4)-β- d-mannanase converted 67% of the total polysaccharide into oligosaccharides within one hour. The β- d-mannanase-derived oligosaccharides had a monosaccharide composition of mannose-glucose of 1.6:1.0. Analysis of the oligosaccharides by TLC and high-performance anion exchange chromatography revealed products characteristic of a glucomannan similar to those found in hardwoods. The presence of β-linked d-glucose residues was confirmed by the removal of glucose from the β- d-mannanase-derived oligosaccharides by a purified β- d-glucosidase. A final extraction of the fibre with aqueous 90% Me 2SO containing 4% (w/v) of boric acid removed a further pectic fraction (Hc3) enriched in neutral sugars. Preliminary analyses suggest that this fraction may contain a polysaccharide akin to rhamnogalacturonan I.

A prominent antigenic surface polypeptide involved in the biogenesis and function of the vaccinia virus envelope

Polypeptides of the vaccinia virus envelope exposed on the surface were identified by means of sulfo- N-hydroxysuccinimidobiotin as a surface tag. Among surface expressed polypeptides is the 35-kDa antigen, previously designated Ag35. Both monoclonal (mAb) and monospecific affinity pure antibodies directed against Ag35 neutralized vaccinia infectiousness, indicating that this prominent surface antigen has a function during early virus-host cell interactions. The binding of several monoclonal antibodies to various regions of Ag35 was tested by reacting CNBr fragments, derived from the polypeptide, employing Western blotting. All mAbs tested reacted with the same region of Ag35. Estimation of the molecular weights (MW), based on migration of the CNBr peptides in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that those partial digestion products which contained a proline-rich 99 amino acid limit digest fragment were present at a position approximately 12.5 kDa larger than that predicted from the DNA sequence. By contrast, partial and limit digest products lacking the proline-rich fragment migrated to the MW position expected from the length of the DNA sequence. This observation demonstrates that departure from a predicted 22.3 kDa to an anomalous MW of Ag35 is conferred by the proline-rich peptide. The surface location of Ag35 was confirmed by immune electron microscopy. In a competition test the binding specificity of mAb and affinity-purified antibodies at the surface of virions could be demonstrated. Evidence for an association of Ag35 with the virus envelope at various stages during biogenesis of vaccinia was obtained by immune electron microscopy of whole mounts and thin sections. Presence of Ag35 as an early component of immature and mature virions, probably residing in the bilayer membrane structure was detected. A distinction can, therefore, be made between Ag35 and several other vaccinia envelope polypeptides which are synthesized as late functions and added during late stages of envelope assembly.

Localization of the membrane-associated region of vesicular stomatitis virus M protein at the N terminus, using the hydrophobic, photoreactive probe 125I-TID

The membrane-reactive, photoactivatable probe 125I-TID [3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-3H-diazirine] was found to label the M protein of vesicular stomatitis virus about 40% as much as G protein in intact virions, in agreement with labeling studies with other probes. By analyzing limited tryptic digestion and specific chemical cleavage products, the label was essentially entirely localized within the first 19, and probably within the first 5 to 10, amino acid residues at the N terminus, identifying this short amphipathic segment as the likely site of interaction of M protein with the viral bilayer.

Purification and properties of a bacteriophage-induced endo-N-acetylneuraminidase specific for poly-alpha-2,8-sialosyl carbohydrate units.

The soluble form of a bacteriophage-induced endo-N-acetylneuraminidase (Endo-N) specific for hydrolyzing oligo- or poly-alpha-2,8-linked sialosyl units in sources as disparate as bacterial and neural membrane glycoconjugates was purified approximately 10,000-fold and characterized. The enzyme appears homogenous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a subunit Mr 105,000. This corresponds to one of the higher Mr phage proteins which comprises 7.5% (by weight) of the total phage protein. The holoenzyme is active at neutral pH and has a Mr by gel filtration of 328,000, suggesting that the active enzyme is a trimer. Endo-N requires a minimum of 5 sialyl residues (DP5, where DP represents degree of polymerization) for activity. The limit digest products from the alpha-2,8-linked polysialic acid capsule of Escherichia coli K1 are DP4 with some DP3 and DP1,2. DP2-4 do not appear to inhibit depolymerization of polysialic acid. Endo-N digestion of the polysialosyl moiety on neural cell adhesion molecules yields sialyl oligomers with DP3 and DP4. The presence of a terminal sialitol changes both the distribution of limit digestion products and the apparent minimum substrate size. Higher Mr alpha-2,8-linked sialyl polymers (approximately DP200) are better substrates (Km 50-70 microM) than sialyl oligomers of approximately DP10-20 (Km 1.2 mM). Endo-N activity is inhibited by DNA and several other poly-anions tested. An examination of the distribution of intermediate products shows that Endo-N binds and cleaves at random sites on the polysialosyl chains, in contrast to initiating cleavage at one end and depolymerizing processively. Endo-N can serve as a specific molecular probe to detect and selectively modify poly-alpha-2,8-sialosyl carbohydrate units which have been implicated in bacterial meningitis and neural cell adhesion.

Native species of helix destabilizing protein-1 in mouse myeloma identified by antibody probing of Western blots

Antibody probing of Western blots is a method for analyzing the apparent M r of a protein in any given preparation (Renart, J., Reiser, J., and Stark, G., Proc. Natl. Acad. Sci. USA 76: 3116, 1979). We prepared a rabbit antiserum to purified mouse myeloma helix destabilizing protein-1 and used this antiserum in Western blotting experiments with a crude homogenate of mouse myeloma. The results indicated that the native species of helix destabilizing protein-1 can be degraded during purification. This in vitro proteolysis results in complete loss of the native species and accumulation of lower M r proteins that represent limit digestion products. These findings have identified the true native form of mouse myeloma HDP-1 as a protein of apparent M r=36,000 to 38,000, instead of the M r=24,000 and 27,000 proteins obtained by routine purification.

Degradation of pigeon liver fatty acid synthetase in the absence of exogenous proteinases

The homogeneity of pigeon liver fatty acid synthetase has been rigorously tested by physicochemical techniques and crossed-rocket immunoelectrophoresis. The enzyme has also been incubated for 1 h at 100°C in 2% sodium dodecyl sulfate and 0.1 M dithiothreitol. The number of protein components on gel electrophoresis and of dansylated amino acids increased as a function of incubation time. Furthermore, the minor proteins observed after gel electrophoresis cross-reacted with antibody raised to the synthetase. Proteolysis was not chemically mediated by the detergent, the reducing agent or the buffer conditions chosen. Several commercially prepared proteins were not degraded by this procedure, and two proteins were recalcitrant to hydrolysis when included in the same incubation mixture as the synthetase. The inclusion of certain microbial proteinase inhibitors decreased the amount of degradation. This demonstrated that hydrolysis of the synthetase is mediated by a specific vertebrate enzyme which retains activity under denaturing conditions at 100°C. Further degradation is also observed after individual treatment of four limited digestion products from the pigeon liver fatty acid synthetase, suggesting the possibility of an inherent proteolytic activity within the complex.

Localization of the gene for cytochrome f in pea chloroplast DNA

Pea chloroplast DNA and cloned restriction fragments of pea chloroplast DNA have been used as templates in a cell-free coupled transcription-translation system from E. coli and have been shown to direct the synthesis of a 39 kD polypeptide related to cytochrome f (37.3 kD). The product was shown to be similar to cytochrome f by immunoprecipitation with specific antibodies and by peptide mapping of the products of limited chymotrypsin digestion. The structural gene for this higher molecular weight form of cytochrome f has been located within a 3.3 kbp BglII fragment situated 18 kbp from the single 16S rRNA gene.

A method to sharply delimit a yeast nuclear gene in a cloned DNA fragment

We have developped a procedure to delimit the boundaries of a cloned gene carried on a DNA fragment as large as 4 to 5 kilobases. The method consists in the following. Two series of limit digest products generated with a tetranucleotide recognition sequence endonuclease and originating from either of the two ends of this DNA segment are tested for their complementing capacity by yeast transformation. The gene is then delimited by the overlap of the two shortest complementing fragments.

Differential proteolytic susceptibility of laminin alpha and beta subunits

Laminin, the glycoprotein of basement membranes, consists of two subunits of 200,000 (α) and 400,000 (β) M r on gel electrophoresis after reduction. We evaluated the relative proteolytic susceptibility of the two subunits using a variety of serine proteases. Human α-thrombin degraded the β subunit without altering the density or size of the α subunit. Chymotrypsin, plasmin, and cathepsin G all degraded both the β and α subunits producing limited digestion products. Chymotrypsin and cathepsin G both produced two major fragments of 160,000 and 130,000 M r whereas plasmin produced two fragments of 180,000 and 140,000 M r. Time course digestion studies demonstrated that the 400-kd β subunit was digested much more rapidly than the α subunit, and suggested that the major fragments (greater than 100,000 M r) produced by chymotrypsin, plasmin, and cathepsin G were derived from the α subunit. The latter supposition was confirmed by first digesting laminin with thrombin to completely remove the β subunit, followed by digestion with chymotrypsin, cathepsin G, or plasmin. We conclude that the β subunit of laminin is highly protease labile. In contrast, the α subunit contains a large region resistant to serine proteases. Electron microscopic studies of the purified fragment of laminin derived from digestion with cathepsin G demonstrated that the protease resistant region of the α subunit contained three arms of similar appearance (32 nm) and included the intersection of the three short arms of the laminin molecule.

A sequence of the yeast 2 μm DNA plasmid chromosome near the origin of replication is exposed to restriction endonuclease digestion

The yeast 2 μm DNA plasmid nucleoprotein complex was subjected to restriction endonuclease digestion to ascertain whether all possible sites are equally accessible to hydrolysis. When plasmid nucleoprotein complexes which had been fixed with formaldehyde were exhaustively digested with restriction endonucleases HinfI or CfoI, only a few of the limit digest products were produced. Furthermore, the limited set of restriction endonuclease sites exposed in formaldehyde-treated plasmid chromosomes could be shown to be preferentially hydrolyzed when plasmid chromosomes which had not been treated with formaldehyde were digested with the same restriction endonucleases. Mapping of the preferred sites revealed that they mapped to the region of the plasmid near the replication origin. These results demonstrate that the protection of DNA from nuclease activity is not constant along the plasmid chromatin, and that a region near the replication origin is preferentially exposed to endonuclease hydrolysis.

Properties of disulfide-linked tubulin purified on hydroxyapatite and its comparison with intact and dissociated microtubules using limited tryptic digestion

This investigation pursued aspects of tubulin structure using limited tryptic digestion as a sensitive monitor of conformational state. A novel form of tubulin was analyzed that had been purified on hydroxyapatite in sodium phosphate buffer and exhibited interchain disulfide crosslinking. Circular dichroism spectra of this protein were highly reproducible and indicate it assumes a stable native state. Limited proteolysis of bovine brain microtubule protein in its assembled or disassembled states yielded a prominent 41,000-molecular-weight product as long as it was stabilized by GTP-containing buffers. Hydroxyapatite-purified tubulin yielded entirely different limited digestion products, with species having molecular weights of 48,000 and several between 35,000 and 19,000. Limited proteolysis was found to offer a useful probe of tubulin structure and to produce fragments which might prove valuable in analyzing its protein substructure. Discussion is given on the relevance of these results to the structural state assumed by tubulin and to the conformation of disulfide-linked tubulin reported to reside in the postsynaptic density.

Mechanism of action of Moloney murine leukemia virus RNase H III.

The mechanism of action of Moloney murine leukemia virus RNase H III was studied, utilizing the model substrate (A)n. (dT)n and polyacrylamide gel electrophoresis to assay enzyme activity. Examination by electrophoresis on 15% polyacrylamide gels in 7 M urea and on DEAE-cellulose paper in 7 M urea revealed that, early in a reaction with [3H](A)n. (dT)n as substrate, RNase H III generated products ranging in length from 80 to 90 nucleotides to less than 10 nucleotides and that after extended incubation the limit digest products generated were 3 to 15 nucleotides long. Product oligomers were of the following configuration: [5'-P, 3'-OH](A)n. RNase H III was shown to be an exonuclease requiring free ends in its substrate for activity by the inability to degrade RNA inserted in Escherichia coli ColE1 plasmid DNA. The enzyme was capable of attacking RNA in RNA-DNA hybrids in the 5' to 3' and 3' to 5' directions as demonstrated by the use of [3H, 5'-32P](A)600. (dT)n and cellulose-[3H](A)n. (dT)n. Rnase H III was random in its mode of action because addition of excess unlabeled (A)n. (dT)n to an ongoing reaction with [3H](A)n. (dT)n as substrate resulted in immediate inhibition of enzyme activity.

Nucleotide sequence of wheat chloroplastid 4.5 S ribonucleic acid. Sequence homologies in 4.5 S RNA species.

The nucleotide sequence of wheat (Triticum aestivum L.) chloroplastid ribosome-associated 4.5 S RNA is U-A-A-G-G-U-G-A-G-C-G-G-C-G-A-G-A-C-G-A-G-C-C-G-U-U-U-A-A-A-U-A-G-G-U-G-U-C-A-A -G-U-G-G-A-A-G-U-G-C-A-G-U-G-A-U-G-U-A-U-G-C-A-G-C-U-G-A-G-G-C-A-U-C-C-U-A-A-C- G-A-A-C-G-A-A-C-G-A-U-U-U-G-A-A-COH. The sequence is highly conserved among chloroplastid 4.5 S RNAs but not related to either the 5 S or 5.8 S rRNA species. When compared with 4.5 S RNAs from other origins, the results indicate that this molecule bears little sequence homology to the mammalian nuclear 4.5 S RNA but suggest that chloroplastid 4.5 S RNA may be related to the bacerial 4.5 S species. An estimate for the secondary structure of the wheat 4.5 S molecule was deduced from products of limited pancreatic and T1 ribonuclease digestion, and similarities with the Escherichia coli 4.5 S RNA are discussed.

Properties of a purified nucleolar ribonuclease from Ehrlich ascites carcinoma cells.

A nucleolar ribonuclease specific for single-stranded ribonucleic acid (RNA) has been isolated and extensively purified from Ehrlich ascites carcinoma cells. The enzyme is optimally active at neutral pH and degrades RNA via a 2',3'-cyclic intermediate leaving 3'- or 2',3'-cyclic terminated oligonucleotides. The ribonuclease has an apparent molecular weight of 38 500 as judged by sedimentation equilibrium and is a basic protein having an isoelectric point greater than 9.0. The enzyme preferentially cleaves poly(C) over poly (U), poly(A), or poly(C).poly(I). Limit digestion products of poly(C) degratation are on the average tri-, tetra-, and pentanucleotides. In the partial digestion of yeast 5.8S rRNA, the nucleolar ribonuclease cleaves only CpA phosphodiester bonds. Spermidine, spermine, and histone I inhibit the activity of nucleolar ribonuclease. Antibodies directed toward pancreatic RNase do not cross-react with the Ehrlich nucleolar ribonuclease.

Amino acid sequence of the sulfate-binding protein from Salmonella typhimurium LT2.

The amino acid sequence of the sulfate-binding protein from Salmonella typhimurium LT2 was determined by automated sequenator analysis of whole protein and fragments derived by chemical and enzymatic cleavage of whole protein. The fragments were products of limited trypsin digestion at arginine, cleavage at tryptophan by BrNps-skatole and o-iodosobenzoic acid, digestion with the protease from Staphylococcus aureus V8 at Glu-X bonds, cleavage by hydroxylamine at Asn-Gly bonds, and subdigestion with trypsin, chymotrypsin, and the Staphylococcus protease. The COOH-terminal sequence was confirmed using carboxypeptidase B and amino acid analysis. The sulfate-binding protein was determined to contain a single polypeptide of 310 residues with a molecular weight of 34,667 calculated from the sequence.