Ajit S. Bhown

Bunyavirus Nucleoprotein, N, and a Non-structural Protein, NSS, Are Coded by Overlapping Reading Frames in the S RNA

It has been shown previously, by sequence analysis of the S RNA segment of snowshoe hare (SSH) bunyavirus, that two overlapping open reading frames in the viral complementary sequence code for proteins with molecular weights of 26.8 X 10(3) and 10.5 X 10(3) respectively. In addition to the viral nucleocapsid (N) protein, which is coded by the S RNA, analyses of parental and reassortant bunyavirus-infected cell extracts have shown that the viral S RNA and M RNA species each code for non-structural proteins (NSS and NSM, respectively). In the present report, in vitro translation analyses of the S mRNA species recovered from virus-infected cells indicate that a single size class of mRNA directs the synthesis of N and NSS. Compositional analyses of selected tryptic peptides of N and NSS have provided proof that N is the product of the first open reading frame, and NSS the product of the second.

High-sensitivity sequence determination of proteins quantitatively recovered from sodium dodecyl sulfate gels using an improved electrodialysis procedure

Abstract An improved electrodialysis procedure has been developed to recover quantitatively proteins from sodium dodecyl sulfate (SDS)-polyacrylamide gels. The efficiency of the method has been demonstrated successfully on H-2K k , β 2 -microglobulin, complement factor D, and viral structural protein p27. The results indicate that yields exceeding 93% are obtainable, and that extended amino acid sequences of the eluted proteins in microgram quantities can be obtained in the presence of SDS without intrinsic and/or extrinsic labeling with radioisotopes.

Isolation and structural analysis of influenza C virion glycoproteins

Abstract Influenza C virions possess a single glycoprotein that is cleaved into two disulfide-linked subunits, gp65 and gp30. When analyzed under nonreducing conditions, the uncleaved (gpI) and cleaved (gpI) glycoproteins differ significantly in apparent molecular weight; however, we observed no difference in their tryptic peptide patterns. We have isolated the glycoproteins by selective solubilization with Triton X-100 or octylglucoside; only preparations obtained with the latter detergent showed hemagglutinating activity. In purified glycoprotein samples, gp65 was routinely observed as a doublet on SDS-polyacrylamide gels. Analysis of tryptic peptides by ion-exchange chromatography demonstrated that the two gp65 bands have indistinguishable polypeptide backbones; they appear to differ, however, in carbohydrate content. The uncleaved glycoprotein as well as gp65 were resistant to Edman degradation indicating the presence of blocked amino termini, whereas gp30 was observed to have the N-terminal tripeptide sequence NH2-Ile-Phe-Gly. This sequence is homologous to a sequence at the N termini of influenza A and B HA2 glycoproteins, except for the presence of an additional terminal glycine residue in these viruses. The influenza C glycoproteins form a regular hexagonal lattice on the viral envelope. This arrangement is sometimes maintained in disrupted virus preparations and in glycoprotein subunits released from the envelope by limited proteolysis, indicating that direct interactions between the glycoprotein molecules are responsible, at least in part, for the observed arrangement. Observations of clustered surface projections on plasma membranes of infected cells, and of released virus particles apparently devoid of internal nucleoproteins, are consistent with the suggestion that lateral interactions between the influenza C glycoproteins may be important in virus assembly.

Isolation, characterization, and amino-terminal amino acid sequence analysis of human neutrophil cathepsin G from normal donors☆

Human neutrophil cathepsin G from normal donors has been purified 82-fold using an isolation procedure which included sequential sodium chloride extraction, Aprotonin-Sepharose affinity chromatography, CM-cellulose ion-exchange chromatography, and AcA44 gel filtration chromatography. The inclusion of this last purification step was crucial for separating inactive lower molecular weight species from the active forms of neutrophil cathepsin G and resulted in a higher specific activity of the final preparation. SDS polyacrylamide gradient gel electrophoresis of the purified reduced protein demonstrated three discrete polypeptides of Mr 31,000, 30,000, and 29,500. Peptide analysis of tryptic digests indicated that these three polypeptides are structurally related to each other and represent microheterogeneity of the purified protein. The cathepsin G peptide maps were distinctly different from the peptide maps of neutrophil elastase. The apparent isoelectric points of these forms as determined by two-dimensional electrophoresis was approximately 8.0. Utilizing microsequencing techniques, the first 25 residues of normal neutrophil cathepsin G have been determined and shown to be identical (except for residue 11) with the sequence of 21 residues of cathepsin G isolated from leukemic myeloid cells. A high degree of homology was found when the amino-terminal regions of neutrophil cathepsin G, rat mast cell protease II (65%) and two human serine proteinases, factor D (52%) and neutrophil elastase (48%), were compared. A precipitating monospecific antiserum to cathepsin G was produced by repeated immunizations of guinea pigs. This antiserum has been used in immunoblotting experiments to demonstrate that the intracellular form(s) of this enzyme is the same approximate Mr as the purified enzyme, and to develop a solid-phase radioimmunoassay for measuring neutrophil cathepsin G in the range 5-50 ng/ml.

Identification of Clostridium histolyticum Collagenase Hyperreactive Sites in Type I, II, and III Collagens: Lack of Correlation with Local Triple Helical Stability

The class I and IIClostridium histolyticum collagenases (CHC) have been used to identify hyperreactive sites in rat type I, bovine type II, and human type III collagens. The class I CHC attack both collagens at loci concentrated in the N-terminal half of these collagens starting with the site closest to the N-terminus. The class II CHC initiate collagenolysis by attacking both collagens in the interior to produce a mixture of C-terminal 62,000 and a N-terminal 36,000 fragments. Both fragments are next shortened by removal of a 3000 fragment. These results are very similar to those reported earlier for the hydrolysis of rat type I collagen by these CHC, indicating that the three collagens share many hyperreactive sites. Similar reactions carried out with the respective gelatins show that they are cleaved at many sites at approximately the same rate. Thus, the hyperreactivity of the sites identified must be attributed to their environment in the native collagens. N-terminal sequencing of the fragments produced in these reactions has allowed the identification of 16 cleavage sites in the α1(I), α2(I), α1(II), and α1(III) collagen chains. An analysis of the triple helical stabilities of these cleavage site regions as reflected by their imino acid contents fails to yield a correlation between reactivity and triple helical stability. The existence of these hyperreactive CHC cleavage sites suggests that type I, II, and III collagens contain regions that have specific nontriple helical conformations. The sequence of these sites presented here now makes it possible to investigate these conformations by computational and peptide mimetic techniques.

An improved procedure for high-sensitivity microsequencing: Use of aminoethyl aminopropyl glass beads in the beckman sequencer and the Ultrasphere ODS column for PTH amino acid identification

An improved procedure for high-sensitivity microsequencing is described. The method employs (i) aminoethyl aminopropyl glass beads for in situ purification of quadrol by absorbing α-amino group reactive impurities commonly found even in highly purified quadrol and (ii) an Altex 5-μm Ultrasphere ODS column and highly purified methanol which is available commercially to identify phenylthiohydantoin derivatives of amino acids by high-pressure liquid chromatography. The efficiency of the method has been demonstrated by the results of sequence analyses of peptides obtained by acid and cyanogen bromide digestions of viral proteins. A significant increase in the initial and repetitive yields has been consistently observed by this procedure.

A simple modification on the vacuum system of the Beckman automated sequencer to improve the efficiency of edman degradation

Abstract A simple modification of the high-vacuum system of the Beckman automated sequencer is described. The air cylinder controlling the high vacuum to the reaction cup has been replaced with an electrically operated stainless-steel solenoid valve. The system maintains better high vacuum without the necessity for frequent oil changes. The efficiency has been demonstrated by the results of extended amino acid sequence analysis of various unknown proteins and peptides.

Use of fluorescamine as an effective blocking reagent to reduce the background in protein sequence analyses by the beckman automated sequencer

As an effective aid to further develop the strategies for extended amino acid sequence determinations, the use of fluorescamine (fluram) as a novel blocking reagent to chemically reduce the newly generated amino termini responsible for the progressively increasing background is described. The method involves interruption of the run when proline has been determined to be the amino terminus, deletion of PITC delivery, in situ treatment of the sample with fluram within the spinning cup extraction of the reaction products using an ethyl acetate: benzene mixture, HFBA delivery, and second extraction with 1-chlorobutane. The normal program is then allowed to continue until the next proline is reached and the entire extended sequence information on nanomole amounts of proteins. The efficiency of the method has been demonstrated by direct comparison of the results of sequence analysis of amyloid P-component with (63 residues) and without (46 residues) fluram treatment.

A comparison of fluorescamine and o-phthaldialdehyde as effective blocking reagents in protein sequence analyses by the Beckman sequencer

Use of o-phthaldialdehyde to chemically reduce the newly generated amino termini responsible for the progressively increasing background during an extended amino acid sequence analysis in a liquid phase sequencer has been described. The results have been compared with Fluram blocking using apomyoglobin and rabbit C-reactive protein as standard and unknown samples, respectively.

A modified system for thiazolinone conversion to thiohydantoin derivatives and their separation by high-pressure liquid chromatography

An improved and very simple procedure for thiazolinone conversion to thiohydantoin derivatives and their separation by reverse-phase high-pressure liquid chromatography is described. Trifluoroacetic acid (10%) in ethyl acetate has been employed as a conversion reagent to circumvent the deamidation of acid amides and methylation of acidic amino acids, with a concomitant increase in the detection limits of these residues. Additionally, a very simple procedure has been developed for the separation of phenylthiohydantoin (PTH) derivatives of amino acids. The system takes advantage of the computer-controlled precise mixing of the solvents A and B to achieve accurate pH and thus avoid the necessity of pH adjustment of a buffer. The procedure is simple and highly reproducible, and separates all the 20 known PTH amino acids. The efficiency of the method has been examined on synthetic and natural proteins/peptides, in manual and autoconversion systems, over a period of more than 18 months.