Identification of a precursor form of cathepsin D in microsomal lumen: Characterization of enzymatic activation and proteolytic processing in vitro

1987 ◽  
Vol 148 (1) ◽  
pp. 335-343 ◽  
Author(s):  
Yukio Nishimura ◽  
Masahide Higaki ◽  
Keitaro Kato
Keyword(s):  
Cathepsin D ◽  
Proteolytic Processing ◽  
Enzymatic Activation

Characterization of in vitro proteolytic processing of β-endorphin by reversed-phase HPLC

Peptides ◽  
1984 ◽  
Vol 5 (6) ◽  
pp. 1037-1042 ◽  
Author(s):  
Thomas P. Davis ◽  
Hans Schoemaker ◽  
Alison J. Culling-Berglund
Keyword(s):  
Reversed Phase ◽  
Proteolytic Processing ◽  
Reversed Phase Hplc

Biological characterization of charge isomers of human growth hormone

1988 ◽  
Vol 118 (1) ◽  
pp. 14-21 ◽  
Author(s):  
A. Skottner ◽  
A. Forsman ◽  
B. Skoog ◽  
J. L. Kostyo ◽  
C. M. Cameron ◽  
...  
Keyword(s):  
Biological Activity ◽  
Biological Activities ◽  
Human Growth ◽  
Proteolytic Processing ◽  
Biological Characterization ◽  
Growth Promoting ◽  
Rat Adipose Tissue ◽  
Insulin Like Activity

Abstract. Since deamidation of the human GH molecule may alter the manner and extent to which the hormone is cleaved by proteases, and since it has been repeatedly suggested that proteolytic processing is required for the expression of certain of the activities of GH, the present study was conducted to determine whether the biological activity profiles of more acidic forms of human GH are altered. Three charge isomers, GH-b, GH-c and GH-d, representing primarily deamidated forms, were isolated from a native human GH preparation (Crescormon®) in amounts adequate for characterization of their biological activities. All three were essentially equipotent in a radioimmunoassay for human GH. When assessed for growth-promoting activity in the hypophysectomized rat, the isomers were again equipotent with each other and with the GH preparation from which they were derived. The charge isomers also had significant in vitro insulin-like activity on isolated rat adipose tissue and diabetogenic activity in the ob/ob mouse. Thus, the biological activity profiles of these charge isomers of human GH do not differ greatly from one another.

scholarly journals Biochemical Characterization of Signal Peptidase I from Gram-Positive Streptococcus pneumoniae

2001 ◽  
Vol 183 (2) ◽  
pp. 621-627 ◽  
Author(s):  
Sheng-Bin Peng ◽  
Li Wang ◽  
John Moomaw ◽  
Robert B. Peery ◽  
Pei-Ming Sun ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Serine Proteases ◽  
Active Sites ◽  
Biochemical Characterization ◽  
Proteolytic Processing ◽  
Signal Peptidase ◽  
Gram Positive ◽  
Signal Peptidase I

ABSTRACT Bacterial signal peptidase I is responsible for proteolytic processing of the precursors of secreted proteins. The enzymes from gram-negative and -positive bacteria are different in structure and specificity. In this study, we have cloned, expressed, and purified the signal peptidase I of gram-positive Streptococcus pneumoniae. The precursor of streptokinase, an extracellular protein produced in pathogenic streptococci, was identified as a substrate of S. pneumoniae signal peptidase I. Phospholipids were found to stimulate the enzymatic activity. Mutagenetic analysis demonstrated that residues serine 38 and lysine 76 of S. pneumoniae signal peptidase I are critical for enzyme activity and involved in the active site to form a serine-lysine catalytic dyad, which is similar to LexA-like proteases andEscherichia coli signal peptidase I. Similar to LexA-like proteases, S. pneumoniae signal peptidase I catalyzes an intermolecular self-cleavage in vitro, and an internal cleavage site has been identified between glycine 36 and histidine 37. Sequence analysis revealed that the signal peptidase I and LexA-like proteases show sequence homology around the active sites and some common properties around the self-cleavage sites. All these data suggest that signal peptidase I and LexA-like proteases are closely related and belong to a novel class of serine proteases.

scholarly journals Expression of Murine Coronavirus Recombinant Papain-Like Proteinase: Efficient Cleavage Is Dependent on the Lengths of both the Substrate and the Proteinase Polypeptides

1999 ◽  
Vol 73 (4) ◽  
pp. 2658-2666 ◽  
Author(s):  
Henry Teng ◽  
Josefina D. Piñón ◽  
Susan R. Weiss
Keyword(s):  
Hepatitis Virus ◽  
In Vitro Transcription ◽  
Proteolytic Processing ◽  
Replicase Gene ◽  
Cleavage Activity ◽  
Viral Sequences ◽  
Rna Polymerase Promoter

ABSTRACT Proteolytic processing of the replicase gene product of mouse hepatitis virus (MHV) is essential for viral replication. In MHV strain A59 (MHV-A59), the replicase gene encodes two predicted papain-like proteinase (PLP) domains, PLP-1 and PLP-2. Previous work using viral polypeptide substrates synthesized by in vitro transcription and translation from the replicase gene demonstrated both cisand trans cleavage activities for PLP-1. We have cloned and overexpressed the PLP-1 domain in Escherichia coli by using a T7 RNA polymerase promoter system or as a maltose-binding protein (MBP) fusion protein. With both overexpression systems, the recombinant PLP-1 exhibited trans cleavage activity when incubated with in vitro-synthesized viral polypeptide substrates. Subsequent characterization of the recombinant PLP-1 revealed that in vitrotrans cleavage is more efficient at 22°C than at higher temperatures. Using substrates of increasing lengths, we observed efficient cleavage by PLP-1 requires a substrate greater than 69 kDa. In addition, when PLP-1 was expressed as a polypeptide that included additional viral sequences at the carboxyl terminus of the predicted PLP-1 domain, a fivefold increase in proteolytic activity was observed. The data presented here support previous data suggesting that in vitro and in vivo cleavage of the ORF 1a polyprotein by PLP-1 can occur in both in cis and in trans. In contrast to the cleavage activity demonstrated for PLP-1, no in vitro cleavage incis or in trans could be detected with PLP-2 expressed either as a polypeptide, including flanking viral sequences, or as an MBP fusion enzyme.

scholarly journals A Glutaredoxin, Encoded by the G4L Gene of Vaccinia Virus, Is Essential for Virion Morphogenesis

2000 ◽  
Vol 74 (19) ◽  
pp. 9175-9183 ◽  
Author(s):  
Christine L. White ◽  
Andrea S. Weisberg ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Virus Replication ◽  
Proteolytic Processing ◽  
Rnase Protection ◽  
Virus Particles ◽  
Late Protein ◽  
Virion Morphogenesis ◽  
Core Components

ABSTRACT Vaccinia virus encodes two glutaredoxins, O2L and G4L, both of which exhibit thioltransferase and dehydroascorbate reductase activities in vitro. Although O2L was previously found to be dispensable for virus replication, we now show that G4L is necessary for virion morphogenesis. RNase protection and Western blotting assays indicated that G4L was expressed at late times after infection and was incorporated into mature virus particles. Attempts to isolate a mutant virus with a deleted G4L gene were unsuccessful, suggesting that the protein was required for virus replication. This interpretation was confirmed by the construction and characterization of a conditional lethal recombinant virus with an inducible copy of the G4L gene replacing the original one. Expression of G4L was proportional to the concentration of inducer, and the amount of glutaredoxin could be varied from barely detectable to greater than normal amounts of protein. Immunogold labeling revealed that the induced G4L protein was associated with immature and mature virions and adjacent cytoplasmic depots. In the absence of inducer, the production of infectious virus was severely inhibited, though viral late protein synthesis appeared unaffected except for decreased maturation-dependent proteolytic processing of certain core components. Electron microscopy of cells infected under nonpermissive conditions revealed an accumulation of crescent membranes on the periphery of electron-dense globular masses but few mature particles. We concluded that the two glutaredoxin homologs encoded by vaccinia virus have different functions and that G4L has a role in virion morphogenesis, perhaps by acting as a redox protein.

Assays for Phospholipid-Dependent Formation of Thrombin and Xa: A Potential Method for Quantifying Lupus Anticoagulant Activity

1991 ◽  
Vol 66 (04) ◽  
pp. 453-458 ◽  
Author(s):  
John T Brandt
Keyword(s):  
Specific Activity ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Clinical Importance ◽  
Potential Method ◽  
Quantitative Expression ◽  
Increased Risk ◽  
Apparent Association

SummaryLupus anticoagulants (LAs) are antibodies which interfere with phospholipid-dependent procoagulant reactions. Their clinical importance is due to their apparent association with an increased risk of thrombo-embolic disease. To date there have been few assays for quantifying the specific activity of these antibodies in vitro and this has hampered attempts to purify and characterize these antibodies. Methods for determining phospholipid-dependent generation of thrombin and factor Xa are described. Isolated IgG fractions from 7 of 9 patients with LAs were found to reproducibly inhibit enzyme generation in these assay systems, permitting quantitative expression of inhibitor activity. Different patterns of inhibitory activity, based on the relative inhibition of thrombin and factor Xa generation, were found, further substantiating the known heterogeneity of these antibodies. These systems may prove helpful in further purification and characterization of LAs.

Characterization of the Original Christmas Disease Mutation (Cysteine 206 → Serine): From Clinical Recognition to Molecular Pathogenesis

1992 ◽  
Vol 67 (01) ◽  
pp. 063-065 ◽  
Author(s):  
Sherryl A M Taylor ◽  
Jacalyn Duffin ◽  
Cherie Cameron ◽  
Jerome Teitel ◽  
Bernadette Garvey ◽  
...  
Keyword(s):  
Nucleotide Substitution ◽  
Novel Mutation ◽  
Factor Ix ◽  
Causative Mutation ◽  
Coding Region ◽  
Body Of Knowledge ◽  
Polymerase Chain ◽  
Splice Junctions

SummaryChristmas disease was first reported as a distinct clinical entity in two manuscripts published in 1952 (1, 2). The eponym associated with this disorder, is the surname of the first patient examined in detail and reported by Biggs and colleagues in a paper describing the clinical and laboratory features of seven affected individuals (3). This patient has severe factor IX coagulant deficiency (less than 0.01 units/ml) and no detectable circulating factor IX antigen (less than 0.01 units/ml). Coding sequence and splice junctions of the factor IX gene from this patient have been amplified in vitro through the polymerase chain reaction (PCR). One nucleotide substitution was identified at nucleotide 30,070 where a guanine was replaced by a cytosine. This mutation alters the amino acid encoded at position 206 in the factor IX protein from cysteine to serine. The non conservative nature of this substitution, the absence of this change in more than 200 previously sequenced factor IX genes and the fact that the remainder of the coding region of this gene was normal, all provide strong circumstantial evidence in favour of this change being the causative mutation in this patient. The molecular characterization of this novel mutation in the index case of Christmas disease, contributes to the rapidly expanding body of knowledge pertaining to Christmas disease pathogenesis.

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