scholarly journals Biochemical characterization of the arginine-specific proteases of Porphyromonas gingivalis W50 suggests a common precursor

1997 ◽  
Vol 323 (3) ◽  
pp. 701-709 ◽  
Author(s):  
Minnie RANGARAJAN ◽  
Susan J. M. SMITH ◽  
Sally U ◽  
Michael A. CURTIS
Keyword(s):  
Molecular Mass ◽  
Porphyromonas Gingivalis ◽  
Extracellular Enzymes ◽  
Biochemical Characterization ◽  
Disease Process ◽  
High Molecular Mass ◽  
Kinetic Properties ◽  
Peptidase Activity ◽  
Post Translational Modification ◽  
Peptide Bonds

Extracellular proteases of Porphyromonas gingivalis specific for arginyl peptide bonds are considered to be important virulence factors in periodontal disease. In order to determine the number, inter-relationship and kinetic properties of these proteases, extracellular enzymes with this peptide-bond specificity were purified and characterized from P. gingivalis W50. Three forms, which we denote RI, RI-A and RI-B, accounted for all of the activity in the supernatant. All three enzymes contain an α chain of ∼54 kDa with the same N-terminal amino acid sequence. RI is a heterodimer of non-covalently linked α and β chains which migrate to the same position on SDS/PAGE but which can be resolved by 8 M urea/PAGE. RI-A and RI-B are both monomeric, but the molecular mass of RI-B (70–80 kDa) is significantly increased due to post-translational modification with lipopolysaccharide. All forms show absolute specificity for peptide bonds with Arg in the P1 position and are also capable of hydrolysing N-terminal Arg and C-terminal Arg–Arg peptide bonds. Thus they show limited amino- and carboxy-peptidase activity. For the hydrolysis of Nα-benzoyl-l-Arg-p-nitroanilide, the pH optimum is 8.0 at 30 °C. The Vmax for all three enzymes is controlled by ionization of two residues with apparent pKas at 30 °C of 6.5±0.05 and 9.7±0.05, and ΔH values of ∼29 kJ/mol and ∼ 24 kJ/mol in the enzyme–substrate complex. By analogy with papain, the pKa of 6.5 could be ascribed to a Cys and the pKa of 9.7 to a His residue. E-64 [l-trans-epoxysuccinyl-leucylamide-4-(4-guanidino)butane] is a competitive inhibitor of RI, RI-A and RI-B. Based on physical properties and kinetic behaviour, RI-A appears to be analogous to gingipain from P. gingivalis HG66. However the α/β structure of RI differs significantly from that of the high-molecular-mass multimeric complex of gingipain containing four haemagglutinins described by others. Since the genes for RI and high-molecular-mass gingipain are identical, the data indicate that an alternative processing pathway is involved in the formation of RI from the initial precursor. Furthermore, the identical N-termini and enzymic properties of the catalytic component of RI, RI-A and RI-B suggest that the maturation pathway of the RI precursor may also give rise to RI-A and RI-B. The physiological functions of these isoforms and their role in the disease process may become more apparent through examination of their interactions with host proteins.

scholarly journals All Detectable High-Molecular-Mass Penicillin-Binding Proteins Are Modified in a High-Level β-Lactam-Resistant Clinical Isolate of Streptococcus mitis

2001 ◽  
Vol 45 (7) ◽  
pp. 2075-2081 ◽  
Author(s):  
Ana Amoroso ◽  
Diego Demares ◽  
Marta Mollerach ◽  
Gabriel Gutkind ◽  
Jacques Coyette
Keyword(s):  
Molecular Mass ◽  
Clinical Isolate ◽  
Active Site ◽  
Binding Proteins ◽  
High Molecular Mass ◽  
Mosaic Structure ◽  
Kinetic Properties ◽  
Streptococcus Mitis ◽  
Penicillin Binding Proteins ◽  
High Level

ABSTRACT All detectable high-molecular-mass penicillin-binding proteins (HMM PBPs) are altered in a clinical isolate of Streptococcus mitis for which the β-lactam MICs are increased from those previously reported in our region (cefotaxime MIC, 64 μg/ml). These proteins were hardly detected at concentrations that saturate all PBPs in clinical isolates and showed, after densitometric analysis, 50-fold-lower radiotracer binding. Resistance was related to mosaic structure in all HMM PBP-coding genes, where critical region replacement was complemented not only by substitutions already reported for the closely related Streptococcus pneumoniae but also by other specific replacements that are presumably close to the active-site serine. Mosaic structure was also presumed in apbp1a-sensitive strain used for comparison, confirming that these structures do not unambiguously imply, by themselves, detectable critical changes in the kinetic properties of these proteins.

Alkaline phosphatase of high and low molecular mass in human serum and bile: a comparative study of kinetic properties.

1980 ◽  
Vol 26 (3) ◽  
pp. 451-456
Author(s):  
P M Crofton ◽  
A F Smith
Keyword(s):  
Alkaline Phosphatase ◽  
Molecular Mass ◽  
High Molecular Mass ◽  
Magnesium Ion ◽  
Kinetic Properties ◽  
Zinc Ions ◽  
Magnesium Ions ◽  
Optimum Conditions ◽  
Ion Concentrations ◽  
Low Magnesium

Abstract We studied the kinetic properties of high- and low-molecular-mass forms of alkaline phosphatase purified from serum and bile, to clarify their interrelationships. They were found to share virtually identical kinetic properties, and to obey the same general kinetics as the liver-derived isoenzyme from serum and the low-molecular-mass isoenzyme from bile with regard to optimum conditions of assay, activation by magnesium ions, inhibition by L-homoarginine, inhibition by nickel and zinc ions, and inactivation by urea. Most of the characteristics such as Km (at low magnesium ion concentrations), Ki for L-homoarginine, and half-life for urea inactivation, were closely similar for low- and high-molecular-mass alkaline phosphatase. We conclude that these forms of alkaline phosphatase in plasma and bile are closely related. We discuss the possible nature of this relationship.

Alkaline phosphatase of high and low molecular mass in human serum and bile: a comparative study of kinetic properties.

1980 ◽  
Vol 26 (3) ◽  
pp. 451-456 ◽  
Author(s):  
P M Crofton ◽  
A F Smith
Keyword(s):  
Alkaline Phosphatase ◽  
Molecular Mass ◽  
High Molecular Mass ◽  
Magnesium Ion ◽  
Kinetic Properties ◽  
Zinc Ions ◽  
Magnesium Ions ◽  
Optimum Conditions ◽  
Ion Concentrations ◽  
Low Magnesium

Abstract We studied the kinetic properties of high- and low-molecular-mass forms of alkaline phosphatase purified from serum and bile, to clarify their interrelationships. They were found to share virtually identical kinetic properties, and to obey the same general kinetics as the liver-derived isoenzyme from serum and the low-molecular-mass isoenzyme from bile with regard to optimum conditions of assay, activation by magnesium ions, inhibition by L-homoarginine, inhibition by nickel and zinc ions, and inactivation by urea. Most of the characteristics such as Km (at low magnesium ion concentrations), Ki for L-homoarginine, and half-life for urea inactivation, were closely similar for low- and high-molecular-mass alkaline phosphatase. We conclude that these forms of alkaline phosphatase in plasma and bile are closely related. We discuss the possible nature of this relationship.

Overexpression, purification and biochemical characterization of a class A high-molecular-mass penicillin-binding protein (PBP), PBP1∗ and its soluble derivative from Mycobacterium tuberculosis

2002 ◽  
Vol 361 (3) ◽  
pp. 635-639
Author(s):  
Sanjib BHAKTA ◽  
Joyoti BASU
Keyword(s):  
Molecular Mass ◽  
Inclusion Bodies ◽  
Biochemical Characterization ◽  
Binding Protein ◽  
High Molecular Mass ◽  
Proteinase K ◽  
Amino Acid Residues ◽  
Penicillin Binding Protein ◽  
E Coli ◽  
Class A

The product of the gene ponA present in cosmid MTCY21D4, one of the collection of clones representing the genome of Mycobacteriumtuberculosis, has been named penicillin-binding protein 1∗ (PBP1∗), by analogy to the previously characterized PBP1∗ of M. leprae. This gene has been overexpressed in Escherichia coli. His6-tagged PBP1∗ localizes to the membranes of induced E. coli cells. Its susceptibility to degradation upon proteinase K digestion of spheroplasts from E. coli expressing the protein supports the view that the majority of the protein translocates to the periplasmic side of the membrane. Recombinant PBP1∗ binds benzylpenicillin and several other β-lactams, notably cefotaxime, with high affinity. Truncation of the N-terminal 64 amino acid residues results in an expressed protein present exclusively in inclusion bodies and unable to associate with the membrane. The C-terminal module encompassing amino acids 272–663 can be extracted from inclusion bodies under denaturing conditions using guanidine/HCl and refolded to give a protein fully competent in penicillin-binding. Deletion of Gly95—Gln143 results in the expression of a protein, which is localized in the cytosol. The soluble derivative of PBP1∗ binds benzylpenicillin with the same efficiency as the full-length protein. This is the first report of a soluble derivative of a class A high-molecular-mass PBP.

Macroenzymes: biochemical characterization, clinical significance, and laboratory detection.

1989 ◽  
Vol 35 (12) ◽  
pp. 2261-2270 ◽  
Author(s):  
A T Remaley ◽  
P Wilding
Keyword(s):  
Liver Disease ◽  
Autoimmune Diseases ◽  
Molecular Mass ◽  
Clinical Significance ◽  
Biochemical Characterization ◽  
Clinical Chemistry ◽  
Diagnostic Markers ◽  
High Molecular Mass ◽  
Serum Enzyme ◽  
Serum Components

Abstract "Macroenzymes" are enzymes in serum that have formed high-molecular-mass complexes, either by self-polymerization or by association with other serum components. Many enzymes in serum that are measured in clinical chemistry laboratories can occur in a macroenzyme form. Macroenzymes are interesting clinically because of their association with several diseases, including autoimmune diseases and liver disease, and are being investigated as possible diagnostic markers. Most importantly, macroenzymes frequently interfere with the interpretation of serum enzyme results, and as a result can cause diagnostic and therapeutic errors. We review the biochemical characterization, clinical significance, and laboratory detection of macroenzymes.

scholarly journals 16S rRNA Is Bound to Era ofStreptococcus pneumoniae

1999 ◽  
Vol 181 (17) ◽  
pp. 5242-5249 ◽  
Author(s):  
Timothy I. Meier ◽  
Robert B. Peery ◽  
S. Richard Jaskunas ◽  
Genshi Zhao
Keyword(s):  
16S Rrna ◽  
Molecular Mass ◽  
Affinity Purification ◽  
High Molecular Mass ◽  
Kinetic Properties ◽  
Gtpase Activity ◽  
Thrombin Cleavage ◽  
Bacterial Cell Cycle ◽  
Similar Kinetic ◽  
Mass Form

ABSTRACT Era is an essential membrane-associated GTPase that is present in bacteria and mycoplasmas. Era appears to play an important role in the regulation of the bacterial cell cycle. In this study, we expressed the native and glutathione S-transferase (GST) fusion forms ofStreptococcus pneumoniae Era in Escherichia coli and purified both proteins to homogeneity. We showed that RNA was copurified with the GST-Era protein of S. pneumoniae during affinity purification and remained associated with the protein after removal of the GST tag by thrombin cleavage. The thrombin-treated and untreated GST-Era proteins could bind and hydrolyze GTP and exhibited similar kinetic properties (dissociation constant [kD ], Km , and V max). However, the native Era protein purified by using different chromatographic columns had a much lower GTPase activity than did GST-Era, although it had a similarkD . In addition, RNA was not associated with the protein. Purified GST-Era protein was shown to be present as high (600-kDa)- and low (120-kDa)-molecular-mass forms. The high-molecular-mass form of GST-Era was associated with RNA and exhibited a very high GTPase activity. Approximately 40% of purified GST-Era protein was associated with RNA, and removal of the RNA resulted in a significant reduction in GTPase activity. The RNA associated with GST-Era was shown to be predominantly 16S rRNA. The native Era protein isolated directly from S. pneumoniae was also present as a high-molecular-mass species (600 kDa) complexed with RNA. Together, our results suggest that 16S rRNA is associated with Era and might stimulate its GTPase activity.

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