scholarly journals Functional characterization of constituent enzyme fractions of mycobacillin synthetase

1985 ◽  
Vol 230 (3) ◽  
pp. 785-789 ◽  
Author(s):  
S K Ghosh ◽  
S Majumder ◽  
N K Mukhopadhyay ◽  
S K Bose
Keyword(s):  
Amino Acids ◽  
Functional Characterization ◽  
Enzyme Fraction ◽  
Sequential Activation

The enzyme fraction A, a constituent enzyme of the three-fraction enzyme mycobacillin synthetase, independently and sequentially activated five amino acids starting from L-proline, producing the pentapeptide Pro(Asp1,Glu1,Tyr1)Asp. The fractions B and C were unable to function independently. However, the fraction B synthesized the nonapeptide Pro(Asp3,Glu1,Tyr2,Ser1)Leu, sequentially activating the pentapeptide and next four amino acids, whereas the fraction C synthesized mycobacillin by the sequential activation of the nonapeptide and the remaining four amino acids. The pH optima of the above enzymes are almost identical (pH 7.8), but their Km values are a little different.

scholarly journals Human xylosyltransferase I and N-terminal truncated forms: functional characterization of the core enzyme

2006 ◽  
Vol 394 (1) ◽  
pp. 163-171 ◽  
Author(s):  
Sandra Müller ◽  
Jennifer Disse ◽  
Manuela Schöttler ◽  
Sylvia Schön ◽  
Christian Prante ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Binding Capacity ◽  
Functional Characterization ◽  
Terminal Region ◽  
Binding Motif ◽  
Heparin Binding ◽  
Loss Of Function ◽  
The Impact

Human XT-I (xylosyltransferase I; EC 2.4.2.26) initiates the biosynthesis of the glycosaminoglycan linkage region and is a diagnostic marker of an enhanced proteoglycan biosynthesis. In the present study, we have investigated mutant enzymes of human XT-I and assessed the impact of the N-terminal region on the enzymatic activity. Soluble mutant enzymes of human XT-I with deletions at the N-terminal domain were expressed in insect cells and analysed for catalytic activity. As many as 260 amino acids could be truncated at the N-terminal region of the enzyme without affecting its catalytic activity. However, truncation of 266, 272 and 273 amino acids resulted in a 70, 90 and >98% loss in catalytic activity. Interestingly, deletion of the single 12 amino acid motif G261KEAISALSRAK272 leads to a loss-of-function XT-I mutant. This is in agreement with our findings analysing the importance of the Cys residues where we have shown that C276A mutation resulted in a nearly inactive XT-I enzyme. Moreover, we investigated the location of the heparin-binding site of human XT-I using the truncated mutants. Heparin binding was observed to be slightly altered in mutants lacking 289 or 568 amino acids, but deletion of the potential heparin-binding motif P721KKVFKI727 did not lead to a loss of heparin binding capacity. The effect of heparin or UDP on the XT-I activity of all mutants was not significantly different from that of the wild-type. Our study demonstrates that over 80% of the nucleotide sequence of the XT-I-cDNA is necessary for expressing a recombinant enzyme with full catalytic activity.

Functional characterization of the transmembrane segment VII of the NHE1 isoform of the Na+/H+ exchangerThis paper is one of a selection of papers published in this Special Issue, entitled The Cellular and Molecular Basis of Cardiovascular Dysfunction, Dhalla 70th Birthday Tribute.

2007 ◽  
Vol 85 (3-4) ◽  
pp. 319-325 ◽  
Author(s):  
Jie Ding ◽  
Raymond W.P. Ng ◽  
Larry Fliegel
Keyword(s):  
Amino Acids ◽  
70Th Birthday ◽  
Functional Characterization ◽  
Integral Membrane Protein ◽  
Transmembrane Segment ◽  
Transmembrane Segments ◽  
Pore Lining ◽  
Positively Charged ◽  
Selection Of

The Na+/H+ exchanger isoform 1 is an integral membrane protein that regulates intracellular pH. It extrudes 1 intracellular H+ in exchange for 1 extracellular Na+. It has 2 large domains, an N-terminal membrane domain of 12 transmembrane segments and an intracellular C-terminal regulatory domain. We characterized the cysteine accessibility of amino acids of the critical transmembrane segment TM VII. Residues Leu 255, Leu 258, Glu 262, Leu 265, Asn 266, Asp 267, Val 269, Val 272, and Leu 273 were all mutated to cysteine residues in the cysteineless NHE1 isoform. Mutation of amino acids E262, N266, and D267 caused severe defects in activity and targeting of the intact full length protein. The balance of the active mutants were examined for sensitivity to the sulfhydryl reactive reagents, positively charged MTSET ((2- (trimethylammonium)ethyl)methanethiosulfonate) and negatively charged MTSES ((2-sulfonatoethyl)methanethiosulfonate). Leu 255 and Leu 258 were sensitive to MTSET but not to MTSES. The results suggest that these amino acids are pore-lining residues. We present a model of TM VII that shows that residues Leu 255, Leu 258, Glu 262, Asn 266, and Asp 267 lie near the same face of TM VII, lining the ion transduction pore.

Cloning and characterization of the heart muscle isoform of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) from crayfish

2002 ◽  
Vol 205 (17) ◽  
pp. 2677-2686 ◽  
Author(s):  
Dongdong Chen ◽  
Zhiping Zhang ◽  
Michele G. Wheatly ◽  
Yongping Gao
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Heart Muscle ◽  
Transmembrane Domain ◽  
Expression Patterns ◽  
Functional Characterization ◽  
Northern Analysis ◽  
Specific Probe ◽  
Tail Muscle

SUMMARY This paper describes the cloning and functional characterization of the heart muscle isoform of Sarco/endoplasmic reticulum Ca2+-ATPase(SERCA) from crayfish Procambarus clarkii. The complete crayfish heart SERCA, identified by reverse transcription-polymerase chain reaction(RT-PCR) and rapid amplification of cDNA ends (RACE), consists of 4495 bp with a 3060 bp open reading frame, coding for 1020 amino acids. This isoform differs from the previously identified axial abdominal (tail) muscle SERCA solely in its C-terminal amino acids. The last nine amino acids of the tail muscle isoform are replaced by 27 hydrophobic amino acids in the heart isoform that have the potential to form an additional transmembrane domain. Consistent with other invertebrate studies, Southern blot analysis suggested that the heart and tail muscle isoforms are encoded from the same gene that is equally related to SERCA-1, -2 and -3 of vertebrates. The tissue distributions of these two isoforms have been assessed using isoform-specific probes and northern analysis. A cardiac-specific probe bound only to a 5.8 kb species in heart and had minimal cross-hybridization with 7.6 and 5.8 kb species in eggs and no hybridization with tail muscle. A tail-isoform-specific probe hybridized with a 4.5 kb species in tail muscle and cross-hybridized with a 4.5 kb species in eggs and 8.8 kb in heart muscle. Both isoforms are expressed in eggs suggesting that transcripts are formed early in development and are subsequently broadly expressed in all tissue types. Expression of the cardiac muscle SERCA isoform varied with the stage of moulting. Expression was high in intermoult and decreased in premoult. However, expression was restored rapidly in postmoult (within 2 days) unlike expression of tail muscle SERCA,which remained downregulated for weeks. Differences in contractility between the two muscle types in the postmoult period may explain these expression patterns.

scholarly journals Functional characterization of alternatively spliced human SERCA3 transcripts

1998 ◽  
Vol 275 (6) ◽  
pp. C1449-C1458 ◽  
Author(s):  
Esteban Poch ◽  
Stephen Leach ◽  
Susan Snape ◽  
Tasha Cacic ◽  
David H. MacLennan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Functional Characterization ◽  
Lymphoid Tissues ◽  
Gene Products ◽  
Alternatively Spliced ◽  
Lower Capacity

The sarcoplasmic (or endoplasmic) reticulum Ca2+-ATPase (SERCA)-3 has been implicated in the possible dysregulation of Ca2+ homeostasis that accompanies the pathology of hypertension and diabetes. We report the molecular cloning of two alternatively spliced transcripts from the human SERCA3 gene, ATP2A3, that encode proteins that differ at their carboxy termini by 36 amino acids. SERCA3 transcripts were most abundantly expressed in lymphoid tissues, intestine, pancreas, and prostate. The two human SERCA3 proteins encoded by alternatively spliced transcripts were recognized by the monoclonal antibody PL/IM430 and demonstrated Ca2+ uptake and ATPase activity with an apparent Ca2+ affinity 0.5 pCa unit lower than that of other SERCA gene products. The subcellular distribution of SERCA3 protein was indistinguishable from that of SERCA2b, with expression in the nuclear envelope and in the endoplasmic reticulum throughout the cell. Two variant SERCA3 constructs, huS3-I and huS3-II, were isolated that encode proteins with three amino acid differences: Ala-673 (in huS3-I) substituted for Thr (in huS3-II), Ile-817 substituted for Met, and an insertion of Glu-994. huS3-I displayed a 10-fold lower capacity to transport Ca2+ than huS3-II.

scholarly journals Chemical, Microbiological, and Functional Characterization of Kefir Produced from Cow’s Milk and Soy Milk

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Raúl Ricardo Gamba ◽  
Shihori Yamamoto ◽  
Mahmoud Abdel-Hamid ◽  
Tetsuya Sasaki ◽  
Toshihide Michihata ◽  
...  
Keyword(s):  
Amino Acids ◽  
Glutamic Acid ◽  
Free Amino Acids ◽  
Free Amino ◽  
Functional Characterization ◽  
Acetic Acid Bacteria ◽  
Cow’S Milk ◽  
Soy Milk ◽  
Cow's Milk

Kefir is a functional beverage that contains lactic and acetic acid bacteria (LAB, AAB) and yeasts. This work’s aim was to study the chemical, microbial, and functional characteristics of kefir produced from cow’s milk and soy milk. After fermentation, free amino acids were 20.92 mg 100 mL−1 and 36.20 mg 100 mL−1 for cow’s milk and soy milk kefir, respectively. Glutamic acid was majority in both, suggesting that microbial proteolysis leads to an increase in free amino acids including glutamic acid. 108–109 CFU mL−1 LAB, 106–107 CFU mL−1 AAB, and 106–107 CFU mL−1 yeasts were counted in cow’s milk kefir, whereas soy milk kefir contained greatly lower yeasts and AAB. Lactococcus lactis, Kazachstania unispora, and Saccharomyces cerevisiae were isolated as major microorganisms in both kefirs. Acetobacter orientalis only existed in cow’s milk kefir. Cow’s milk and soy milk showed ACE inhibitory activity, which significantly increased after fermentation. Both kefirs also exhibited antioxidant activity and bactericidal activity against Escherichia coli, Salmonella Typhimurium, and Staphylococcus aureus.

Structural and functional characterization of the RBBP4–ZNF827 interaction and its role in NuRD recruitment to telomeres

2018 ◽  
Vol 475 (16) ◽  
pp. 2667-2679 ◽  
Author(s):  
Sile F. Yang ◽  
Ai-ai Sun ◽  
Yunyu Shi ◽  
Fudong Li ◽  
Hilda A. Pickett
Keyword(s):  
Amino Acids ◽  
Electrostatic Interactions ◽  
Zinc Finger Protein ◽  
Functional Characterization ◽  
Vital Role ◽  
Telomere Maintenance ◽  
Nurd Complex ◽  
Nucleosome Remodeling ◽  
Alternative Lengthening

The nucleosome remodeling and histone deacetylase (NuRD) complex is an essential multi-subunit protein complex that regulates higher-order chromatin structure. Cancers that use the alternative lengthening of telomere (ALT) pathway of telomere maintenance recruit NuRD to their telomeres. This interaction is mediated by the N-terminal domain of the zinc-finger protein ZNF827. NuRD–ZNF827 plays a vital role in the ALT pathway by creating a molecular platform for recombination-mediated repair. Disruption of NuRD binding results in loss of ALT cell viability. Here, we present the crystal structure of the NuRD subunit RBBP4 bound to the N-terminal 14 amino acids of ZNF827. RBBP4 forms a negatively charged channel that binds to ZNF827 through a network of electrostatic interactions. We identify the precise amino acids in RBBP4 required for this interaction and demonstrate that disruption of these residues prevents RBBP4 binding to both ZNF827 and telomeres, but is insufficient to decrease ALT activity. These data provide insights into the structural and functional determinants of NuRD activity at ALT telomeres.

scholarly journals A Practical Guide to Small Protein Discovery and Characterization using Mass Spectrometry

2021 ◽  
Author(s):  
Christian H. Ahrens ◽  
Joseph T. Wade ◽  
Matthew M. Champion ◽  
Julian D. Langer
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Functional Characterization ◽  
Model Organisms ◽  
Small Protein ◽  
Targeted Analysis ◽  
Small Proteins ◽  
Domains Of Life ◽  
Protein Discovery

Small proteins of up to ∼50 amino acids are an abundant class of biomolecules across all domains of life. Yet, due to the challenges inherent in their size, they are often missed in genome annotations, and are difficult to identify and characterize using standard experimental approaches. Consequently, we still know few small proteins even in well-studied prokaryotic model organisms. Mass spectrometry (MS) has great potential for the discovery, validation, and functional characterization of small proteins. However, standard MS approaches are poorly suited to the identification of both known and novel small proteins due to limitations at each step of a typical proteomics workflow, i.e., sample preparation, protease digestion, liquid chromatography, MS data acquisition, and data analysis. Here, we outline the major MS-based workflows and bioinformatic pipelines used for small protein discovery and validation. Special emphasis is placed on highlighting the adjustments required to improve detection and data quality for small proteins. We discuss both the unbiased detection of small proteins and the targeted analysis of small proteins of interest. Finally, we provide guidelines to prioritize novel small proteins, and an outlook on methods with particular potential to further improve comprehensive discovery and characterization of small proteins. IMPORTANCE Small proteins of up to ∼50 amino acids play important physiological roles across all domains of life. Mass spectrometry is an ideal approach to detect and characterize small proteins, but many aspects of standard mass spectrometry workflows are biased against small proteins due to their size. Here, we highlight applications of mass spectrometry to study small proteins, emphasizing modifications to standard workflows to optimize the detection of small proteins.

scholarly journals The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids

2014 ◽  
Vol 25 (19) ◽  
pp. 3017-3027 ◽  
Author(s):  
Olivia Tort ◽  
Sebastián Tanco ◽  
Cecilia Rocha ◽  
Ivan Bièche ◽  
Cecilia Seixas ◽  
...  
Keyword(s):  
Amino Acids ◽  
Posttranslational Modification ◽  
Protein Modification ◽  
Functional Characterization ◽  
Protein Family ◽  
Terminal Protein ◽  
Microtubule Cytoskeleton ◽  
Acidic Amino Acids ◽  
Carboxy Terminal

The posttranslational modification of carboxy-terminal tails of tubulin plays an important role in the regulation of the microtubule cytoskeleton. Enzymes responsible for deglutamylating tubulin have been discovered within a novel family of mammalian cytosolic carboxypeptidases. The discovery of these enzymes also revealed the existence of a range of other substrates that are enzymatically deglutamylated. Only four of six mammalian cytosolic carboxypeptidases had been enzymatically characterized. Here we complete the functional characterization of this protein family by demonstrating that CCP2 and CCP3 are deglutamylases, with CCP3 being able to hydrolyze aspartic acids with similar efficiency. Deaspartylation is a novel posttranslational modification that could, in conjunction with deglutamylation, broaden the range of potential substrates that undergo carboxy-terminal processing. In addition, we show that CCP2 and CCP3 are highly regulated proteins confined to ciliated tissues. The characterization of two novel enzymes for carboxy-terminal protein modification provides novel insights into the broadness of this barely studied process.

scholarly journals Structural and functional characterization of Mpp75Aa1.1, a putative beta-pore forming protein from Brevibacillus laterosporus active against the western corn rootworm

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0258052
Author(s):  
Jean-Louis Kouadio ◽  
Stephen Duff ◽  
Michael Aikins ◽  
Meiying Zheng ◽  
Timothy Rydel ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Amino Acids ◽  
Receptor Binding ◽  
Functional Characterization ◽  
Diabrotica Virgifera Virgifera ◽  
The United States ◽  
Western Corn Rootworm ◽  
Corn Rootworm ◽  
Insecticidal Proteins

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a major corn pest of significant economic importance in the United States. The continuous need to control this corn maize pest and the development of field-evolved resistance toward all existing transgenic maize (Zea mays L.) expressing Bacillus thuringiensis (Bt) insecticidal proteins against WCR has prompted the development of new insect-protected crops expressing distinct structural classes of insecticidal proteins. In this current study, we describe the crystal structure and functional characterization of Mpp75Aa1.1, which represents the first corn rootworm (CRW) active insecticidal protein member of the ETX_MTX2 sub-family of beta-pore forming proteins (β-PFPs), and provides new and effective protection against WCR feeding. The Mpp75Aa1.1 crystal structure was solved at 1.94 Å resolution. The Mpp75Aa1.1 is processed at its carboxyl-terminus by WCR midgut proteases, forms an oligomer, and specifically interacts with putative membrane-associated binding partners on the midgut apical microvilli to cause cellular tissue damage resulting in insect death. Alanine substitution of the surface-exposed amino acids W206, Y212, and G217 within the Mpp75Aa1.1 putative receptor binding domain I demonstrates that at least these three amino acids are required for WCR activity. The distinctive spatial arrangement of these amino acids suggests that they are part of a receptor binding epitope, which may be unique to Mpp75Aa1.1 and not present in other ETX_MTX2 proteins that do not have WCR activity. Overall, this work establishes that Mpp75Aa1.1 shares a mode of action consistent with traditional WCR-active Bt proteins despite significant structural differences.

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