scholarly journals QUANTITATIVE AND HISTOCHEMICAL STUDIES ON THE DESORPTION AND READSORPTION OF ALDOLASE IN CROSS-STRIATED MUSCLE

1969 ◽  
Vol 17 (5) ◽  
pp. 314-320 ◽  
Author(s):  
H. ARNOLD ◽  
J. NOLTE ◽  
D. PETTE
Keyword(s):  
Enzyme Activity ◽  
Actin Filaments ◽  
Phosphate Buffer ◽  
Striated Muscle ◽  
Psoas Muscle ◽  
Histochemical Staining ◽  
Intracellular Location ◽  
Successive Extraction ◽  
Complete Extraction

Complete extraction of aldolase from minced rabbit psoas muscle was achieved by successive extraction steps in 0.1 M phosphate buffer. Aldolase was then readsorbed quantitatively to the depleted myofibrils. Extraction, readsorption and a final redsorption of the enzyme were followed quantitatively by enzyme activity determinations and qualitatively by histochemical staining of aldolase. The intracellular location of the readsorbed enzyme was found to be identical with that of aldolase in native muscle. In both cases, aldolase was localized within the isotropic bands. These results as well as the previously demonstrated binding of the enzyme to F-actin suggest that aldolase is located within the interfilamentary sarcoplasm of the isotropic bands and is probably also bound in vivo to the actin filaments.

scholarly journals Analysis of myofibrillar structure and assembly using fluorescently labeled contractile proteins.

1984 ◽  
Vol 98 (3) ◽  
pp. 825-833 ◽  
Author(s):  
J W Sanger ◽  
B Mittal ◽  
J M Sanger
Keyword(s):  
Actin Filaments ◽  
Striated Muscle ◽  
Contractile Proteins ◽  
Actin Binding ◽  
Thin Filaments ◽  
In Vitro System ◽  
Self Association ◽  
Cross Bridges

To study how contractile proteins become organized into sarcomeric units in striated muscle, we have exposed glycerinated myofibrils to fluorescently labeled actin, alpha-actinin, and tropomyosin. In this in vitro system, alpha-actinin bound to the Z-bands and the binding could not be saturated by prior addition of excess unlabeled alpha-actinin. Conditions known to prevent self-association of alpha-actinin, however, blocked the binding of fluorescently labeled alpha-actinin to Z-bands. When tropomyosin was removed from the myofibrils, alpha-actinin then added to the thin filaments as well as the Z-bands. Actin bound in a doublet pattern to the regions of the myosin filaments where there were free cross-bridges i.e., in that part of the A-band free of interdigitating native thin filaments but not in the center of the A-band which lacks cross-bridges. In the presence of 0.1-0.2 mM ATP, no actin binding occurred. When unlabeled alpha-actinin was added first to myofibrils and then labeled actin was added fluorescence occurred not in a doublet pattern but along the entire length of the myofibril. Tropomyosin did not bind to myofibrils unless the existing tropomyosin was first removed, in which case it added to the thin filaments in the l-band. Tropomyosin did bind, however, to the exogenously added tropomyosin-free actin that localizes as a doublet in the A-band. These results indicate that the alpha-actinin present in Z-bands of myofibrils is fully complexed with actin, but can bind exogenous alpha-actinin and, if actin is added subsequently, the exogenous alpha-actinin in the Z-band will bind the newly formed fluorescent actin filaments. Myofibrillar actin filaments did not increase in length when G-actin was present under polymerizing conditions, nor did they bind any added tropomyosin. These observations are discussed in terms of the structure and in vivo assembly of myofibrils.

scholarly journals Some properties of the uridine diphosphate glucuronyltransferase activity synthesizing thio-β-d-glucuronides

1973 ◽  
Vol 131 (1) ◽  
pp. 139-147 ◽  
Author(s):  
H. P. A. Illing ◽  
G. J. Dutton
Keyword(s):  
Enzyme Activity ◽  
Phenolic Compounds ◽  
Organ Culture ◽  
Tissue Distribution ◽  
Glucuronic Acid ◽  
Uridine Diphosphate ◽  
Intracellular Location ◽  
Gunn Rats ◽  
Optimum Ph

1. Some properties of the UDP-glucuronyltransferase synthesizing thio-β-d-glucuronides were investigated and compared with those of the enzyme synthesizing the O-glucuronides of analogous phenols. 2. Enzyme activity was generally similar for both classes of substrate in tissue distribution, intracellular location, optimum pH, perinatal development and induction by organ culture or by phenobarbital. 3. Certain differences were noted between the two types of activity in behaviour on storage and on activation, in kinetic behaviour and in distribution between Wistar and Gunn rats; the Gunn rats were not deficient in hepatic UDP-glucuronyltransferase activity towards o-aminothiophenol. 4. These differences are no greater than those exhibited in the synthesis of various O-glucuronides; therefore thiolic substrates could compete in vivo with phenolic compounds for access to the UDP-glucuronyltransferase complex as well as for UDP-glucuronic acid.

Orientation of actin filaments during motion in motility assay

1995 ◽  
Vol 53 ◽  
pp. 52-53
Author(s):  
J. Borejdo ◽  
S. Burlacu
Keyword(s):  
Actin Filaments ◽  
Thin Filament ◽  
Striated Muscle ◽  
Myosin Head ◽  
Classical Method ◽  
Polarization Of Fluorescence ◽  
Single Protein ◽  
Intracellular Organelles ◽  
Change Orientation ◽  
Active Entity

Polarization of fluorescence is a classical method to assess orientation or mobility of macromolecules. It has been a common practice to measure polarization of fluorescence through a microscope to characterize orientation or mobility of intracellular organelles, for example anisotropic bands in striated muscle. Recently, we have extended this technique to characterize single protein molecules. The scientific question concerned the current problem in muscle motility: whether myosin heads or actin filaments change orientation during contraction. The classical view is that the force-generating step in muscle is caused by change in orientation of myosin head (subfragment-1 or SI) relative to the axis of thin filament. The molecular impeller which causes this change resides at the interface between actin and SI, but it is not clear whether only the myosin head or both SI and actin change orientation during contraction. Most studies assume that observed orientational change in myosin head is a reflection of the fact that myosin is an active entity and actin serves merely as a passive "rail" on which myosin moves.

scholarly journals A Region of the Myosin Rod Important for Interaction With Paramyosin in Caenorhabditis elegans Striated Muscle

Genetics ◽  
2000 ◽  
Vol 156 (2) ◽  
pp. 631-643
Author(s):  
Pamela E Hoppe ◽  
Robert H Waterston
Keyword(s):  
Caenorhabditis Elegans ◽  
Heavy Chain ◽  
Molecular Interaction ◽  
Striated Muscle ◽  
Genetic Interaction ◽  
Specific Interaction ◽  
Thick Filament ◽  
Parallel Arrangement ◽  
Myosin Rod

Abstract The precise arrangement of molecules within the thick filament, as well as the mechanisms by which this arrangement is specified, remains unclear. In this article, we have exploited a unique genetic interaction between one isoform of myosin heavy chain (MHC) and paramyosin in Caenorhabditis elegans to probe the molecular interaction between MHC and paramyosin in vivo. Using chimeric myosin constructs, we have defined a 322-residue region of the MHC A rod critical for suppression of the structural and motility defects associated with the unc-15(e73) allele. Chimeric constructs lacking this region of MHC A either fail to suppress, or act as dominant enhancers of, the e73 phenotype. Although the 322-residue region is required for suppression activity, our data suggest that sequences along the length of the rod also play a role in the isoform-specific interaction between MHC A and paramyosin. Our genetic and cell biological analyses of construct behavior suggest that the 322-residue region of MHC A is important for thick filament stability. We present a model in which this region mediates an avid interaction between MHC A and paramyosin in parallel arrangement in formation of the filament arms.

scholarly journals Topological Data Analysis Approaches to Uncovering the Timing of Ring Structure Onset in Filamentous Networks

2021 ◽  
Vol 83 (3) ◽  
Author(s):  
Maria-Veronica Ciocanel ◽  
Riley Juenemann ◽  
Adriana T. Dawes ◽  
Scott A. McKinley
Keyword(s):  
Time Series ◽  
Data Analysis ◽  
Actin Filaments ◽  
Time Series Data ◽  
Polymer Networks ◽  
Topological Data Analysis ◽  
Series Data ◽  
Topological Features ◽  
Topological Data

AbstractIn developmental biology as well as in other biological systems, emerging structure and organization can be captured using time-series data of protein locations. In analyzing this time-dependent data, it is a common challenge not only to determine whether topological features emerge, but also to identify the timing of their formation. For instance, in most cells, actin filaments interact with myosin motor proteins and organize into polymer networks and higher-order structures. Ring channels are examples of such structures that maintain constant diameters over time and play key roles in processes such as cell division, development, and wound healing. Given the limitations in studying interactions of actin with myosin in vivo, we generate time-series data of protein polymer interactions in cells using complex agent-based models. Since the data has a filamentous structure, we propose sampling along the actin filaments and analyzing the topological structure of the resulting point cloud at each time. Building on existing tools from persistent homology, we develop a topological data analysis (TDA) method that assesses effective ring generation in this dynamic data. This method connects topological features through time in a path that corresponds to emergence of organization in the data. In this work, we also propose methods for assessing whether the topological features of interest are significant and thus whether they contribute to the formation of an emerging hole (ring channel) in the simulated protein interactions. In particular, we use the MEDYAN simulation platform to show that this technique can distinguish between the actin cytoskeleton organization resulting from distinct motor protein binding parameters.

scholarly journals SPEG binds with desmin and its deficiency causes defects in triad and focal adhesion proteins

2020 ◽  
Author(s):  
Shiyu Luo ◽  
Qifei Li ◽  
Jasmine Lin ◽  
Quinn Murphy ◽  
Isabelle Marty ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Skeletal Muscles ◽  
Striated Muscle ◽  
Calcium Handling ◽  
Intermediate Filament Protein ◽  
Β1 Integrin ◽  
Adhesion Proteins ◽  
Focal Adhesion Proteins ◽  
Early Endosomes

Abstract SPEG, a member of the myosin light chain kinase family, is localized at the level of triad surrounding myofibrils in skeletal muscles. In humans, SPEG mutations are associated with centronuclear myopathy and cardiomyopathy. Using a striated muscle specific Speg-knockout (KO) mouse model, we have previously shown that SPEG is critical for triad maintenance and calcium handling. Here we further examined the molecular function of SPEG and characterized the effects of SPEG deficiency on triad and focal adhesion proteins. We used yeast two-hybrid assay, and identified desmin, an intermediate filament protein, to interact with SPEG and confirmed this interaction by co-immunoprecipitation. Using domain-mapping assay, we defined that Ig-like and fibronectin III domains of SPEG interact with rod domain of desmin. In skeletal muscles, SPEG depletion leads to desmin aggregates in vivo and a shift in desmin equilibrium from soluble to insoluble fraction. We also profiled the expression and localization of triadic proteins in Speg-KO mice using western blot and immunofluorescence. The amounts of RyR1 and triadin were markedly reduced, whereas DHPRα1, SERCA1, and triadin were abnormally accumulated in discrete areas of Speg-KO myofibers. In addition, Speg-KO muscles exhibited internalized vinculin and β1 integrin, both of which are critical components of the focal adhesion complex. Further, β1 integrin was abnormally accumulated in early endosomes of Speg-KO myofibers. These results demonstrate that SPEG-deficient skeletal muscles exhibit several pathological features similar to those seen in MTM1 deficiency. Defects of shared cellular pathways may underlie these structural and functional abnormalities in both types of diseases.

Controlled Nano–Bio Interface of Functional Nanoprobes for in Vivo Monitoring Enzyme Activity in Tumors

ACS Nano ◽  
2019 ◽  
Author(s):  
Ziyan Sun ◽  
Kai Cheng ◽  
Yuyu Yao ◽  
Fengyu Wu ◽  
Jonathan Fung ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
In Vivo Monitoring

Serum Cholinesterase Variants: Examination of Several Differential Inhibitors, Salts, and Buffers Used to Measure Enzyme Activity

1971 ◽  
Vol 17 (3) ◽  
pp. 183-191 ◽  
Author(s):  
Philip J Garry
Keyword(s):  
Enzyme Activity ◽  
Sodium Fluoride ◽  
Phosphate Buffer ◽  
Divalent Cations ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Serum Cholinesterase ◽  
Low Concentrations

Abstract Dibucaine, used as a differential inhibitor with acetyl-, propionyl-, and butyrylthiocholine as substrate, clearly identified the "usual" and "atypical" serum cholinesterases. Succinylcholine was also used successfully as a differential inhibitor with butyrylthiocholine as substrate. Sodium fluoride, used as a differential inhibitor, gave conflicting results, depending on whether Tris or phosphate buffer was used in the assay. Mono- and divalent cations (NaCl, KCl, MgCl2, CaCl2, and BaCl2) activated the "usual" and inhibited the "atypical" enzyme at low concentrations. The "usual" enzyme had the same activity in 0.05 mol of Tris or phosphate buffer per liter, while the heterozygous and "atypical" enzymes showed 12 and 42% inhibition, respectively, when assayed in the phosphate buffer. Kinetic studies showed the phosphate acted as a competitive inhibitor of "atypical" enzyme. Km values, determined for "usual" and "atypical" enzymes, were 0.057 and 0.226 mmol/liter, respectively, with butyrylthiocholine as substrate.

Nutrient uptake and enzyme activity of the preattachment goat embryo developed in vivo

1994 ◽  
Vol 41 (1) ◽  
pp. 204 ◽  
Author(s):  
D.K. Gardner ◽  
M. Lane ◽  
P.A. Batt
Keyword(s):  
Enzyme Activity ◽  
Nutrient Uptake
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