Silver-stained fibrin zymography: separation of proteases and activity detection using a single substrate-containing gel

A new zymogram method, silver-stained fibrin zymography, for separation of protease bands and activity detection using a single substrate gel, was developed. The method takes advantage of the nanoscale sensitivity of both zymography and silver staining. After SDS-PAGE in a gel containing fibrin, the gel was incubated in enzyme reaction buffer and the zymogram was silver-stained. Bands with protease activity were stained with silver in clear areas where the protein substrate had been degraded. The molecular sizes of proteases were accurately determined. Furthermore, proteases of high molecular weight were clearly and sharply resolved.     

Relationship between enzymatic activity and oligomerization state of tyrosine hydroxylase

The native form of tyrosine hydroxylase (TH) is a homotetramer which consists of four identical subunits each with an MW of approximately 60 kD. The relationships between the catalytic activity of TH and oligomerization of the enzyme have not yet been characterized. We have investigated, by deletion and/or substitution mutagenesis, the involvement of the leucine zipper (LZ) motifs in the oligomer formation of TH and its relation to catalytic activity. Our results demonstrate that deletion of the carboxyl-terminal LZ (LZ-C) abolishes tetramer formation. Interruption of the other two LZ motifs (LZ-A and LZ-B), located in a central region of the catalytic domain by substitution of Leu to Pro at residues 294 and 301 or 386 and 393 has no effect on the tetramer formation of TH. However, the interruption of LZ-A and LZ-B abolishes TH enzymatic activity. The substitution of Leu residues 188 and 190 with Pro at the regulatory domain of TH reduces enzymatic TH activity without affecting tetramer formation. Thus, LZ-C is required for tetramer formation, while LZ-A and LZ-B seem to be involved in the catalytic activity without affecting the tetramer formation of TH.     

Isoelectric focusing of multiple forms of DNase in thin layers of polyacrylamide gel and detection of enzymatic activity with a zymogram method following separation

Multiple forms of bovine pancreatic DNase (DNases A, B, C, and D) are separated by isoelectric focusing in thin layers of polyacrylamide gel with a carrier ampholyte in the pH range 4–6. The isoelectric points of DNases A, B, C, and D are 5.22, 4.96, 5.06, and 4.78, respectively. A zymogram method for detecting DNase activity as bands in the gel following isoelectric focusing is described. The method detects microgram amounts of DNase and has only one step. It can be used with the parified cazyme as well as with crude extracts of tissues containing DNase. By this method, two major components of DNase in ovine pancreas and at least three in malted barley as well as two previously unideatified forms of DNase in bovine pancreas with isoelectric points of 5.12 and 5.48 (DNases E and F) are observed.     

Relationship between bisphosphonate concentration and osteoclast activity and viability

Summary Difluoromethylidene bisphosphonate (F₂MBP) is one of the many bisphosphonates known to inhibit bone resorption in vitro and in vivo. We have developed an analytical method, employing anion exchange and postcolumn indirect fluorescence detection, by which F₂MBP can be quantified in bone samples. The objective of this study was to relate the concentration of F₂MBP in embryonic bones treated in organ culture to the physiological effects of the compound, such as bone resorption (i.e., the amount of ⁴⁵Ca released into the medium from prelabeled bones) and viability of the osteoclast population (i.e., the incidence of abnormal osteoclasts). Osteoclasts in bones treated with F₂MBP exhibited morphological features of apoptosis, such as nuclear fragmentation. Both the number and percentage of these abnormal cells increased with dose of F₂MBP and duration of incubation. The decrease in normal osteoclasts was correlated with the decreased amount of ⁴⁵Ca released into the medium. Bones treated with F₂MBP for only the first 5 min of the 48-h incubation period had similar numbers of abnormal osteoclasts and amounts of ⁴⁵Ca released, as had bones incubated with F₂MBP continuously for 48 h. The uptake of F₂MBP into the bone was rapid. Bones treated with F₂MBP for 6 h were similar to bones treated with F₂MBP for the entire 48-h incubation period, both in F₂MBP concentration and the ⁴⁵Ca release ratios. These relationships between concentrations of F₂MBP within bone and osteoclast activity and viability implicate apoptosis in the mechanism by which this bisphosphonate inhibits bone resorption.     

Detection of elastase activity with a zymogram method after isoelectric focusing in polyacrylamide gel

A zymogram method for detecting elastase activity following isoelectric focusing in polyacrylamide gel is described. After enzyme activity has been visualized, the gel itself is available for protein staining and for analysis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in second dimension. The zymogram method is suitable for detecting microgram amounts of elastase and has one step only. It can be used with the purified enzyme as well as with crude extracts of tissue containing elastases showing activity toward succinyl-(Ala)₃-p-nitroanilide. By this method a major component of elastase in both porcine and rat pancreas was detected. In addition, two forms of elastase with isoelectric points of 8.2 and 8.8, respectively, were identified in rat leukocyte extracts.     

Three-dimensional cell and tissue patterning in a strained fibrin gel system

Techniques developed for the in vitro reproduction of three-dimensional (3D) biomimetic tissue will be valuable for investigating changes in cell function in tissues and for fabricating cell/matrix composites for applications in tissue engineering techniques. In this study, we show that the simple application of a continuous strain to a fibrin gel facilitates the development of fibril alignment and bundle-like structures in the fibrin gel in the direction of the applied strain. Myoblasts cultured in this gel also exhibited well-aligned cell patterning in a direction parallel to the direction of the strain. Interestingly, the direction of cell proliferation was identical to that of cell alignment. Finally, the oriented cells formed linear groups that were aligned parallel to the direction of the strain and replicated the native skeletal muscle cell patterning. In addition, vein endothelial cells formed a linear, aligned vessel-like structure in this system. Thus, the system enables the in vitro reproduction of 3D aligned cell sets replicating biological tissue patterns.     

Further development of a versatile system for preparative electrophoresis in acrylamide gel

Modifications to a preparative scale acrylamide gel electrophoresis apparatus enable better resolution to be obtained by reducing band distortion due to heating effects. A scaled-up (14-cm diameter) version of the apparatus is described, and also a system for automatic recycling of material only partially purified after one passage through the gel.     

Relationship between the oligomeric status of HIV-1 integrase on DNA and enzymatic activity

The 3'-processing of the extremities of viral DNA is the first of two reactions catalyzed by HIV-1 integrase (IN). High order IN multimers (tetramers) are required for complete integration, but it remains unclear which oligomer is responsible for the 3'-processing reaction. Moreover, IN tends to aggregate, and it is unknown whether the polymerization or aggregation of this enzyme on DNA is detrimental or beneficial for activity. We have developed a fluorescence assay based on anisotropy for monitoring release of the terminal dinucleotide product in real-time. Because the initial anisotropy value obtained after DNA binding and before catalysis depends on the fractional saturation of DNA sites and the size of IN.DNA complexes, this approach can be used to study the relationship between activity and binding/multimerization parameters in the same assay. By increasing the IN:DNA ratio, we found that the anisotropy increased but the 3'-processing activity displayed a characteristic bell-shaped behavior. The anisotropy values obtained in the first phase were predictive of subsequent activity and accounted for the number of complexes. Interestingly, activity peaked and then decreased in the second phase, whereas anisotropy continued to increase. Time-resolved fluorescence anisotropy studies showed that the most competent form for catalysis corresponds to a dimer bound to one viral DNA end, whereas higher order complexes such as aggregates predominate during the second phase when activity drops off. We conclude that a single IN dimer at each extremity of viral DNA molecules is required for 3'-processing, with a dimer of dimers responsible for the subsequent full integration.     

Relations between enzymatic and association reactions in the development of bovine fibrin clot structure

Development of fibrin clot structure was examined at pH 7.0, Γ/2 0.15, and 29 °C as a function of thrombin and fibrinogen concentrations. Parameters for the release of Apeptides, to give $\text{?}{}_{\mathrm{<mtext>A</mtext>}}$ were evaluated. Characteristics of time dependencies of development of turbidity, 90 ° light scattering, network, and compactible network were established. Mean mass/length ratios of fibrin in developing and mature networks were determined. Relationships between results combined with an inferred dependence of lateral interaction on release of B-peptides are used to disclose a model in which a protofibril network is formed first and the intrinsic length of this network (i.e., length exclusive of overlap or loose ends) determines network length, thus mean mass/length ratio, at maturity. Statements regarding initial protofibril network are: (i) A dominant group of $\text{?}{}_{\mathrm{<mtext>A</mtext>}}$-protofibrils appears first. With decreasing rate of production of $\text{?}{}_{\mathrm{<mtext>A</mtext>}}$ their average length increases and number decreases. (ii) Slower release of B-peptides produces $\text{?}{}_{\mathrm{<mtext>AB</mtext>}}$ whose fraction $\text{θ}{}_{\mathrm{<mtext>AB</mtext>}}\text{=}\text{?}{}_{\mathrm{<mtext>AB</mtext>}}\text{(?}{}_{\mathrm{A}}\text{+?}{}_{\mathrm{AB}}$) determines the occurrence of protofibril regions capable of contributing to a lateral interaction sufficiently stable for the formation of network. (iii) When dominant protofibrils attain a minimum combination of average length, number concentration, and frequency of occurrence of capable regions, an initial protofibril network is rapidly generated. (iv) Capable regions near protofibril ends are preferentially involved in initial network formation. (v) The initial network mesh size is large compared to average concomitant free protofibril length. (vi) With B-peptide release dependent on prior A-peptide release, protofibrils in the initial network have the highest capable region frequency, and this is maintained as lateral interaction progresses. Then, fibrin which is free at initial network formation and fibrin which is produced subsequently interact mainly to increase the mean mass/length ratio of initial network elements.     

Formation of fibrin gel in fibrinogen-thrombin system: static and dynamic light scattering study

The dynamics of thrombin-induced fibrin gel formation was investigated by means of static and dynamic light scattering. The decay time distribution function, obtained by the dynamic light scattering, clearly revealed a stepwise gelation process: the formation of fibrin and protofibril from fibrinogen followed by the lateral aggregation of protofibrils to form fibrin fibers and the formation of a three-dimensional network consisting of fibers. This conversion process was correlated with the angular dependence of the scattered light intensity (static light scattering). The correlation function of dynamic light scattering was analyzed in terms of sol-gel transition and gel structure. The correlation function showed a stretched exponential type behavior before the sol to gel transition point, and it showed a power law behavior at the gelation point.     

Development and validation of an improved diced electrophoresis gel assay cutter-plate system for enzymomics studies

Diced electrophoresis gel (DEG) assay is a methodology to identify enzymes with a specified activity in complex cell or tissue lysates by means of two-dimensional separation using isoelectric focusing and native PAGE, followed by dicing of the gel into small pieces that are assayed separately, and digestion and peptide fingerprinting to identify the protein(s) of interest in positive wells. The existing hand-made system has some disadvantages, and here we describe the development and validation of an improved cutter-plate system that enables simple, reliable and reproducible DEG assay in a 384-well plate-based format with signal readout using fluorometric or LC-MS-based reaction monitoring. To illustrate the usefulness of this system, we describe its application to profile esterase activities in ovarian adenocarcinoma SKOV3 cell lysate and mouse liver lysate that activate a fluorogenic substrate, fluorescein dibutyrate (FDBu), as well as esterase activities in mouse liver lysate that activate S-bromobenzylglutathione dicyclopentyl ester (BBGDC), a prodrug of anti-tumor agent S-bromobenzylglutathione. The activity spot patterns detected for FDBu and BBGDC were completely different, indicating that different metabolic systems are involved in hydrolysis of these substrates. The major detected spot in each case was identified. The developed system provides a highly reproducible general assay platform that should be useful for characterizing novel protein functions in complex bio-samples, as well as enzymomics studies.