Use Of Two-dimensional Electrophoresis Research Articles

The effect of exogenous factors on the polyenzymatic activity of RuBisCO and ATP synthase of chloroplasts from pea leaves

Aim. To isolate and purify protein complexes – ATP synthase and RuBisCO – from pea leaf chloroplasts and study the effect of a microbiological fertilizer “Extracon” and sulfonamide inhibitors acetazolamide and ethoxyzolamide on the enzymatic activity of these proteins.Materials and methods. Chloroplasts were isolated from the leaves of two-week-old pea sprouts, protein complexes of purified thylakoid membranes were solubilized with digitonin (10 mg of digitonin per 1 mg of protein), the protein concentration was determined according to Lowry. Native electrophoresis with displacement of the charge of the soluble protein fraction from the chloroplast stroma, as well as membrane proteins, was carried out in the modified system of Anderson et al., Kolisnichenko et al. A modified Lemmley system was applied to the protein electrophoresis in the polyacrylamide gel in the presence of sodium dodecyl sulfate. The methods of Alain and Hintsik, as well as Gomorrah were used to determine the ATPase activity in the polyacrylamide gel. Visualization of the carbonic anhydrase activity in the polyacrylamide gel was performed by the method of Edwards and Petton. Results and discussion. Using physicochemical methods of potentiometry, spectrophotometry the ATPase, carbonic anhydrase and esterase activities of the enzymes were studied. The results obtained indicate that specific carbonic anhydrase inhibitors (acetazolamide and ethoxyzolamide) also block the esterase and ATPase activity of the enzyme complexes. “Extracon” (a multifunctional microbiological preparation) almost 1.5 times increases the activity of the enzymes, showing a complex activating effect of the fertilizer on both light and dark reactions of photosynthesis.Conclusions. The method of identification and isolation of RuBisCO and ATP synthase on the basis of two-dimensional electrophoresis and electrophoretic elution has been proposed. It allows determining the presence of certain enzyme activity of complexes at first in SDS plates (express analysis) and further to study the effect of various factors of endogenous and exogenous origin on the enzymatic properties of electrophoretically pure enzymes. The use of two-dimensional electrophoresis as a tool for assessing the impact of various factors of endogenous and exogenous origin on the plant cell and the plant as a whole through constant monitoring of the work and activity of enzyme systems of the plant cell is promising.

Primary Impacts of the Fungal Toxin Sporidesmin on HepG2 Cells: Altered Cell Adhesion without Oxidative Stress or Cell Death.

The fungal metabolite sporidesmin is responsible for severe necrotizing inflammation of biliary tract and liver of livestock grazing on pasture containing spores of Pithomyces chartarum that synthesizes the toxin. The toxin is secreted into bile causing the erosion of the biliary epithelium accompanied by inflammation and damage to surrounding tissues. Toxicity has been suggested to be due to cycles of reduction and oxidation of sporidesmin leading to oxidative damage from the formation of reactive oxygen species. The current work is the first test of the oxidative stress hypothesis using cultured cells. Oxidative stress could not be detected in HepG2 cells incubated with sporidesmin using a dichlorodihydrofluorescein diacetate assay or by use of two-dimensional electrophoresis to search for oxidized peroxiredoxins. There was also no evidence for necrosis or apoptosis, although there was a loss of cell adhesion that was accompanied by the disruption of intracellular actin microfilaments that have known roles in cell adhesion. The results are consistent with a model in which altered contact between cells in situ leads to altered permeability and subsequent inflammation and necrosis, potentially from the leakage of toxic bile into surrounding tissues. There is now a need for the further characterization of the damage processes in vivo, including the investigation of altered permeability and mechanisms of cell death in the biliary tract and other affected organs.

Use of Two-Dimensional Electrophoresis to Explore Foodborne Bacterial Pathogen Responses to Gastrointestinal Stress.

Proteomics was applied here to study Listeria monocytogenes response to gastrointestinal stress. It separated extracted proteins by their isoelectric point (pI) in the first dimension followed by separation by molecular weight in the second dimension on a polyacrylamide gel. L. monocytogenes was grown in an appropriate culture medium after which it was transferred to a simulated cheese medium for 2h. Bacteria were exposed to gastric stress using artificial saliva and gastric fluid for 5min and 2h, respectively. After each step samples were taken for protein extraction and a two-dimensional electrophoresis approach. Proteins were separated on 18cm Immobiline DryStrip gels with a pH range of 4-7 and the protein pattern analyzed.

Protein profile of the seminal plasma of collared peccaries (Pecari tajacu Linnaeus, 1758)

This study was conducted to characterize the major proteins of the peccary seminal plasma, based on the semen samples collected from nine adult and reproductively sound animals. Our approach included the use of two-dimensional electrophoresis followed by Coomassie blue staining and analysis of polypeptide maps with PDQuest Software (Bio-Rad). Proteins were identified by tandem mass spectrometry (LC-MS/MS). We detected 179 protein spots per gel and 98 spots were identified by mass spectrometry, corresponding to 23 different proteins. The combined intensity of those spots accounted for 56.2±6% of the intensities of all spots and 60.9% of the intensities of spots presented in every protein map. Protein spots identified as clusterin represented 19.7±8.3% of the integrated optical densities of all spots detected in the seminal plasma maps. There was a negative association (r=-0.87; P<0.05) between the intensity of a clusterin spot and the percentage of sperm with functional membrane. Spermadhesin porcine seminal plasma protein 1 and bodhesin 2 comprised 5.4±1.9 and 8.8±3.9% of the total intensity of all spots respectively. Many proteins appeared in a polymorphic pattern, such as clusterin (27 spots), epididymal secretory glutathione peroxidase (ten spots), inter-α-trypsin inhibitor (12 spots), and IgG-binding protein (ten spots), among others. In conclusion, we presently describe the major seminal plasma proteome of the peccary, which exhibits a distinct high expression of clusterin isoforms. Knowledge of wild species reproductive biology is crucial for an understanding of their survival strategies and adaptation in a changing environment.

Comprehensive proteomic analysis of human dentin

Proteomic analysis of the human body is a significant recent scientific endeavour. In this study, we investigated the proteomic profile of human dentin using modern analytical and mass spectrometric techniques. Five healthy permanent human molars from five adults were cut, pulverized, denaturated with guanidine buffer, and demineralized with EDTA buffer. The extracted proteins were analysed by gel electrophoresis (SDS-PAGE and two-dimensional gel electrophoresis), digested with trypsin, and separated by liquid chromatography/high-resolution tandem mass spectrometry. We identified 289 proteins with high confidence, 90 of which had not been previously detected in human dentin. Nine (currently hypothetical) proteins were identified for the first time in an actual human sample. The proteins have a variety of functions, including calcium-ion binding, formation of the extracellular matrix, formation of the cytoskeleton, cytoskeletal protein binding, immune response, and transport. In conclusion, this is the first use of two-dimensional electrophoresis for investigating human dentin.

Proteomic analysis of Trichoderma atroviride reveals independent roles for transcription factors BLR-1 and BLR-2 in light and darkness.

The genus Trichoderma is one of the most widely used biological control agents of plant-pathogenic fungi. The main mechanism for survival and dispersal of Trichoderma is through the production of asexual spores (conidia). The transition from filamentous growth to conidiation can be triggered by light, nutrient deprivation, and mechanical damage of the mycelium. We conducted proteomic profiling analyses of Trichoderma atroviride after a blue light pulse. The use of two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) analysis allowed us to identify 72 proteins whose expression was affected by blue light. Functional category analysis showed that the various proteins are involved in metabolism, cell rescue, and protein synthesis. We determined the relationship between mRNA levels of selected genes 30 min after a light pulse and protein expression levels at different times after the pulse and found this correlation to be very weak. The correlation was highest when protein and mRNA levels were compared for the same time point. The transcription factors BLR-1 and BLR-2 are vital to the photoconidiation process; here we demonstrate that both BLR proteins are active in darkness and affect several elements at both the transcript and protein levels. Unexpectedly, in darkness, downregulation of proteins prevailed in the Δblr-1 mutant, while upregulation of proteins predominated in the Δblr-2 mutant. Our data demonstrate that the BLR proteins play roles individually and as a complex.

In the Eye of the Beholder: Does the Master See the SameSpots as the Novice?

Historically, the use of two-dimensional electrophoresis (2-DE) in quantitative proteomics has been hampered by significant technical variance. Over the past decade, a range of technological leaps have reduced the overall variance of 2-DE, thus turning the technology into a robust platform for quantitative intact proteomics. However, as the confounding gel-to-gel variation improves, the variance arising from the subsequent image analysis becomes more prominent. Limitations in image alignment and spot detection of previous generations of 2-DE analysis software have demanded considerable user-intervention and manual editing, resulting in introduction of a large degree of subjectivity and software-induced variance. We evaluated the performance of SameSpots, representing a new generation of 2-DE image analysis software, using both DIGE and traditional single-stain 2-DE approaches. Evaluations of the software-induced variance in relation to other sources of variance, as well as the subjectivity through comparison of analyses performed by an expert user and a novice lab-user, were performed. In terms of statistical power, the less-experienced user achieved the better results, but no discernible difference was detected in multivariate comparisons between the users. In conclusion, we found that SameSpots represents improvements both in reproducibility and objectivity in relation to previous generations of 2-DE analysis software.

Identification of Protein Clusters Predictive of Response to Chemotherapy in Breast Cancer Patients

An attempt for the identification of potential biomarkers predictive of response to chemotherapy (CHT) in breast cancer patients has been performed by the use of two-dimensional electrophoresis and mass spectrometry analysis. Since growth and progression of tumor cells depend also on stromal factors in the microenvironment, we choose to investigate the proteins secreted in Tumor Interstitial Fluid (TIF) and in Normal Interstitial Fluids (NIF). One-hundred and twenty-two proteins have been analyzed and a comparison was also made between the proteomic profile of responders versus nonresponders to CHT. At baseline, proteins isolated in TIF and NIF of all the 28 patients show significant differences in expression. Two clusters of proteins, differentially expressed in TIF with respect to NIF were found. Most significant is the decreased expression in TIF of CRYAB. In the protein metabolism group, also FIBB was found decreased. Some proteins involved in energy pathways were overexpressed (PGAM-1, ALDO A, PGK1, G3Pcn), while some other were down-regulated (CAH2, G3Pdx, PRDX6, TPIS). The same trend was observed for signal transduction proteins, with 14-3-3-Z overexpressed, and ANXA2 and PEBP 1 down-regulated. Moreover, an analysis has been conducted comparing protein expression in interstitial fluids of responders and nonresponders, irrespective of TIF or NIF source. This analysis lead us to identify two clusters of proteins with a modified expression, which might be predictive of response to CHT. In responders, an increase in expression of LDHA, G3Pdx, PGK1sx (energy pathways), VIME (cell growth and maintenance) and 14-3-3-Z (signal transduction), coupled with a decreased expression of TPIS, CAH 2, G3Psx, PGK 1dx (energy pathways), TBB5 (cell growth and maintenance), LDHB and FIBB (protein metabolism), was found. We observed that CHT modifies the expression of these cluster proteins since, after treatment, their expression in TIF of responder is generally decreased. Patients not responding to CHT show an unchanged expression pattern in TIF, with the exception of protein 14-3-3-Z, which is overexpressed, and a decreased expression in NIF of several cluster proteins. In conclusion, the identification of protein clusters associated with response to CHT might be important for predicting the efficacy of a specific antineoplastic drug and for the development of less empiric strategies in choosing the therapy to be prescribed to the single patient.

A simple and reliable protocol for mouse serum proteome profiling studies by use of two-dimensional electrophoresis and MALDI TOF/TOF mass spectrometry

BackgroundUnravelling the serum proteome is the subject of intensified research. In this regard, two-dimensional electrophoresis coupled with MALDI MS analysis is still one of the most commonly used method. Despite some improvements, there is the need for better protocols to enable comprehensive identification of serum proteins.Here we report a combination of two proteomic strategies, zoom in acidic and neutral part of 2-D gels and an application of two optimised matrix preparations for MALDI-MS analyses to simplify serum proteome mapping.ResultsMouse serum proteins were separated by 2-D electrophoresis at the pH ranges 3–10 and 4–7, respectively. Then in gel tryptic digests were analysed by MALDI-MS. Notably, sample-matrix preparations consisted of either a thin-layer α-ciano-4-hydroxycinnamic acid (CHCA) matrix deposition or a matrix-layer 2,5-dihydroxybenzoic acid (DHB). This enabled an identification of 90 proteins. The herein reported method enhanced identification of proteins by 32% when compared with previously published studies of mouse serum proteins, using the same approaches. Furthermore, experimental improvements of matrix preparations enabled automatic identification of mouse proteins, even when one of the two matrices failed.ConclusionWe report a simple and reliable protocol for serum proteome analysis that combines an optimized resolution of 2-D gels spots and improved sample-matrix preparations for MALDI-MS analysis. The protocol allowed automated data acquisition for both CHCA and DHB and simplified the MS data acquisition therefore avoiding time-consuming procedures. The simplicity and reliability of the developed protocol may be applied universally.

Column chromatographic prefractionation leads to the detection of 543 different gene products in human fetal brain

In a previous publication a large series of proteins were identified in fetal human brain by the use of two-dimensional electrophoresis (2-DE) with subsequent matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and MALDI-tandem time-of-flight (TOF/TOF) analysis. Further identification of many more different spots by traditional 2-DE without additional step such as narrow immobilized ph gradient (IPG) strips or prefractionation seems unlikely and we therefore decided to separate extracted brain proteins by ion-exchange chromatography using a TSK gel DEAE-5PW column followed by 2-DE of individual fractions and analysis by MALDI-TOF/TOF with LIFT technology in fetal brain of the early second trimester. About 1880 protein spots corresponding to 543 different gene products were identified. These proteins included housekeeping, signaling, cytoskeletal, metabolic, antioxidant, and neuron/synaptosomal specific proteins. Among these, 314 gene products (314/543, 57.8%), which have never been detected in traditional 2-DE of human fetal brain, were observed by this method. This updated map of fetal brain proteins may serve as data base and reference map for fetal brain proteins, and the methodology applied may be used as a valuable analytical tool for the basis of protein expressional studies in health and disease.

Persistence ofStreptococcus pyogenesin Stationary-Phase Cultures

In addition to causing fulminant disease, Streptococcus pyogenes may be asymptomatically carried between recurrent episodes of pharyngitis. To better understand streptococcal carriage, we characterized in vitro long-term stationary-phase survival (>4 weeks) of S. pyogenes. When grown in sugar-limited Todd-Hewitt broth, S. pyogenes cells remained culturable for more than 1 year. Both Todd-Hewitt supplemented with excess glucose and chemically defined medium allowed survival for less than 1 week. After 4 weeks of survival in sugar-limited Todd-Hewitt broth, at least 10(3) CFU per ml remained. When stained with fluorescent live-dead viability stain, there were a number of cells with intact membranes that were nonculturable. Under conditions that did not support persistence, these cells disappeared 2 weeks after loss of culturability. In persistent cultures, these may be cells that are dying during cell turnover. After more than 4 weeks in stationary phase, the culturable cells formed two alternative colony phenotypes: atypical large colonies and microcolonies. Protein expression in two independently isolated microcolony strains, from 14-week cultures, was examined by use of two-dimensional electrophoresis. The proteomes of these two strains exhibited extensive changes compared to the parental strain. While some of these changes were common to the two strains, many of the changes were unique to a single strain. Some of the common changes were in metabolic pathways, suggesting a possible alternate metabolism for the persisters. Overall, these data suggest that under certain in vitro conditions, S. pyogenes cells can persist for greater than 1 year as a dynamic population.

Recombinant bovine somatotropin misuse in cattle: Evaluation of Western blotting and 2D electrophoresis methods on biological samples for the demonstration of its administration

Somatotropins (growth hormone, GH) which are used in cattle for growth and lactating performances are difficult to reliably detect since no direct method exists. Until now, the detection of GH in bovine milk, plasma and tissue has been based on radioimmunoassays and enzyme-linked immunosorbent assay (ELISA) tests that are unable to distinguish between endogenous and recombinant hormones. Attempt to directly detect recombinant GH administration is an analytical challenge which has never been successfully reported. The aim of the present work was to develop an original method, based on the detection in serum of antibodies raised against growth hormones as a consequence of their administration to animals. A second approach was developed with the characterisation of recombinant bovine somatotropin (rbST) in milk through the use of two-dimensional electrophoresis coupled to in-gel tryptic digestion followed by tandem mass spectrometry coupled to liquid chromatography (LC-ESI-MS/MS) measurements of the produced peptides. Results obtained showed the relevance of the two approaches. The Western blot one, carried out in this work on real biological blood samples collected on treated animals, would fit as a rapid and rather sensitive screening method, which can further more be achieved more over a long period after the administration of recombinant hormone. The second method, which consisted in a proteomic approach, dealt with the development of a confirmatory technique. Results on spiked milk samples were very promising and showed the need of further developments in this way.

The use of two-dimensional electrophoresis in the characterization of the water-soluble protein fraction of commercial flat fish species

The water-soluble proteins of nine flat fish species of high commercial value, belonging to the Pleuronectidae, Scophtalmidae, and Soleidae families, were analyzed by two-dimensional (2D) electrophoresis, this being carried out by nondenaturing isoelectric focusing (IEF) in the 3.5–9.5 pH range and gradient SDS-PAGE in the 12–14% range. Most of the major proteins fell in the 3.5–6.9 pH range. From these, the most specific proteins were in the acidic fraction (pI<5.2; MW<16 kDa). Species-specific 2D protein patterns were observed in all the nine species, and more than 25 proteins could be individualized. The combination of nondenaturing IEF and gradient SDS-PAGE, both in the absence of urea, allowed detailed characterization of both the isoelectric points and molecular weights of the major water-soluble proteins and proved to be a valuable tool for the differential characterization of the flat fish species studied.

Plasticity and stressor specificity of osmotic and heat shock responses of Gillichthys mirabilis gill cells.

Short-term effects of osmotic and heat shock on proteins of Gillichthys mirabilis gill cells were analyzed. The protein synthesis rate (PSR) of gill cells was influenced by hyperosmotic shock (335-->635 mosmol/kgH2O) and heat shock (25-->37 degrees C), but not by hyposmotic shock (335-->190 mosmol/kgH2O). Between 4 and 6 h after hyperosmotic shock, gill cell protein synthesis was inhibited relative to controls in serum-free medium but increased threefold over control values in medium supplemented with 10% serum. Serum-dependent stimulation of PSR was also observed after heat shock. By use of two-dimensional electrophoresis, 21 proteins, induced after hyperosmotic shock, 14 after hyposmotic shock, and 16 after heat shock were found. The osmotic shock response of gill cells was highly stressor specific because only five or three proteins that were induced after hyperosmotic or hyposmotic shock, respectively, were identical to proteins induced in response to heat shock. Heat shock protein 70 isoforms were only induced after heat shock, but not in response to osmotic shock. In gill and kidney epithelium, the transcription factor c-Jun was modified within 30 min after transfer of whole fish from 1,086 mosmol/kgH2O to 5 or 2,172 mosmol/kgH2O, but osmotic shock in vitro had no effect on c-Jun in isolated gill cells. Ion-substitution experiments revealed that the increase of PSR after hyperosmotic shock in serum-supplemented medium significantly depended on an elevation of extracellular Na+ concentration. These data provide evidence for the plasticity and stressor specificity of osmotic and heat shock responses of fish gill cells.

Comparative analysis of polypeptides in the heat shock proteins synthesized in the myocardium of Wistar and August rats

The use of two-dimensional electrophoresis to study the polypeptide composition of heat shock proteins 70 synthesized in the myocardium of Wistar and August rats in response to thermal stress and repetitive immobilization stress revealed interstrain differences in the composition of stimulated polypeptides in these proteins. The differences are mainly due to differential expression of the heat shock protein gene(s) encoding polypeptides with pI 6.2–6.0 in the two strains. The drastically reduced levels of the synthesis and/or accumulation of some heat shock proteins 70 in the hearts of August rats are associated with their failure to develop cardioprotective phenomena in response to repetitive immobilization stress.

Alteration of the biochemical valves in the central metabolism of Escherichia coli.

Although E. coli central metabolism has been studied for several decades, many regulatory features are still unknown. To achieve the goal of rational manipulation of cellular metabolism, it is important to understand how E. coli responds to overexpressed enzymes. By studying the biochemical control of fluxes between PEP, pyruvate, and OAA, we have addressed some fundamental questions that may prove to be essential for applications in metabolic engineering. First, we found that simultaneous overexpression of Pck and Ppc, or Pps alone in the presence of glucose leads to phenotypes consistent with futile cycline. In contrast to our expectation, futile cycling per se does not affect the growth rate significantly. However, excessive futile cycling may cause competitive disadvantage in the natural environment. Overexpression of Pck caused growth inhibition but no futile cycling. Therefore, E. coli controls the expression of gluconeogenic enzymes not only to avoid excessive futile cycling, but also to prevent toxicity effects. In metabolic engineering, futile cycling may be used as a strategy to stimulate metabolism for either production of metabolites or digestion of toxic wastes. Second, we found that the expression levels of Pps and Pck in E. coli are not optimal for growth on pyruvate and succinate, respectively. Overexpression of these enzymes increases the growth rate on pyruvate and on succinate, respectively, indicating that the slow growth rates on these substrates are at least partially caused by the insufficient supply of PEP and its derivatives. Moreover, E. coli also has not optimized the Ppc level for optimal growth yield on glucose in uncontrolled batch cultures. These results demonstrate that the central metabolism is not optimized for growth under defined laboratory conditions. Thus, the possibility exists that adjustment of native enzyme levels in the central metabolism can improve bioreactor performance. Third, we found that overexpression of Pck affects the transcriptional levels of unrelated genes. This example indicates that physiological responses to enzyme (over)expression should be interpreted cautiously, as changing the expression level of a specific enzyme may affect many unlinked genes. Similar results have also been obtained by use of two-dimensional electrophoresis of proteins from E. coli. Although more questions remain to be answered, fast progress in the area of metabolic engineering can be expected in the near future.

Genetic variation detected by quantitative analysis of end-labeled genomic DNA fragments.

The continuing efforts to evaluate specific human populations for altered germinal mutation rates would profit from more efficient and more specific approaches than those of the past. To this end, we have explored the potential usefulness of two-dimensional electrophoresis of DNA fragments obtained from restriction-enzyme-digested genomic DNA. This permits the analysis, on a single preparation, of approximately 2000 DNA fragments varying in size from 1.0 to 5.0 kb in the first dimension and from 0.3 to 2.0 kb in the second dimension. To enter into a genetic analysis, these fragments must exhibit positional and quantitative stability. With respect to the latter, if spots that are the product of two homologous DNA fragments are to be distinguished with the requisite accuracy from spots that are the product of only one fragment, the coefficient of variation of spot intensity should be approximately < or = 0.12. At present, 482 of the spots in our preparations meet these standards. In an examination of preparations based on three Japanese mother/father/child trios, 43 of these 482 spots were found to exhibit variation that segregated within families according to Mendelian principles. We have established the feasibility of cloning a variant fragment from such gels and establishing its nucleotide sequence. This technology should be highly efficient in monitoring for mutations resulting in loss/gain/rearrangement events in DNA fragments distributed throughout the genome.

Two-dimensional electrophoresis as a complementary method of isolating peptide fragments of cleaved proteins for internal sequencing

To determine the primary structure of proteins, usually proteolytic enzyme digests are separated by reversed-phase high-performance liquid chromatography (HPLC) and each fraction is collected and sequenced. The results obtained by different cleavages are combined to reveal the entire sequence. However, there are many N-terminal-blocked proteins and/or intact proteins or their particular fragments that are not eluted from HPLC columns. Internal fragments of such proteins were successfully isolated by the use of two-dimensional electrophoresis, after digestion. Electroblotted spots were easily sequenced to identify those difficult fragments which could not be obtained using HPLC.

Molecular evolution and phylogeny of theDrosophila virilis species group as inferred by two-dimensional electrophoresis

Systematic relationship among the 12 species of the Drosophila virilis species group, and Drosophila robusta, were investigated by the use of two-dimensional electrophoresis (2-DE). A total of 389 protein characters (about 200 loci) were scored and analyzed both phylogenetically and phenetically. The resulting phylogeny was found to be largely concordant with the current views of evolution among these species based on other independent morphological, chromosomal, electrophoretic, and immunological data sets, although some notable differences were observed. The 2-DE data also appeared to be useful for constructing a molecular clock to date the absolute times of divergence among the species. It appears from this analysis that the evolution of the major clades within the species group occurred about 20 million years ago. Previous suggestions that the rate of molecular evolution was different between the virilis and montana phylads was not confirmed. The technique of 2-DE seems to be an excellent tool for reconstructing phylogenies and should be particularly valuable for examining relatively closely related species.

Purification, characterization, and biosynthesis of bovine enamelins.

Enamelins were extracted from developing bovine enamel with 0.5 M EDTA, 4 M guanidine HCl, and purified by DEAE-Sephacel, Sephacryl S-200, and high-performance gel filtration chromatography. Four distinct enamelins having molecular weights of 70, 45, 30, and 28 K daltons were isolated. Their amino acid compositions were found to be rich in Pro, Glu, Gly, and Asp. Low molecular weight enamelins (45, 30, and 28 K) were more abundant in Pro, Gly, and Phe. Two-dimensional electrophoretic pattern of enamelins revealed several spots that immunoreacted to monoclonal anti-enamelin antibody raised in mice. Enamelins were found to be comprised of heterogeneous proteins as well as amelogenins. Biosynthesis of enamelins was investigated by incubating the bovine ameloblast cell layer, and several radioactive enamelins were identified by the use of two-dimensional electrophoresis. The data in this study suggest that enamelins were synthesized by the ameloblasts.