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PubMed1991

Purification and characterization of a bovine pregnancy-associated glycoprotein.

A 67000 Mr bovine pregnancy-associated glycoprotein (bPAG) has been isolated from fetal cotyledons and purified to homogeneity by HPLC. The purification was monitored by a double immunodiffusion test and by RIA in conjunction with an antiserum raised against a crude fraction of placenta-specific antigens. The molecular weight of bPAG was estimated to be 67000 by SDS-PAGE. The isoelectric points (pI) of the four isoforms, determined by high-resolution analytical electrofocusing in polyacrylamide gel, were 4.4, 4.6, 5.2, and 5.4. The carbohydrate content of the bPAG consisted of approximately 10.02 +/- 1.09% neutral sugar and variant amounts of sialic acid (from 0.29 +/- 0.06% in the most basic isoform to 2.1 +/- 0.31% in the most acidic isoform). A specific antiserum was raised against the purified bPAG. A specific RIA showed that the bPAG was antigenically unrelated to BSA, alphafetoprotein (AFP), and human schwangerschafts-spezifischen (pregnancy-specific) beta 1 glycoprotein (SP1). According to some characteristics (e.g. the molecular weight), the purified bPAG may correspond to a form of the pregnancy-specific protein B previously described by Sasser and colleagues (Biol Reprod 1986; 35:936-942).

Amino Acid Sequence↗
PubMed1979

Purification and characterization of an extracellular beta-n-acetylhexosaminidase from Paecilomyces persicinus.

Both beta-N-acetylglucosaminidase nad beta-N-acetylgalactosaminidase activities were detected in the culture fluids of Paecilomyces persicinus P-10 after growth in a soybean meal-corn meal medium. The active material was purified by means of protamine sulfate fractionation and ultrafiltration, followed by ion exchange and gel chromatography. The ratio of the two activities remained constant throughout the purification, and the final product was shown to migrate as a single band by using gel isoelectric focusing, disc electrophoresis, and detergent gel electrophoresis. Temperature, pH, inhibition, and kinetic studies were performed to characterize both activities. The molecular weight of the enzyme was estimated to be about 100,000 by high-resolution gel chromatography. Based on the data obtained, it is suggested that both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities reside in the same protein.

Acetylglucosaminidase↗
PubMed1991

Purification of the gamma-aminobutyric acidA/benzodiazepine receptor complex by immunoaffinity chromatography.

The bovine gamma-aminobutyric acidA/benzodiazepine receptor complex has been purified by a novel immunoaffinity chromatography method on immobilized monoclonal antibody 62-3G1. Immunopurification of the complex was achieved in a single step with an improved yield over affinity chromatography on the benzodiazepine Ro 7-1986/1. High-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the immunoaffinity-purified receptor revealed three major peptide bands of 51,000, 55,000, and 57,000 Mr which were also present in the Ro 7-1986/1 affinity-purified receptor. Peptide mapping, immunoblotting with subunit specific antibodies, and photoaffinity labeling with [3H]flunitrazepam and [3H]muscimol have been used for the identification of receptor subunits, including several which comigrated in a single band in SDS-PAGE.

Affinity Labels↗
PubMed1976

Thiol-protein disulphide oxidoreductases. Differences between protein disulphide-isomerase and glutathione-insulin transhydrogenase activities in ox liver.

1. Protein disulphide-isomerase and glutathione-insulin transhydrogenase activities were assayed in parallel through a conventional purification of protein disulphide-isomerase from ox liver. 2. Throughout a series of purification steps (differential centrifugation, acetone extraction, (NH4)2SO4 precipitation and ion-exchange chromatography), the two activities appeared in the same fractions but were purified to different extents. 3. The final sample was 143-fold purified in protein disulphide-isomerase but only 10-fold purified in glutathione-insulin transhydrogenase; nevertheless the two activities in this preparation were not resolved by high-resolution isoelectric focusing and both showed pI4.65. 4. In a partially purified preparation containing both activities, glutathione-insulin transhydrogenase was far more sensitive to heat denaturation than was protein disulphide-isomerase; conversely protein disulphide-isomerase was more sensitive to inactivation by deoxycholate. 5. The data are inconsistent with a single enzyme being responsible for all the protein disulphide-isomerase and glutathione-insulin transhydrogenase activity of ox liver. It is suggested that several similiar thiol-protein disulphide oxidoreductases of overlapping specificities may better account for the data.

Animals↗
PubMed1991

Metal-affinity separations: a new dimension in protein processing.

Rapid growth in the preparative and high-resolution analytical applications of metal-affinity chromatography demonstrate the appeal of metal recognition as a basis for protein separations. Stable, inexpensive chelated metals effectively mimic biospecific interactions, providing selective ligands for protein binding. This article reviews recent progress in understanding the mechanisms of metal-protein recognition that underlie metal-affinity separations. Also discussed are schemes for integrating metal-affinity purifications into the expression and bioprocessing of recombinant proteins. Promising future developments include new metal-affinity processes for analytical and preparative-scale separations and a range of techniques for enhancing the selectivity of metal-affinity separations.

Animals↗
PubMed1977

Scope and mechanism of carbohydrase action: stereospecific hydration of D-glucal catalyzed by alpha- and beta-glucosidase.

A unique demonstration is presented of the capacity of glycosidases to create anomeric configuration de novo. Purifed Candida tropicalis alpha-glucosidase and sweet almond beta-glucosidase have been found to attack the same substrate, D-glucal, and to convert this unusual glycosyl substrate (which lacks alpha or beta anomeric configuration) to 2-deoxy-alpha-(or beta-) D-glucose, respectively. The stereospecificity of the hydration reaction catalyzed by each enzyme in D2O was revealed by the use of high-resolution (270 MHz) 1H magnetic resonance spectroscopy. The alpha-glucosidase caused a specific axial protonation (deuteration) of D-glucal at C-2, and formation of 2-deoxy-alpha-D-[2(a)-2H]glucose. The beta-glucosidase catalyzed an oppositely directed axial protonation at C-2 and formation of 2-deoxy-beta-D-[2(e)-2H]glucose. These results are not accounted for by the generally accepted mechanisms of carbohydrase action derived from studies with glycosidically linked substrates alone. D-Glucal apparently binds to the enzymes with essentially the same overall orientation as the D-glucosyl moiety of glycosidically linked substrates (with the double bond of D-glucal lying essentially in the plane of the similarly bound D-glucosyl group). Thus, the alpha-glucosidase evidently protonates D-glucal from above the double bond and alpha-D-glucosidic substrates from below the glycosidic oxygen; beta-glucosidase apparently protonates D-glucal from below the double bond and beta-D-glucosides from above the glycosidic oxygen. A detailed mechanism is proposed for the hydration of D-glucal by each enzyme, involving an incipient glycosyl carbonium ion and assuming the presence at the active site of two carboxyl groups arranged to account for catalysis of glycosylations from glycosidically linked substrates. That D-glucal serves as a glycosyl substrate for these enzymes strongly supports the concept that glycosidases and glycosyltransferases are catalysts of glycosylation (i.e., glycosylases), since this concept does not make the usual assumption that carbohydrases are restricted to acting on substrates having a glycosidic bond and either alph- or beta-anomeric configuration.

Candida↗
PubMed1992

Purification and characterization of an enterotoxin from Bacteroides fragilis.

An enterotoxin produced by Bacteroides fragilis was purified to homogeneity and characterized as to its biological activity and basic molecular properties. Toxin preparations were prepared by growing B. fragilis VPI 13784 in brain heart infusion broth to early stationary phase, immediately precipitating the culture supernatant fluid with 70% ammonium sulfate, and stabilizing the precipitate with the protease inhibitor TPCK (tolylsulfonyl phenylalanyl chloromethyl ketone). The toxin was sequentially purified by anion-exchange chromatography on Q-Sepharose, hydrophobic interaction chromatography on phenyl-agarose, and high-resolution ion-exchange chromatography on Mono Q. The toxin appeared homogeneous as judged by polyacrylamide gel electrophoresis. The estimated molecular weight of the highly purified toxin as determined by gel filtration chromatography on Superose-12 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 19,000. It has an isoelectric point of approximately 4.5 and is stable at pHs 5 to 10. The purified toxin is stable at -20 and 4 degrees C and upon freeze-drying, but it is unstable at temperatures above 55 degrees C. It is sensitive to proteinase K and Streptomyces protease but is resistant to trypsin and chymotrypsin. The activity of the purified toxin is neutralized by antiserum to a toxigenic strain of B. fragilis but not by antiserum to nontoxigenic strains. N-terminal amino acid analysis reveal an unambiguous sequence of Ala-Val-Pro-Ser-Glu-Pro-Lys-Thr-Val-Tyr-Val-Ile-Xxx-Leu-Arg-Glu-Asn-Gly- Ser-Thr . The highly purified toxin induced a strong fluid accumulation response in the lamb ileal-loop assay as well as a cytotoxic response (cell rounding) on HT-29 colon carcinoma cells. Thus, the purified toxin can cause both enterotoxic and cytotoxic activities.

Animals↗
PubMed1991

Mechanisms of mutagenesis by exocyclic DNA adducts. Construction and in vitro template characteristics of an oligonucleotide bearing a single site-specific ethenocytosine.

By using a gene-targeted random DNA adduction approach, we have recently shown that chloroacetaldehyde, a metabolite of vinyl chloride, induces mutations predominantly at cytosines under conditions in which both ethenoadenine (epsilon A) and ethenocytosine (epsilon C) are formed. Although the observed mutational specificity of epsilon C suggested that it was a noninstructional lesion, the high efficiency of mutagenesis and an apparent lack of SOS dependence were reminiscent of mispairing lesions. To obtain more direct evidence showing that epsilon C has properties of a noninstructional mutagenic lesion, we have examined the in vitro template properties of a single epsilon C residue at a unique position in a synthetic oligonucleotide. The oligonucleotide was constructed by use of the following steps: (a) in vitro treatment of the pentameric oligodeoxyribonucleotide TTCTT with chloroacetaldehyde to convert the central cytosine to ethenocytosine; (b) purification and characterization of TT epsilon CTT; and (c) ligation of purified TT epsilon CTT to two decamers to create a 25 nt long oligodeoxyribonucleotide with a centrally located epsilon C residue. The template characteristics of epsilon C were examined by the annealing of end-labeled primers to the purified epsilon C-containing oligonucleotide and primer elongation by Escherichia coli DNA polymerase I in the presence of one or more nucleotide precursors. The elongation products were analyzed by high-resolution gel electrophoresis followed by autoradiography and quantitated by computing densitometry.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylating Agents↗
PubMed1992

Secretion of T cell receptor fragments from recombinant Escherichia coli cells.

This study describes the secretion and purification of T cell receptor (TCR) V alpha, V beta domains and single chain V alpha-V beta fragments (scTCRs) from recombinant Escherichia coli cells. The TCR V alpha and V beta genes are derived from a T cell hybridoma that is associated with disease pathogenesis in murine experimental allergic encephalomyelitis (EAE). Circular dichroism (c.d.) analyses of the single domains and the scTCR indicate that they are folded into beta-pleated sheet structures similar to those of immunoglobulin variable domains. The secreted TCR fragments can be purified in milligram quantities, and could therefore be used in high-resolution structural studies, in immunization to generate anti-clonotypic antibodies or in vaccination.

Animals↗
PubMed1992

Identification and purification of calcium-independent phospholipase A2 from bovine brain cytosol.

Substantial amounts of phospholipase A2 activity were detected in bovine brain cytosol. The major phospholipase A2 activity was present in the precipitate at 40% saturation with solid ammonium sulfate. After the desaltate of the precipitate was loaded onto an Ultrogel AcA 54 gel filtration column, almost all the activity eluted in the void volume when chromatographed without 1 M KCl. However, when buffer with 1 M KCl was used as the eluent, two active peaks were obtained. One peak (peak I) eluted in the void volume, and the other (peak II) eluted with an apparent molecular mass of 39 kDa as compared with standards. The former was active with diacylglycero-3-phosphoethanolamine, whereas the latter was active with both diacylglycero-3-phosphoethanolamine and 1-alk-1'-enyl-2-acylglycero-3-phosphoethanolamine (plasmenylethanolamine). The apparent molecular mass of peak I was estimated to be 110 kDa as compared with standards on an Ultrogel AcA 34 gel filtration column. Both peaks were purified further with a hydrophobic chromatography column (AffiGel 10 coupled with plasmenylethanolamine) and then by high-resolution liquid chromatography on an MA7Q column. The phospholipase A2 obtained from peak II migrated as one main band with a 40-kDa molecular mass and two minor bands with 14- and 25-kDa molecular masses. Phospholipase A2 obtained from peak I eluted as a single peak on high-resolution liquid chromatography but contained two bands with apparent molecular masses of 100 and 110 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Wild-type and mutant bacteriorhodopsins D85N, D96N, and R82Q: purification to homogeneity, pH dependence of pumping, and electron diffraction.

Bacterioopsin, expressed in Escherichia coli as a fusion protein with 13 heterologous residues at the amino terminus, has been purified in the presence of detergents and retinylated to give bacteriorhodopsin. Further purification yielded pure bacteriorhodopsin, which had an absorbance ratio (A280/A lambda max) of 1.5 in the dark-adapted state in a single-detergent environment. This protein has a folding rate, absorbance spectrum, and light-induced proton pumping activity identical with those of bacteriorhodopsin purified from Halobacterium halobium. Protein expressed from the mutants D85N, D96N, and R82Q and purified similarly yielded pure protein with absorbance ratios of 1.5. Proton pumping rates of bacteriorhodopsins with the wild-type sequence and variants D85N, D96N, and R82Q were determined in phospholipid vesicles as a function of pH. D85N was inactive at all pH values, whereas D96N was inactive from pH 7.0 to pH 8.0, where wild type is most active, but had some activity at low pH. R82Q showed diminished proton pumping with the same pH dependence as for wild type. Bacteriorhodopsin purified from E. coli crystallized in two types of two-dimensional crystal lattices suitable for low-dose electron diffraction, which permit detailed analysis of structural differences in site-directed variants. One lattice was trigonal, as in purple membrane, and showed a high-resolution electron diffraction pattern from glucose-sustained patches. The other lattice was previously uncharacterized with unit cell dimensions a = 127 A, b = 67 A, and symmetry of the orthorhombic plane group pgg.

Bacteriorhodopsins↗
PubMed1992

Metal ion specificity at the catalytic site of yeast enolase.

A new, more gentle enzyme purification for yeast enolase was developed. A series of kinetic experiments was performed with yeast enolase where the concentration of Mg(II) is kept constant and at the Km' level; the addition of Mn(II), Zn(II), or Cu(II) gives a hyperbolic decrease in the enzyme activity. The final velocity of these mixed-metal systems is the same as the velocity obtained only with Mn(II), Zn(II), or Cu(II), respectively. The concentration of the second metal that gives half-maximal effect in the presence of Mg(II) is approximately the same as the apparent Km (Km') value measured for that cation alone. Direct binding of Mn(II) to apoenolase in the absence and presence of Mg(II) shows that Mn(II) and Mg(II) compete for the same metal site on enolase. In the presence of D-2-phosphoglycerate (PGA) and Mg(II), only a single cation site per monomer is occupied by Mn(II). Water proton relaxation rate (PRR) studies of enzyme-ligand complexes containing Mn(II) and Mn(II) in the presence of Mg(II) are consistent with Mn(II) binding at site I under both conditions. PRR titrations of ligands such as the substrate PGA or the inhibitors orthophosphate or fluoride to the enolase-Mn(II)-Mg(II) complex are similar to those obtained for the enolase-Mn(II) complex, also indicating that Mn(II) is at site I in the presence of Mg(II). High-resolution 1H and 31P NMR was used to determine the paramagnetic effect of enolase-bound Mn(II) on the relaxation rates of the nuclei of the competitive inhibitor phosphoglycolate. The distances between the bound Mn(II) and the nuclei were calculated.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1979

High-resolution flow-zonal centrifuge system.

A modified CF-32 Beckman flow centrifuge rotor has been developed that provides a long sedimentation path length with high gravitational force at the gradient sample interface. The modified rotor exhibits excellent separative capability and extraction efficiency when applied to purification of human influenza B and herpes simplex viruses.

Centrifugation, Zonal↗
PubMed1992

Safety aspects in the manufacturing of plasma-derived coagulation factor concentrates.

Plasma-derived coagulation factor concentrates, prepared using traditional manufacturing processes, have transmitted viral diseases, especially AIDS, hepatitis B and hepatitis C to patients. To date, more extensive selection of blood donors, improved screening procedures of each plasma donation for direct and indirect viral markers, and newly developed virucidal procedures, especially pasteurization and solvent-detergent, together with extraction technologies of plasma proteins based on high-resolution chromatographic separations, have diminished considerably the risks of transmitting these pathogenic agents. To ensure safety, each production process must be carefully validated, following a rigorous scientific approach to assess its ability to inactivate or eliminate viruses. In addition, Good Manufacturing Practices must avoid any variation from these validated viral inactivation processes and must eliminate risks of potential downstream contamination of purified plasma fractions following viral inactivation or elimination steps. Other side-effects associated with conventional low-purity preparations, such as acute haemolytic anemia due to contamination by isohaemagglutinins, or immunosuppression possibly due to an overload in fibrinogen and immunoglobulins, have not been reported following infusion of highly purified coagulation factor concentrates. Present state-of-the-art virus inactivation and protein-purification technologies have significantly improved the safety of plasma coagulation factor concentrates.

Acquired Immunodeficiency Syndrome↗
PubMed2026

Comparative performance of portable DNA extraction protocols and bioinformatics workflows for rapid detection of gram-negative bacteria and antimicrobial resistance using Oxford Nanopore sequencing.

Oxford Nanopore Technology (ONT) enables rapid, portable pathogen identification and antimicrobial resistance (AMR) detection, but the reliability of downstream genomic analyses is highly dependent on DNA extraction quality, particularly in resource-limited settings. This study comparatively evaluated four portable bacterial DNA extraction protocols derived from three commercial kits to determine their impact on nanopore sequencing performance, bioinformatics workflow completion, and field deployability. Six gram-negative bacterial isolates (Escherichia coli, n = 4; Pseudomonas sp., n = 1; and Salmonella sp., n = 1) were processed using four extraction protocols: SwiftX DNA, SwiftX DNA with proteinase K (ProtK), SwiftX ParaBact, and NucleoSpin Microbial. Twenty-four resulting DNA extracts were sequenced on a single multiplexed MinION R10.4.1 flow cell. Sequencing data were analyzed using validated Galaxy-based generic and species-specific pipelines. Workflow completion was defined as successful progression through quality control, assembly, virulence, plasmid, and AMR detection modules. DNA purity varied substantially by extraction protocol and was strongly associated with successful workflow completion (Kruskal-Wallis, P = 0.0006). Accordingly, NucleoSpin Microbial achieved 100% workflow completion, and SwiftX ParaBact achieved 83%, while both SwiftX DNA-based protocols failed to complete full workflows. Importantly, key AMR genes required to classify isolates as multidrug-resistant were consistently detected using both NucleoSpin Microbial and SwiftX ParaBact extractions. However, NucleoSpin Microbial assemblies showed significantly higher contiguity and enabled a broader, more complete detection of virulence factors, pathogenicity islands, plasmid replicons, and accessory AMR genes, reflecting enhanced genomic resolution.IMPORTANCERapid whole-genome sequencing is increasingly used to detect antimicrobial resistance and guide public health responses, but its reliability depends strongly on how bacterial DNA is extracted. In this study, we have shown that DNA extraction method choice has a major impact on Oxford Nanopore sequencing performance across clinically relevant gram-negative bacteria. While silica column-based extraction maximized genomic completeness and analytical depth, paramagnetic bead-based reverse purification offered superior portability with sufficient resolution for frontline AMR surveillance. These findings highlight a practical trade-off between field deployability and high-resolution genomic characterization in low-resource settings.

DNA extraction↗
PubMed1991

Purification of secreted recombinant proteins from Escherichia coli.

Secretion systems engineered for the expression of heterologous protein in E. coli provide several advantages for subsequent isolation of purified product. Proteins released from the periplasmic space, which represent a small fraction (i.e., 4-10%) of total cell protein, can readily be separated from other cellular proteins by centrifugation of the remaining cellular debris or cross-flow ultrafiltration. The starting material derived from secretion systems is generally of higher purity than comparable material produced from strains expressing cytoplasmically for systems exhibiting similar expression levels. The available evidence suggests that recombinant proteins derived from the periplasm are generally, but not always (44-46), soluble in a nonaggregated form. Consequently, simple purification protocols can be effectively employed for producing homogeneous product with a high yield. The majority of the secreted recombinant proteins reviewed in this chapter were purified by simple one- or two-step chromatography procedures. High-resolution techniques such as reversed phase HPLC were found necessary only in cases where the secreted polypeptides were contaminated with proteolytic degradation variants, e.g., hirudin (51) and beta-endorphin (22). The fact that a high level of biological activity has been shown to be characteristic of purified recombinant proteins secreted into the periplasmic space suggests the presence of a native conformation stabilized by the expected disulfide linkages. Intramolecular disulfide bonds most probably form either as the polypeptide is translocated through the cytoplasmic membrane into the periplasm or within the periplasmic compartment, which has a higher oxidation potential than that found in the cytoplasm (57). Studies performed with hGH (31) and muIL-2 (35) provide excellent examples of differences observed in protein folding and disulfide bond formation between heterologous proteins expressed in the cytoplasmic and periplasmic compartments. Thus, hGH and muIL-2 extracted from the cytoplasm of E. coli have been characterized as high molecular weight disulfide-bonded oligomers. It is likely that oligomerization occurs as the polypeptides are released from the reducing environment of the cytoplasm. In contrast, secreted hGH and muIL-2 extracted from the periplasm of E. coli by osmotic shock displayed the properties of a property folded native protein with correct disulfide pairing. In the case of muIL-2 only a small residual fraction (approximately 15%) of the purified secreted protein exhibited incomplete oxidation of cysteine (35). Secretion of heterologous proteins into the periplasm prevents their exposure to the action of proteases located in the cytoplasm of E. coli (58). The smaller polypeptides such as somatostatin (59), IGF-1 (46), and hEGF (54) are known to be particularly susceptible to intracellular degradation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Preparative peptide purification by cation-exchange and reversed-phase perfusion chromatography.

Cation exchange was compared to reversed-phase chromatography for the preparative purification of a 28-residue peptide (vasoactive intestinal polypeptide) on the 100-mg scale. Optimized high-speed, high-resolution methods were developed for both chromatographic modes on POROS Perfusion Chromatography flow-through particle chromatography columns. While both methods appeared to provide similar purity, the cation exchange column had approximately ten times the loading capacity per unit column volume as the reversed-phase column. Five-minute methods for desalting the cation exchange-purified peptide and analysis of fractions were developed using small reversed-phase columns. The cation-exchange method was scaled up to process 95 mg of crude peptide in a 12-min run.

Amino Acid Sequence↗
PubMed1992

Co-purification of p34cdc2/p58cyclin A proline-directed protein kinase and the retinoblastoma tumor susceptibility gene product: interaction of an oncogenic serine/threonine protein kinase with a tumor-suppressor protein.

Proline-directed protein kinase (PDPK) is characterized as a cytoplasmic oncogenic serine/threonine kinase that is activated by growth factor-mediated mechanisms and is proposed to function in mammalian somatic cells as an S phase promoting factor. The present study was undertaken to assess the hypothesis that p34cdc2/p58cyclinA PDPK is a physiologically relevant form of the p34cdc2 protein kinase that phosphorylates and inactivates the product of the retinoblastoma/osteosarcoma tumor susceptibility gene (Rb protein). In the course of these studies it was determined (fortuitously) that the p34cdc2/p58cyclinA PDPK purified from the cytosol of FM3A mouse mammary carcinoma cells was 'contaminated' by several high molecular weight substrate proteins that essentially co-purified with the protein kinase, one of which was identified as the Rb protein itself (p105Rb). High-resolution fast protein liquid chromatography (FPLC) revealed that the Rb protein co-purified with a particular subset of the PDPK heterodimer, i.e. with a single species of the 58 kDa cyclinA doublet. The subset of PDPK associated with the Rb protein exhibited somewhat lower specific enzyme activity, as judged by in vitro kinase assays and comparative Western blotting. Immunoprecipitation studies confirmed that p105Rb is physically associated with the p34cdc2/p58cyclin A PDPK. Further studies confirmed that the underphosphorylated Rb protein (p105Rb) present in G1 lysates of synchronized human MG63 osteosarcoma cells could be readily phosphorylated by purified PDPK in vitro, resulting in the characteristic shift in the apparent molecular mass (SDS-PAGE) of the Rb protein that is reported to accompany the hyperphosphorylation and functional inactivation of this protein. Moreover, the induction of the cyclin A subunit of PDPK in these synchronized MG63 cells was found to be closely correlated with the cell cycle-dependent phosphorylation of the Rb protein. From these studies it is concluded that the growth factor-sensitive PDPK is a physiological Rb kinase, which may function to inactivate the Rb protein in vivo.

Animals↗