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S R Planck

Publications and source records attributed to S R Planck.

49 records · Page 3Linked to original sources
PubMed1985

Heterogeneity of 125I-labeled epidermal growth factor.

Highly purified epidermal growth factor (EGF) was iodinated, and the labeled product with the same isoelectric point as underivatized EGF was isolated by isoelectric focusing. When the 125I-labeled EGF was analyzed by reverse-phase chromatography, the resulting profile of 125I activity was much broader than the profile obtained with underivatized EGF. Rechromatography of 125I-EGF fractions indicated that our highly-purified labelled EGF was indeed heterogeneous. Analysis of each HPLC column fraction demonstrated that degradation of EGF had not occurred. The column fractions containing 125I-EGF were pooled into five groups for analysis of cell binding characteristics. Scatchard plot analysis of the five 125I-EGF pools revealed markedly different binding behaviors. In contrast, they had equal potency in stimulating DNA synthesis, within the sensitivity of our assay. Specific activity measurements indicated that the five HPLC pools of 125I-EGF had varying numbers of 125I atoms per EGF molecule. The heterogeneity of the highly purified 125I-EGF and the binding characteristics of the 125I-EGF subfractions pose serious implications for all workers who use iodinated ligands for receptor binding studies.

Animals↗
PubMed1985

Amino acid sequence of the UP1 calf thymus helix-destabilizing protein and its homology to an analogous protein from mouse myeloma.

A complete amino acid sequence has been determined for the UP1 single-stranded DNA binding protein from calf thymus that was first described by G. Herrick and B. M. Alberts [(1976) J. Biol. Chem. 251, 2124-2132]. Peptides required to establish the UP1 sequence were isolated by reversed-phase HPLC of digests produced by endoproteinase Lys-C, trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and cyanogen bromide cleavage of UP1. The purified peptides were coupled to aminopolystyrene prior to solid-phase sequencing. UP1 contains 195 amino acids and has a molecular weight of 22,162. UP1 has a blocked NH2 terminus and contains a single NG,NG-dimethylarginine residue near its COOH terminus. Gas-phase sequencing of tryptic peptides derived from an analogous protein from mouse myeloma cells [Planck, S. R. & Wilson, S. H. (1980) J. Biol. Chem. 255, 11547-11556] revealed that this mouse helix-destabilizing protein shares a high degree of sequence homology with UP1. Of the 59 amino acids in the mouse protein that have so far been found to be homologous with UP1, 48 correspond exactly to sequences found in UP1. Most of the 11 differences that have been found between the sequences of these two proteins are conservative in nature, involving primarily the interchange of chemically similar amino acids. One 9-residue mouse sequence that is not obviously homologous to UP1 may be a result of the larger size of the mouse myeloma protein as compared to UP1. Since none of the UP1 or mouse myeloma helix-destabilizing protein sequence appears to be homologous to that of any previously sequenced protein, we presume that these two proteins represent a distinct class of single-stranded nucleic acid binding proteins that probably play a role in metabolism of single-stranded RNA or DNA in vivo.

Amino Acid Sequence↗
PubMed1984

Intracellular processing of epidermal growth factor. I. Acidification of 125I-epidermal growth factor in intracellular organelles.

We previously reported that 125I-labeled epidermal growth factor is processed intracellularly to acidic macromolecules in Rat-1 fibroblasts. The present study defines the precursor-product relationship and localization of the processing steps to subcellular organelles by the use of a single isoelectric species of 125I-epidermal growth factor and Percoll gradient fractionation. The native pI 4.55 125I-epidermal growth factor was rapidly processed to a pI 4.2 species on or near the cell surface and in organelles corresponding to clathrin-coated vesicles, Golgi, and endoplasmic reticulum. This species was then processed to a pI 4.35 species in similar organelles. The pI 4.2 and 4.35 species were converted to a pI 4.0 species in dense, lysosome-like organelles. This species was ultimately degraded and exocytosed from the cell as low molecular weight products.

Animals↗
PubMed1984

Intracellular processing of epidermal growth factor. II. Intracellular cleavage of the COOH-terminal region of 125I-epidermal growth factor.

Epidermal growth factor (EGF) undergoes a specific series of alterations during the course of its binding and internalization into cultured fibroblasts. The modified EGF species can be distinguished from each other and from native EGF by their isoelectric points. We employed peptide mapping techniques to determine the nature of these alterations. We found that 125I-EGF with a pI of 4.55 was converted to a pI 4.2 species by removal of 1 or 2 amino acid moieties from the COOH-terminal end of the protein. A pI 4.35 species was generated by a trypsin-like cut between amino acid residues 48 and 49, for a total of 5 amino acid moieties removed from the native EGF. The pI 4.0 species was formed by removal of at least the COOH-terminal arginine from the pI 4.35 species. Thus, upon binding and internalization, EGF was sequentially cleaved in the COOH-terminal region. Removal of the COOH-terminal polypeptide has been shown to dramatically reduce the affinity of EGF for its receptor, raising the possibility that intracellular dissociation of EGF from its receptor may be a direct result of the intracellular processing of EGF.

Animals↗
PubMed1983

Binding of epidermal growth factor in rat pancreatic acini.

Specific, saturable EGF receptors were demonstrated in isolated rat pancreatic acini. Binding of EGF to these receptors was one-half maximal at 20 min and maximal at 120 min. Scatchard analyses revealed a single order of binding sites with a Kd of 4.90 nM. Following binding, EGF was rapidly internalized and converted to two acidic species. EGF did not alter either basal amylase release or the rate of [3H]phenylalanine incorporation into TCA-precipitable protein. The finding of high affinity EGF receptors in pancreatic acinar cells supports the hypothesis that EGF participates in the long-term regulation of pancreatic exocrine function.

Animals↗
PubMed1983

p-Azidophenylglyoxal-epidermal growth factor: a photoactivatable affinity crosslinking reagent.

A photoaffinity derivative of highly purified 125I-labelled epidermal growth factor (125-I-EGF) has been synthesized. The heterobifunctional crosslinking reagent p-azidophenylglyoxal (PAPG) was bound to arginine residues in 125I-EGF. PAPG-125 I-EGF bound to EGF receptors on rat fibroblasts and human A431 epidermoid carcinoma cells in culture. An apparent decreased affinity of PAPG-125I-EGF for the EGF receptor is in accord with at least one arginine being at or near the EGF receptor binding site. The PAPG-125I-EGF:EGF receptor complexes on rat cells were internalized to the same extent as control EGF:receptor complexes. A431 cells treated with PAPG-125I-EGF were irradiated with ultraviolet light and the labelled proteins were analyzed by SDS-polyacrylamide gel electrophoresis. The 3 major labelled proteins had apparent molecular weights ranging from 75,000 to 200,000. Only the labelling of the 200,000-Mr protein was prevented by the addition of excess unlabelled EGF with the PAPG-125I-EGF. This molecular weight is in agreement with the reported size of the EGF receptor plus EGF. A protein with apparent molecular weight of 100,000 was labelled by 125I-EGF by an unknown mechanism which was dependent on the dose of UV light and blocked by the addition of excess unlabelled EGF.

Aldehydes↗
PubMed1982

Intracellular processing of 125I-epidermal growth factor in rat embryo fibroblasts.

The intracellular fate of endocytosed 125I-epidermal growth factor was examined in Rat-1 fibroblasts. Cells were pulse-labeled for 5 min in 125I-EGF and chased for 3 hr with an excess of unlabeled EGF. At various times after application of the cold chase, cells were harvested and processed for isopycnic gradient centrifugation on Percoll gradients. Within the period of the 125I-EGF pulse, about 50% of the 125I activity appeared in an organelle containing peak in the gradients. By 20 min after application of the cold chase, 125I activity in the organelle peak began to decrease, and the decrease continued over the next few hours. The 125I activity which exited from its organelle-associated location appeared to be present in the cytosol and was apparently not confined within organelles. Lysosomotropic amines inhibited the egress of 125I activity from the organelle compartment. The 125I activity from both organelle and nonorganelle compartments reacted as completely as authentic 125I-EGF with anti-EGF antibodies and was similar in size to authentic 125I-EGF. Little or no intracellular low molecular weight 125I-containing compounds were detected, although they accumulated in the culture medium. Analytical isoelectric focusing revealed that the organelle-bound form of endocytosed 125I-EGF was more acidic than authentic 125I-EGF and, upon exiting from the organelle compartment, was processed to an even more acidic form. It was the second macromolecular form of processed 125I-EGF that was ultimately degraded to low molecular weight compounds which were then externalized from the cells.

Animals↗
PubMed1980

Studies on the structure of mouse helix-destabilizing protein-1. DNA binding and controlled proteolysis with trypsin.

A mouse helix-destabilizing protein (HD protein-1) has been purified and characterized, and controlled tryptic digestion has been used to generate two large fragments of this protein and to study structural changes accompanying DNA binding. HD protein-1, a DNA-binding protein that has higher affinity for single-stranded DNA (ssDNA)-cellulose than for double-stranded DNA (dsDNA)-cellulose and is resistant to a dextran sulfate elution from ssDNA-cellulose, was purified from mouse myeloma by the method described by Herrick and Alberts (Herrick, G., and Alberts, B. M. (1976) J. Biol. Chem. 251, 2124-2132). HD protein-1 was heterogeneous with regard to apparent molecular weight (range of Mr = 24,000 to 33,000), but individual Mr species shared extensive primary structure homology as revealed by tryptic peptide mapping. The predominant species of this protein, Mr = 27,000, was resolved from other species and obtained in nearly homogeneous form by preparative isoelectric focusing. Mouse HD protein-1 was capable of lowering the Tm of poly[d(A-T)] by 25 degrees C, indicating that it is a helix-destabilizing protein. Sedimentation boundary analysis revealed that binding to ssDNA was noncooperative and that the binding site covered about 6 nucleotide residues. There was a 35% increase in the intrinsic tryptophan fluorescence of the protein in the presence of ssDNA, suggesting that structural change accompanies binding. Subcellular localization studies indicated that 75% of mouse HD protein-1 is nuclear, but not chromatin-associated, and primary structure analysis indicated that HD protein-1 is distinct from high mobility group proteins 1 and 2, histones, and P8 protein. Tryptic hydrolysis of HD protein-1 produced discrete, large fragments whose apparent molecular weights ranged from 19,000 to 24,000, and whose relative abundance was changed by the presence of ssDNA during the digestion. Thus, a Mr = 22,000 fragment (22 HDP*) predominated in the absence of ssDNA, and a Mr = 19,000, fragment (19 HDP*) predominated in the presence of ssDNA. Poly(dT) and denatured calf thymus DNA were more effective than were other polynucleotides tested in promoting accumulation of 19 HDP*; (dT)8 was as effective as were longer molecules of (dT)n, but (dT)4 and (dT)6 were much less effective, indicating that the binding site involved in 19 HDP* accumulation covered between 6 and 8 residues of (dT)n. Both 19 HDP* and 22 HDP* have the same COOH-terminal end and the same affinity for ssDNA-cellulose as does the native HD protein-1, indicating that a Mr = 8,000 sequence at the NH2-terminal end of HD protein-1 is not required for binding to ssDNA. Even though 22 HDP* retained the ability to bind to ssDNA, it could not be converted to 19 HDP* by further trypsin digestion.

Amino Acids↗
PubMed1980

Distinction between mouse DNA polymerases alpha and beta by tryptic peptide mapping.

Results presented here and in a previous paper (Tanabe et al. (1979) Biochemistry 18, 3401--3406) indicate that mouse beta-polymerase is a single polypeptide with an apparent molecular weight of 40,000. This polypeptide has now been analyzed by tryptic peptide mapping. Comparison of the results with identical analysis of mouse alpha-polymerase reveals that the tryptic peptides derived from the two enzymes are different. These results indicate that beta-polymerase is neither a subunit of alpha-polymerase nor a proteolytic degradation product of alpha-polymerase.

Animals↗
PubMed1979

Mouse DNA polymerase alpha. Subunit structure and identification of a species with associated exonuclease.

Two species of alpha-polymerase with very similar catalytic properties have been purified to near homogeneity from a soluble protein fraction of mouse myeloma. Sedimentation analysis in 0.5 M salt-containing glycerol gradients indicated that both species had a native Mr of about 190,000. Each species contained nonidentical subunits with apparent molecular weights of about 47,000 and 54,000. Subunits of Mr = approximately 50,000 had been found previously in calf thymus alpha-polymerase (Holmes, A. M., Hesslewood, I. P., and Johnston, I. R. (1974) Eur. J. Biochem. 43, 487-499; (1976) Eur. J. Biochem. 62, 229-235). Tryptic peptide mapping failed to reveal primary structure homology between the subunits of the two enzymes. Thus, the two alpha-polymerases are clearly different species. These two enzymes are further distinguished by the fact that one of them has associated exonuclease activities. One activity degraded single-stranded DNA to mononucleotides in the 3' leads to 5' direction and acted distributively. The other exonuclease activity also degraded single-stranded DNA to mononucleotides, but this degradation was in the 5' leads to 3' direction in a processive fashion. Both exonuclease activities co-migrated with the polymerase activity during the final purification step of polyacrylamide gradient gel electrophoresis, which yielded the essentially homogenous alpha-polymerase, and also during sedimentation of the purified enzyme through a high salt glycerol gradient.

Animals↗
PubMed1977

Deoxyribonucleic acid chain growth and organization of replicating units in HeLa cells.

A method for studying DNA chain growth and chromosomal organization of replicons in HeLa cells has been developed. DNA replication is initiated with bromodeoxyuridine followed by pulse labeling of active replicons with [3H]thymidine and growth of chains for finite intervals in unlabeled thymidine. Photolysis of the bromodeoxyuridine-DNA leader with 313-nm light releases the newly replicated chains that are then analyzed by sedimentation in alkaline sucrose gradients. This method of analysis provides data on the rate of chain growth, the bidirectionality of replication, and the distribution of the active replicons at specific intervals in the S period. Applying this method to cells caused to synthesize DNA at a lowered temperature (27 degrees C) or with protein synthesis restricted by cycloheximide revealed that the immediate reduction in the rate of DNA replication in both instances was due to a decreased rate of chain growth without derangement of the overall process.

Bromodeoxyuridine↗