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P J Isackson

Publications and source records attributed to P J Isackson.

At least 73 records · Page 4Linked to original sources
PubMed1987

A complete cDNA sequence for the major epidermal growth factor binding protein in the male mouse submandibular gland.

The complete cDNA sequence of the major epidermal growth factor binding protein (EGF-BP), isolated from the mouse submandibular gland, has been determined. Oligonucleotide probes complementary to unique, nonconserved, regions of homogeneous preparations of EGF-BP were used to identify the correct cDNA clone from a male mouse submandibular gland cDNA library. The nucleotide sequence codes for a glandular kallikrein that is the main arginine esterase complexed with epidermal growth factor. The mRNA coding for this EGF-BP is estimated at 0.24% of the total mRNA of the adult male mouse submandibular gland, thus representing an abundant member of the kallikrein family in this tissue. In addition, the cDNA sequence defines a putative transcription start site. The reported cDNA sequence is clearly different from, and not an allelic form of, a previously reported cDNA sequence for EGF-BP. The present work reconciles conflicting information in the literature regarding the identity of EGF-BP.

Amino Acid Sequence↗
PubMed1987

Epidermal growth factor binding protein: identification of a different protein.

Partial amino acid sequence analysis of epidermal growth factor binding protein (EGF-BP), an arginine esteropeptidase that specifically associates with EGF to form a high molecular weight complex in male mouse submandibular glands, has revealed a single, distinct protein that is different from three previously reported forms of EGF-BP. This protein shows substantial sequence homology with these other putative forms of EGF-BP as well as with a large family of kallikreins expressed in the mouse submandibular gland. Purified EGF-BP contains three polypeptide chains as a result of two internal cleavages at residues 87-88 and 140-141. These modifications may represent processing events that are critical for determining the binding specificity of EGF-BP, since they occur within regions surrounding the substrate binding site.

Amino Acid Sequence↗
PubMed1987

Role of glandular kallikreins as growth factor processing enzymes: structural and evolutionary considerations.

Hormones and growth factors are generally released from larger precursors by limited proteolysis. The causative agents remain poorly defined with respect to location and properties. One subset of proteases, the glandular kallikreins, have been implicated in a few cases, in part because of their specific association with mature forms of some hormones. However, limited distribution and low copy number in some species cast doubt on this hypothesis, and they may well play other physiological functions that remain to be elucidated.

Amino Acid Sequence↗
PubMed1987

Expression of the alpha subunit of 7S nerve growth factor in the mouse submandibular gland.

alpha-NGF is an inactive serine protease that is associated in the mouse submandibular gland with a closely related serine protease, gamma-NGF, and the neurotrophic factor, beta-NGF. The heterogeneity of purified alpha-NGF has been examined by DEAE-cellulose chromatography and SDS polyacrylamide gel electrophoresis. A possible explanation for the observed heterogeneity is presented. Antibodies have been prepared against alpha-NGF and purified by affinity chromatography so that they do not cross-react with gamma-NGF. This antibody preparation recognizes two very similar proteins in male mouse submandibular gland RNA-directed cell-free translation mixtures. The expression of only one of these forms is regulated by testosterone. Oligonucleotide probes specific for each of the three NGF subunits have been prepared and used for Northern blot analysis of RNA from the mouse submandibular gland. The three subunits were found to be coordinately expressed and each were 30-fold more abundant in male than in female glands.

Amino Acid Sequence↗
PubMed1985

Dominant negative mutations in the Tn10 tet repressor: evidence for use of the conserved helix-turn-helix motif in DNA binding.

The Tn10 tet repressor regulates transcription of the tetracycline-resistance determinant in transposon Tn10. Previous DNA sequencing studies identified a region of tet repressor (amino acids 26-47) that is homologous to the helix-turn-helix regions of lambda Cro, lambda repressor, and catabolite gene activator protein that are implicated in sequence-specific DNA binding. Here we report the isolation of dominant tetR mutations that result in tet repressors deficient in tet operator binding but that retain some capacity to form dimers with, and thereby inactivate, wild-type repressor monomers. The mutations were isolated by transforming a tetR+ tetA-lacZ fusion strain with hydroxylamine-mutagenized tetR plasmid DNA and then screening for increased lacZ expression. DNA sequence analysis of 35 independent isolates identified seven different mutations, five of which are in the region of helix-turn-helix sequence homology. In vitro binding studies indicate that the mutations in this region of tet repressor reduce the affinity of tet repressor for tet operator DNA by at least a factor of 1000 but have no significant effect on the affinity of tet repressor for tetracycline. These results provide strong support for the proposal that tet repressor utilizes the conserved helix-turn-helix structural motif in binding to tet operator DNA.

Amino Acid Sequence↗
PubMed1984

Mouse 7S nerve growth factor: complete sequence of a cDNA coding for the alpha-subunit precursor and its relationship to serine proteases.

Two synthetic oligonucleotides, one 14-mer and one 15-mer, each containing 32 sequences and corresponding to two regions of the partially determined protein sequence, were utilized to identify three cDNA clones coding for the precursor of the alpha-subunit of 7S mouse nerve growth factor (NGF). This library, containing 860 clones, had been preselected from a much larger one by low-stringency hybridization using a cDNA probe corresponding to one of the large family of glandular kallikreins expressed in the adult male mouse submandibular gland. Partial sequence analysis had previously established the alpha-subunit to be a member of this group, albeit with no demonstrable catalytic activity. Nucleotide sequence analysis of the longest of these clones (2A4) predicted the apparent complete amino acid sequence of the 265-residue precursor. One of the other clones (3F2) contained an A----G substitution at position 565 resulting in a Lys----Glu change at position 160 of the mature sequence. These clones probably represent two different alleles. Several amino acid changes, relative to other serine proteases, are evident, which may account for the apparent lack of enzymatic activity. An Arg----Gln substitution at residue -1 would prevent cleavage of the putative activation peptide, and the deletion of residues 2-5 interrupts the highly conserved Ile/Val-Ile/Val-Gly-Gly N-terminal sequence. An Asp----Tyr substitution in the binding pocket and a Gly----His substitution near the active site serine also probably contribute to the inactive structure. The role of this subunit in NGF function remains obscure.

Amino Acid Sequence↗
PubMed1984

The alpha-subunit of mouse 7 S nerve growth factor is an inactive serine protease.

Tryptic and cyanogen bromide peptides accounting for approximately 85% of the amino acid sequence of the alpha-subunit of mouse 7 S nerve growth factor have been isolated and extensively sequenced. The partial structure revealed a high degree of identity with the gamma-subunit (greater than 80%), which is an arginine esteropeptidase of the serine protease family. However, the alpha-subunit does not cleave synthetic arginine ester or peptide substrates nor is it labeled by diisopropylfluorophosphate. The lack of catalytic activity may result from a Gly----His substitution near the active site serine or from the blocked NH2 terminus.

Amino Acid Sequence↗
PubMed1984

Nerve growth factor: mechanism of action.

Although the detailed mechanism of action of NGF as it occurs in vivo still requires considerable clarification, a model in which NGF is generated by end organs of responsive neurons and interacts with these neurons to provide trophic stimulation and maintenance of viability seems highly plausible. The demonstration of NGF synthesis by these cells, including the details of the process, is required, and information regarding the events following the formation of the hormone-receptor complex at the plasma membrane must be obtained. However, the contribution of internalization and the possible role of the internalized receptor as a second messenger for generating the long-term effects of the hormone are particularly appealing. Any findings with NGF will almost certainly provide additional important insights into other polypeptide growth factors, particularly those substances found in the neurotrophic subset.

Affinity Labels↗
PubMed1983

Production of HMG-3 by limited trypsin digestion of purified high-mobility-group nonhistone chromatin proteins.

Three isolated nonhistone proteins (HMG-1, HMG-2 and HMG-E) have been purified from chicken erythrocyte chromatin without exposure to overt denaturing conditions, and subjected to limited proteolysis. When treated with trypsin, the three proteins exhibited similar patterns of degradation, as judged by SDS and acid/urea gel electrophoresis. In particular, the first product, P1 (a relatively stable intermediate in each digestion), was a protein analogous to HMG-3, a principal degradation product in preparations of calf thymus high-mobility-group proteins. At least in the case of HMG-E, the products formed by tryptic attack on P1 are the two individual DNA binding domains of HMG-E. P1 derived from HMG-E and one of the individual DNA binding domains of HMG-E were purified by chromatography on columns containing DNA-cellulose or phosphocellulose. The properties of these two portions of HMG-E are consistent with our recently postulated three-domain structure for HMG-1 and its homologs (Reeck, G.R., Isackson, P.J. and Teller, D.C. (1982) Nature 300, 76-78). Thus, P1 consists of two DNA-binding domains of approximately equal molecular weight covalently linked together. From chromatography on DNA-cellulose columns, it is clear that P1 binds to DNA more tightly than does HMG-E. The highly acidic C-terminal domain of HMG-E (which is removed by trypsin in generating P1) thus counteracts the DNA binding of the two other domains of HMG-E (at least in the protein's interaction with purified DNA).

Amino Acids↗
PubMed1983

Ionic interactions between proteins in nonequilibrium pH gradient electrophoresis: histones affect the migration of high mobility group nonhistone chromatin proteins.

In two-dimensional gel electrophoresis of the high mobility group (HMG) proteins, it has proved necessary to use nonequilibrium pH gradient electrophoresis (NEPHGE) in the first dimension rather than isoelectric focusing, because of the basic character of most of the HMG proteins [D. Tyrell, P. J. Isackson, and G. R. Reeck (1982) Anal. Biochem. 119, 433-439]. In this paper it is reported that in samples that contain histones, the mobilities of HMG proteins (particularly HMG-1, HMG-2, and HMG-E) are severely distorted in NEPHGE. This presumably results from formation of complexes between histones and HMG proteins through ionic interactions. Analysis of HMG proteins by NEPHGE/sodium dodecyl sulfate-gel electrophoresis is thus precluded in samples containing histones. Our results raise the possibility of similar artifacts occurring in NEPHGE (or isoelectric focusing) analysis of other proteins with regions of high charge density.

Animals↗
PubMed1982

Influence of nonhistone chromatin protein HMG-1 on the enzymatic digestion of purified DNA.

The effect of chicken erythrocyte High Mobility Group protein 1 (HMG-1) on the enzymatic hydrolysis of purified double-stranded and single-stranded bacteriophage lambda DNA was studied. HMG-1 was found to inhibit the digestion of single- and double-stranded DNA by S1 nuclease and DNase I, respectively. HMG-I increased the rate of hydrolysis of double-stranded DNA by micrococcal nuclease, particularly at low HMG-1/DNA ratios, and had little effect on the hydrolysis of single-stranded DNA by micrococcal nucleases, even at high HMG-1 DNA ratios. We also present a semi-quantitative estimate that HMG-1 and HMG-2 occur in chromatin from rapidly dividing, cultured rat hepatoma cells at about 8 times the level that they occur in adult rat liver chromatin.

Animals↗
PubMed1982

Removal of degradation products from calf thymus high mobility group non-histone chromatin proteins by chromatography on immobilized double-stranded DNA.

The substantial protease activity in calf thymus chromatin inevitably produces some degradation of high mobility group (HMG) non-histone proteins in NaCl extracts of calf thymus chromatin. We have found that proteins considered to be degradation products can be conveniently and cleanly separated from intact high mobility group proteins 1 and 2 by chromatography on double-stranded DNA-cellulose in 0.2 M NaCl/1 mM Tris-HCl (pH 7.5). Under those conditions, only the presumptive degradation products are retained by the column.

Animals↗
PubMed1981

Nonhistone chromatin proteins HMG-14 and HMG-17 bind preferentially to single-stranded DNA.

Proteins extracted from chicken erythrocyte chromatin with 0.35 M NaCl were subjected to sequential chromatography on columns containing immobilized double-stranded and single-stranded DNA's. Two-dimensional electrophoresis of protein fractions revealed that HMG-14 and HMG-17 are among the proteins that are retained by the single-stranded DNA column in 0.2 M NaCl/l mM Tris-Cl (pH 7.5) after having failed to be retained by the double-stranded column under the same conditions. That suggests that those two proteins possess preferential affinity for single-stranded DNA. Further evidence for that was provided by chromatography of purified HMG-14 and of purified HMG-17 on single-stranded and double-stranded DNA columns. We discuss the possible relevance of our results to suggested functions of HMG-14 and HMG-17.

Animals↗