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P H Johnson

Publications and source records attributed to P H Johnson.

At least 19 recordsLinked to original sources
PubMed1992

The structure of a complex of bovine alpha-thrombin and recombinant hirudin at 2.8-A resolution.

Crystals of the complex of bovine alpha-thrombin with recombinant hirudin variant 1 have space group C222(1) with cell constants a = 59.11, b = 102.62, and c = 143.26 A. The orientation and position of the thrombin component was determined by molecular replacement and the hirudin molecule was fit in 2 magnitude of Fo - magnitude of Fc electron density maps. The structure was refined by restrained least squares and simulated annealing to R = 0.161 at 2.8-A resolution. The binding of hirudin to thrombin is generally similar to that observed in the crystals of human thrombin-hirudin. Several differences in the interactions of the COOH-terminal polypeptide of hirudin, specifically of residues Asp-55h, Phe-56h, Glu-57h, and Glu-58h, and a few differences in the interactions of the hirudin core, specifically of residues Asp-5h, Ser-19h, and Asn-20h, with thrombin from human thrombin-hirudin suggest that there is some flexibility in the binding of these 2 molecules. Most of the residues in the 9 subsites that bind fibrinopeptide A7-16 to thrombin also interact with the NH2-terminal domain of hirudin. The S1 subsite is a notable exception in that only 1 of its 6 residues, namely Ser-214, interacts with hirudin. The only difference between human and bovine thrombins that appears to influence the binding of hirudin is the replacement of Lys-149E by an acidic glutamate in the bovine enzyme.

Amino Acid Sequence↗
PubMed1992

Expression of synthetic genes encoding fused proteins under tight control of modified regulatory regions of the colicin operon.

A versatile expression vector system for construction of gene and protein fusions, specific radiolabeling of gene products and high-level protein production is described. Expression cassettes were constructed containing structural genes encoding native and analog forms of connective tissue-activating peptide-III (CTAP-III), beta-thromboglobulin, neutrophil-activating protein and modified regulatory sequences derived from the colicin E1 operon. Gene expression was enhanced by changes in the colicin promoter that increased the transcription initiation rate both in vivo and in vitro, and by deletion of a sequence affecting catabolite repression. High-level expression, producing recombinant protein up to 30% of the total cellular protein, was induced rapidly after stimulation of the SOS response by using either mitomycin C or nalidixic acid, by temperature shift using temperature-sensitive mutations in the LexA or RecA proteins, or by UV light. The presence of radiolabeled amino acids during induction resulted in greater than 95% preferential labeling of recombinant proteins. CTAP-III remained stable for more than 6 h following decay of the inducing signal. The use of CTAP-III in protein fusions improved stability of several therapeutically useful proteins including the thrombin-specific inhibitor, hirudin and a cell receptor-binding domain of laminin, when they were produced in Escherichia coli.

Amino Acid Sequence↗
PubMed1992

Purification of the Lewis blood-group gene associated alpha-3/4-fucosyltransferase from human milk: an enzyme transferring fucose primarily to type 1 and lactose-based oligosaccharide chains.

A soluble Lewis blood-group gene associated alpha-3/4-L-fucosyltransferase has been purified from human milk by a series of steps involving hydrophobic chromatography on Phenyl Sepharose 4B, ion exchange chromatography on CM-Sephadex C-50, affinity chromatography on GDP-hexanolamine Sepharose 4B and gel filtration on Sephacryl S-200. The first step separated alpha-3-L-fucosyltransferase activity directed towards N-acetylglucosamine in Type 2 (Gal beta 1-4GlcNAc-R) acceptors from an alpha-3/4-fucosyltransferase fraction acting on both Type 1 (Gal beta 1-3GlcNAc-R) and Type 2 acceptors. Further purification of this latter fraction on CM-Sephadex and GDP-hexanolamine Sepharose gave a single peak of fucosyltransferase activity that catalysed the addition of fucose to N-acetylglucosamine in both Type 1 and Type 2 acceptors and to the O-3 position of glucose in lactose-based oligosaccharides. The enzyme preparation at this stage resembled previously described alpha-3/4-fucosyltransferase preparations purified from human milk. However, gel filtration of this preparation on Sephacryl S-200 or Sephadex G-150 separated further amounts of alpha-3-fucosyltransferase activity acting solely on Type 2 acceptors and left a residual alpha-3/4-fucosyltransferase that retained strong alpha-4 activity with the Type 1 acceptor, lacto-N-biose 1, and alpha-3 activity with 2'-fucosyllactose, but had relatively little alpha-3 activity with N-acetyllactosamine and virtually no capacity to transfer fucose to glycoproteins with N-linked oligosaccharide chains having unsubstituted terminal Type 2 structures.

Chromatography, Affinity↗
PubMed1992

Reassessment of the acceptor specificity and general properties of the Lewis blood-group gene associated alpha-3/4-fucosyltransferase purified from human milk.

The acceptor specificity and general properties of a Lewis blood-group gene associated alpha-3/4-L-fucosyltransferase isolated from human milk have been examined at the penultimate purification stage involving affinity chromatography on GDP-hexanolamine Sepharose, and after a subsequent gel filtration step on Sephacryl S-200. Both preparations transferred fucose to the O-4 position of N-acetylglucosamine in Type 1 (Gal beta 1-3GlcNAc-R) acceptors and the O-3 position of glucose in lactose-based (Gal beta 1-4Glc) oligosaccharides, and both used Type 1 sialylated compounds when the terminal N-acetylneuraminic acid was present in alpha-2,3 linkage. The striking difference between the two preparations was in their reactivity with Type 2 (Gal beta 1-4GlcNAc-R) chains; after Sephacryl S-200 chromatography the apparent KM values for the alpha-3/4- preparation with unsubstituted low-molecular-weight Type 2 oligosaccharides were considerably increased. Substitution of the terminal galactose with sialic acid in alpha-2,3 linkage decreased the KM values for low-molecular-weight oligosaccharides but no detectable incorporation of fucose was observed into N-acetyllactosamine end-groups of glycoproteins with N-linked oligosaccharide chains, irrespective of the presence of sialic acid in the terminal sequences.

Carbohydrate Sequence↗
PubMed1992

Detection of ABO blood group polymorphism by denaturing gradient gel electrophoresis.

We report the use of a polymerase chain reaction (PCR) format together with denaturing gradient gel electrophoresis (DGGE) which allows rapid identification of the 6 major genotypes (AA, AO, BB, BO, AB and OO) of the human ABO blood group polymorphism in a single amplification. The procedure also distinguishes hitherto undescribed polymorphisms associated with the O and B alleles. Thus in testing 95 unrelated European individuals 4 different O alleles, 2 B alleles and 1 A allele were identified by DGGE and the level of recognisable heterozygosity, and hence the information content of the locus as a genetic marker, was raised from 3/95 (3%) to 66/95 (70%). The procedure is robust, genotyping is rapid and clear-cut, and has immediate implications for the use of the ABO locus in linkage analysis on chromosome 9q, the investigation of disease associations and forensic identification.

ABO Blood-Group System↗
PubMed1991

Chemical modifications and amino acid substitutions in recombinant hirudin that increase hirudin-thrombin affinity.

Recombinant hirudin (r-hirudin), unlike the naturally occurring leech protein, lacks a sulfate ester on Tyr-63 which reduces its binding affinity to thrombin by 3-10-fold. We demonstrate that nitration or iodination of Tyr-63 restores hirudin-thrombin affinity to levels similar to or exceeding that of the natural inhibitor. In contrast, nitration of Tyr-3 reduces the affinity of hirudin for thrombin. These chemical modifications results in multiple reaction products that are readily separated by reverse-phase HPLC. The mechanism of the observed changes in thrombin affinity may involve a reduction in the pK of the hydroxyl group of tyrosine due to substitution of the electrophilic iodo or nitro group on the phenyl ring, resulting in an increased negative charge at neutral pH. For Tyr-63, this effect mimics the sulfatotyrosine of natural hirudin, leading to an increased thrombin affinity at the anion-binding exosite. For Tyr-3, the increased polarity may destabilize its interaction within the apolar-binding site of thrombin. Substitution of the highly conserved Tyr-3 residue with Phe or Trp not only enables specific and quantitative chemical modification at Tyr-63 but also independently increases hirudin-thrombin affinity. Kinetic analysis of thrombin inhibition showed that enhanced binding by r-hirudin(nitro-Tyr-63) is due to an increase in the association rate between hirudin and thrombin whereas the reduced binding of r-hirudin(nitro-Tyr-3) results from a large increase in the dissociation rate. These observations indicate that specific segments within both the amino- and carboxy-terminal regions of hirudin interact with thrombin.

DNA Mutational Analysis↗
PubMed1991

Hirudin: amino-terminal residues play a major role in the interaction with thrombin.

Amino acid substitutions within the amino-terminal 5 residues of the thrombin-specific inhibitor hirudin dramatically alter its ability to inhibit the thrombin-catalyzed hydrolysis of both a chromogenic substrate and fibrinogen. Replacing the highly conserved Tyr-3 residue with Trp or Phe increases hirudin's affinity for thrombin 3-6-fold (decreases the inhibition constant, Ki) whereas Thr results in a 450-fold increase in Ki. A more extensive modification involving deletion of the amino-terminal Val, and Tyr-3----Val, Thr-4----Gln, and Asp-5----Ile replacement, results in a large reduction in thrombin inhibitory activity corresponding to greater than a 10(7)-fold increase in Ki and a 10(3)-fold increase in IC50, using D-Phe-L-pipecolyl-Arg-p-nitroanilide (S-2238) and fibrinogen, respectively, as substrates. Kinetic analysis of these mutant proteins and synthetic peptide fragments and available structural information on thrombin and hirudin derived from protein crystallography and two-dimensional NMR studies indicate that the amino-terminal region of hirudin binds at the apolar binding/active site region of thrombin, with Tyr-3 occupying the S3 specificity site. The large effect of these modifications on hirudin activity suggests that alteration of the amino-terminal segment can destabilize the interaction of other regions of hirudin with thrombin.

Amino Acid Sequence↗
PubMed1991

Detection of the common alpha-1-antitrypsin variants by denaturing gradient gel electrophoresis.

The well-characterized and highly polymorphic human alpha-1-antitrypsin (AAT) gene was used as a test locus to evaluate the general applicability of denaturing gradient gel electrophoresis (DGGE) for the detection of single base change polymorphisms. We report the resolution of all the major alleles, M1Ala, M1Val, M2, M3, S and Z and the identification of substantial genetic polymorphism in intron 3-4 by this technique. DGGE was found to be a quick and efficient method for the screening of multiple samples for the presence of genetic variation.

Alleles↗
PubMed1991

Structure-function and refolding studies of the thrombin-specific inhibitor hirudin.

We have developed a novel expression and purification system that yields recombinant desulfo-hirudin (HV-1) with high specific activity (10,000 antithrombin units/mg) and an inhibition constant (Ki) for human alpha-thrombin of 0.2 pM. Reduced and denatured hirudin rapidly refolds to the native, fully active conformation at high concentration (greater than 50 mg/ml) by incubation at pH 10. Analytical gel filtration studies at neutral pH suggest that hirudin is a multimer. Initial binding of hirudin to thrombin appears to be followed by dissociation of the hirudin multimer to give a tight-binding 1:1 hirudin:thrombin complex. Thrombin inhibition studies showed that hirudin synthetic peptide fragments 42-65 and 51-65 [but not (Ala22)-6-28, containing two of the three disulfide bonds formed in native hirudin] were similarly effective in inhibiting thrombin cleavage of fibrinogen (IC50 = 4.9 and 6.0 microM, respectively, at a thrombin concentration of 1 microM). We conclude that hirudin has unusual structural and refolding properties and that its mechanism of inhibition involves noncovalent interaction with multiple sites on thrombin. The interaction of hirudin (specifically the region of Lys-47) with the basic specificity pocket of thrombin may contribute to the binding but is not essential for its inhibitory activity.

Amino Acid Sequence↗
PubMed1979

Electrophoretic characterization of intracellular forms of bacteriophage phi X174 DNA: identification of novel intermediate of altered superhelix density.

The replication cycle of bacteriophage phi X174 DNA has been analyzed by agarose gel electrophoresis. The electrophoretic behavior of the predominant species of parental and progeny DNA molecules formed between 5 and40 min after infection was deduced and quantitated. Migration through 1.4% agarose at 5 and 10 V/cm resolved all known viral DNA species as well as fragments of host chromosomal DNA. Among parental replicative form(RF) molecules synthesized, 1 to 3% were full length linear duplexes (RFIII) and approximately 65% were closed circular duplexes (RFI). Most of the input viral strands remained in a duplex structure throughout the period of infection studied here. Among progeny molecules, RFIII was not readily detected unless viral DNA synthesis was inhibited by chloramphenicol. Late in infection, 20% of the progeny RF were found to exist as form I dna. in addition, approximately 1% of the viral DNA was found as unit length linear single strands. Electrophoretic analysis of RF DNA after controlled denaturation suggests the existence of four populations of closed circular RF: (i) molecules of native superhelix density (RFI); (ii) a population of molecules of altered topological linking number, alpha, differing in increments of one superhelical turn (tau) between tau values of 0 and approximately -31; (iii) a superimposed population of topological isomers which under electrophoresis conditions have mean tau value (tau) equal to +5; and (iv) a population of "complexed" molecules with a reduced number of superhelical turns due to their association with single-stranded DNA and RNA. Complexed parental molecules isolated from cells infected at high multiplicity released FRI and homologous single-stranded DNA upon denaturation and are postulated to be intermediates in genetic recombination. Complexed RF DNA isolated from cells infected at low multiplicity release native supercoils upon reaction with RNase H and are observed by electron microscopy to contain displacement loops. Such molecules are likely intermediates in transcription. Our results are consistent with a structure of complexed RFI involving a partially triple-stranded helix in which a covalently closed circular duplex molecule contains a reduced number of superhelical turns due to the unwinding produced by base pairing between one strand of the supercoil and an associated homologous single strand of DNA or RNA.

Bacteriophage phi X 174↗
PubMed1979

The lumbar disc.

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Aging↗
PubMed1977

A restriction endonuclease cleavage map of mouse mitochondrial DNA.

A restriction endonuclease cleavage map is presented for mouse mitochondrial DNA. This map was constructed by electron microscopic measurements on partial digests containing fixed D-loops, and by electrophoretic analysis of partial and complete single enzyme digests, and of double digests. No map differences were detected between mitochondrial DNA from cultured LA9 cells and an inbred mouse line for the six endonucleases used. Three cleavage sites recognized by HpaI, five sites recognized by HincII, two sites recognized PstI and four sites recognized by BamI were located with respect to the origin of replication and the EcoRI and HinIII sites previously determined by others. No cleavages were produced by KpnI or SalI. The migration of linear DNA with a molecular weight greater than 1 X 10(6) was not a linear function of log molecular weight in 1% agarose gels run at 6.6 volts/cm.

Animals↗