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S C Bock

Publications and source records attributed to S C Bock.

33 records · Page 2Linked to original sources
PubMed1989

Protein inhibitors of crystal growth.

Nephrocalcin is a urinary glycopeptide that may be a physiological inhibitor of nephrolithiasis. Monomeric nephrocalcin purified from ethylenediaminetetracetic acid-treated urine is 14,000 daltons. Compositional analyses indicate that nephrocalcin is 10 per cent carbohydrate by weight and that 25 per cent of the amino acid residues are acidic (glutamic acid, aspartic acid and gamma-carboxyglutamic acid). Nephrocalcin binds reversibly to calcium oxalate crystals with a dissociation constant of about 0.5 microM. The high collapse pressure of nephrocalcin, 41.5 dynes per cm., measured for a monolayer at the air-water interface, suggests a highly organized structure in which hydrophilic and hydrophobic regions occupy separate regions on the surface of the inhibitor. Nephrocalcin contains the unusual amino acid, gamma-carboxyglutamic acid. Nephrocalcin isolated from urine of stone formers and from kidney stones does not contain gamma-carboxyglutamic acid and it has altered surface properties compared to normal nephrocalcin. The presence of the gamma-carboxyglutamic acid modification and the ability to form stable films with high collapse pressures may be important factors enabling nephrocalcin to prevent stone formation in vivo. The blood of cold water fishes contains antifreeze glycopeptides and/or peptides to prevent it from freezing. The structure of one such antifreeze peptide and its interactions with the crystal lattice of hexagonal ice are discussed as a model for how nephrocalcin might interact with calcium oxalate crystals and arrest their growth in urine.

Amino Acid Sequence↗
PubMed1988

Antithrombin III Utah: proline-407 to leucine mutation in a highly conserved region near the inhibitor reactive site.

A dysfunctional antithrombin III (ATIII) gene encoding a qualitatively and quantitatively abnormal anticoagulant molecule is responsible for hereditary thrombosis in a Utah kindred [Bock et al. (1985) Am. J. Hum. Genet. 37, 32-41]. Nucleotide sequencing of the entire protein-encoding portion of the cloned ATIII-Utah gene revealed a C to T transitional mutation which converts proline-407 to leucine. Proline-407 is located 14 amino acids C-terminal to the reactive site arginine of ATIII in a core region of the molecule that has been highly conserved during evolution of the serine protease inhibitor (serpin) gene family. The location of this proline in the crystal structure of the homologous serpin alpha 1-antitrypsin suggests that the leucine substitution in ATIII-Utah may interfere with correct folding of the mutant gene product, leading to its rapid turnover and the low antithrombin levels observed in patient plasmas. The Pro-407 to Leu mutation does not interfere with binding of antithrombin III to heparin. Patient antithrombin III, isolated by affinity chromatography on heparin-Sepharose, was reacted with purified thrombin. ATIII encoded by the patient's normal gene formed protease-inhibitor complexes with thrombin, whereas the product of the ATIII-Utah gene did not. The Pro-407 to Leu mutation destroys a restriction site for the enzyme StuI, permitting rapid diagnosis of affected members of the Utah kindred by Southern blotting of genomic DNA.

Amino Acid Sequence↗
PubMed1987

Molecular genetic survey of 16 kindreds with hereditary antithrombin III deficiency.

Molecular genetic techniques were utilized to examine antithrombin III (ATIII) gene status in 16 independently ascertained kindreds with hereditary ATIII deficiency. In one of these families antithrombin III deficiency is caused by hemizygosity of the ATIII locus. In the remaining 15 kindreds, two copies of the ATIII gene are present and appear to be grossly normal at the level of whole genome Southern blotting, suggesting that small deletions, insertions or limited nucleotide substitution(s) in the antithrombin III gene, or "trans-acting" defects at other loci involved in the processing, modification, and secretion of biologically active ATIII are responsible for the observed anticoagulant disorders.

Antithrombin III↗
PubMed1986

Human C1 inhibitor: primary structure, cDNA cloning, and chromosomal localization.

The primary structure of human C1 inhibitor was determined by peptide and DNA sequencing. The single-chain polypeptide moiety of the intact inhibitor is 478 residues (52,869 Da), accounting for only 51% of the apparent molecular mass of the circulating protein (104,000 Da). The positions of six glucosamine-based and five galactosamine-based oligosaccharides were determined. Another nine threonine residues are probably also glycosylated. Most of the carbohydrate prosthetic groups (probably 17) are located at the amino-terminal end (residues 1-120) of the protein and are particularly concentrated in a region where the tetrapeptide sequence Glx-Pro-Thr-Thr, and variants thereof, is repeated 7 times. No phosphate was detected in C1 inhibitor. Two disulfide bridges connect cysteine-101 to cysteine-406 and cysteine-108 to cysteine-183. Comparison of the amino acid and cDNA sequences indicates that secretion is mediated by a 22-residue signal peptide and that further proteolytic processing does not occur. C1 inhibitor is a member of the large serine protease inhibitor (serpin) gene family. The homology concerns residues 120 through the C-terminus. The sequence was compared with those of nine other serpins, and conserved and nonconserved regions correlated with elements in the tertiary structure of alpha 1-antitrypsin. The C1 inhibitor gene maps to chromosome 11, p11.2-q13. C1 inhibitor genes of patients from four hereditary angioneurotic edema kindreds do not have obvious deletions or rearrangements in the C1 inhibitor locus. A HgiAI DNA polymorphism, identified following the observation of sequence variants, will be useful as a linkage marker in studies of mutant C1 inhibitor genes.

Amino Acid Sequence↗
PubMed1986

Modification of the active site of alkaline phosphatase by site-directed mutagenesis.

The catalytically essential amino acid in the active site of bacterial alkaline phosphatase (Ser-102) has been replaced with a cysteine by site-directed mutagenesis. The resulting thiol enzyme catalyzes the hydrolysis of a variety of phosphate monoesters. The rate-determining step of hydrolysis, however, is no longer the same for catalysis when the active protein nucleophile is changed from the hydroxyl of serine to the thiol of cysteine. Unlike the steady-state kinetics of native alkaline phosphatase, those of the mutant show sensitivity to the leaving group of the phosphate ester.

2,4-Dinitrophenol↗
PubMed1985

Intron structure of the human antithrombin III gene differs from that of other members of the serine protease inhibitor superfamily.

Antithrombin III (ATIII) plays an integral role in the coagulation system by inhibiting thrombin and several other activated clotting factors. Inherited deficiency of ATIII is quite common and can result in life-threatening thrombotic complications. In order to understand the basis of ATIII deficiency, we have isolated and characterized the normal human ATIII gene from a recombinant Charon 4A bacteriophage genomic library. The ATIII gene contains six exons and five introns distributed over approximately 19 kilobases of DNA. The positions of introns in the ATIII gene were compared with other members of the serine protease inhibitor family which share 17-31% amino acid homology. When aligned to achieve maximal protein homology, only one of the ATIII introns corresponded to the four introns of rat angiotensinogen or human alpha 1-antitrypsin. Similarly, only one ATIII intron was homologous to the seven introns of chicken ovalbumin. We present two testable models to explain the discrepancy in intron positions among members of the serine protease inhibitor superfamily of genes.

Amino Acid Sequence↗
PubMed1985

Human apolipoprotein B cDNA clone isolation and demonstration that liver apolipoprotein B mRNA is 22 kilobases in length.

An expression library made in plasmids pUC8 and pUC9 with mRNA derived from the human hepatoma cell line HepG2 was screened with a rabbit antiserum to human low density lipoprotein (LDL). Approximately 12,000 clones were screened and five positives were identified. The cDNA inserts were all 1500-1600 base pairs in length. The insert from one clone, pB8, was isolated, labeled by nicktranslation, and found to cross-hybridize strongly with the other four cDNA clones. The pB8 clone produces a fusion protein of approximately equal to 37.5 kDa that reacts in electrophoretic transfer blot analysis with rabbit anti-human LDL. This reactivity can be abolished by pretreatment of the antiserum with purified human LDL, p = 1.025 - 1.050 g/ml. A pB8-derived probe was used to demonstrate that apolipoprotein B (apo B) mRNA is present in HepG2 cells and liver extracts but not in HeLa cells or extracts from small intestine, heart, aorta, spleen, brain, skeletal muscle, lung, kidney, or ovary. RNA transfer blot analysis revealed that HepG2 cell apo B mRNA was approximately equal to 22 kilobases in length. These cDNA clones should allow the isolation of the apo B gene and ultimately the elucidation of the primary structure of this protein.

Apolipoproteins B↗
PubMed1985

Assignment of the human antithrombin III structural gene to chromosome 1q23-25.

The human antithrombin III (ATIII) structural gene was mapped by in situ hybridization and quantitative analysis of ATIII gene dosage in DNA isolated from carriers of chromosome 1 deletions. These studies indicate that the ATIII structural gene maps to human chromosome q23-q25 and so is likely identical to AT3.

Antithrombin III↗
PubMed1985

Hereditary thrombosis in a Utah kindred is caused by a dysfunctional antithrombin III gene.

Maximum likelihood analysis of linkage between antithrombin III (ATIII) DNA polymorphisms and ATIII deficiency in a large Utah kindred suggests that thrombotic disease in this family is caused by a dysfunctional ATIII gene. ATIII-deficient family members were identified on the basis of: (1) reduced anticoagulant activity and (2) the presence of an electrophoretically abnormal inhibitor molecule in their plasmas. Affected individuals have two copies of the ATIII structural gene, and both alleles appear normal at the resolution of whole genome Southern blotting. However, family studies revealed statistically significant cosegregation of ATIII-deficiency trait and a particular ATIII DNA polymorphism haplotype (lod = 3.35; theta = 0.0); this result is consistent with the presence of a dysfunctional ATIII gene on a chromosome of the +, S haplotype.

Alleles↗
PubMed1983

Characterization of an unusual DNA length polymorphism 5' to the human antithrombin III gene.

Nucleotide sequence analysis revealed that a DNA length polymorphism 5' to the human antithrombin III gene is due to the presence of 32bp or 108bp nonhomologous nucleotide sequences (variable segments) 345bp upstream from the translation initiation codon. Sequences at the 3' borders of both variable segments can form intrastrand inverted repeat structures with sequences further downstream. An inverted repeat is also found immediately 5' to the site where the variable segments are located. Thus, cruciform structures may form flanking the variable segments of both alleles of this DNA length polymorphism. DNA secondary structure may be detected with single strand specific nucleases. S1 nuclease sensitive sites were mapped in recombinant plasmids containing the cloned alleles of the ATIII length polymorphism. The site most sensitive to S1 is located upstream from the variable segments in an AT-rich segment flanked by 6bp direct repeats. A region of lesser nuclease sensitivity was also observed in the AT-rich loops formed between the inverted repeats 5' to the variable segments.

Amino Acid Sequence↗
PubMed1982

Cloning and expression of the cDNA for human antithrombin III.

A partial cDNA clone for human antithrombin III (ATIII) was obtained by screening a cDNA library prepared from size fractionated liver RNA with a pool of eight 16-base long synthetic DNA fragments whose sequence was determined from protein sequence data. A fragment of the partial cDNA clone was used to enrich RNA for ATIII messages, and cDNA clones encoding the entire ATIII structural gene were identified. The complete nucleotide and predicted amino acid sequences of human ATIII and its 32 residue signal peptide are reported, and provide further opportunity to compare the ATIII primary structure with corresponding regions from homologous proteins, alpha 1-antitrypsin and ovalbumin. Plasmids in which the structural genes for mature and pre-ATIII were linked to the E. coli trp promoter-operator support the synthesis of human antithrombin III and pre-antithrombin III in bacteria.

Amino Acid Sequence↗
PubMed1982

Hybridization-selected translation of Bombyx mori high-cysteine chorion proteins in Xenopus laevis oocytes.

Xenopus laevis oocytes were injected with poly(A)+-mRNA isolated from chorionating follicular epithelium of the domesticated silk moth (Bombyx mori). On two-dimensional gel electrophoresis, the resultant translation products comigrated with authentic, secreted, chorion standards, demonstrating that the frog oocyte system synthesizes and correctly process virtually all major chorion components. A cDNA clone has been shown to contain sequences complementary to those of mRNAs encoding B mori high-cysteine (Hc) chorion proteins Hc6-Hc11. mRNAs were selected by hybridization to plasmid m5000 DNA bound to diazobenzyloxymethyl-cellulose and subsequently translated in X. laevis oocytes into forms that comigrated with authentic chorion standards. The selection of a distinct subset of Hc mRNAs under stringent hybridization conditions (70% formamide/0.2 M NaCl, 60 degrees C) suggests that they are encoded by related genes. This is consistent with the pattern obtained by hybridizing radioactive m5000 DNA to Southern blots prepared from EcoRI-cleaved B. mori chromosomal DNA.

Animals↗
PubMed1981

The sequence of human serum albumin cDNA and its expression in E. coli.

A recombinant plasmid has been constructed which contains the mature protein coding region of the human serum albumin (HSA) gene. Bacteria containing this plasmid synthesize HSA protein under control of the E. coli trp promoter-operator. The DNA sequence and predicted protein sequence of HSA were determined from the cDNA plasmid and are compared to existing data obtained from direct protein sequencing. The DNA sequence predicts a mature protein of 585 amino acids preceded by a 24 amino acid "prepro" peptide.

Amino Acid Sequence↗
PubMedDate not supplied

Idealization of the hydrophobic segment of the alkaline phosphatase signal peptide.

Proteins secreted by prokaryotic cells are synthesized as precursors containing an amino-terminal extension sequence or signal peptide. Although these signal peptides share little primary sequence homology, recent studies suggest that they function via common pathways during the transport process and that a common element may reside in their secondary structural characteristics. We are investigating the role of an idealized hydrophobic sequence with high potential for alpha-helix formation in the Escherichia coli alkaline phosphatase signal peptide. Here, amino-acid substitutions were made using site-directed mutagenesis to produce a mutant signal sequence containing nine consecutive leucine residues in the hydrophobic core segment. Transport studies with this mutant precursor indicate that mature alkaline phosphatase is correctly targeted to the E. coli periplasm and that processing of the precursor to the mature form of the enzyme is extremely rapid. In contrast, processing is slowed when the mutant signal sequence is lengthened by the insertion of five additional leucine residues and one serine.

Alkaline Phosphatase↗