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J Shafqat

Publications and source records attributed to J Shafqat.

33 records · Page 2Linked to original sources
PubMed1996

Liver class-I alcohol dehydrogenase isozyme relationships and constant patterns in a variable basic structure. Distinctions from characterization of an ethanol dehydrogenase in cobra, Naja naja.

The major ethanol dehydrogenase of cobra liver was characterized in order to clarify isozyme relationships and functional motifs of the vertebrate enzyme. The cobra protein is a class-I form, most related to one of the isozyme subunits (the a form) in Uromastix (lizard) liver. This positions the isozyme duplication and defines the main-line alternative. The new structure also allows extensive correlations with structure/function relationships for alcohol dehydrogenases in general, of which 38 animal variants (still disregarding strain and allelic differences) now have been characterized. Architectural features are discerned, distinguishing the enzyme at large, the classes, and the functional interactions at the sites of substrate binding and coenzyme binding. Variability is greater at the substrate-binding site, with only one of 13 residues strictly conserved (His67, one of the active-site zinc ligands) but all other residues differing among and frequently within classes. However, many substrate-interacting residues are class preferential and may be used in predictive assignments. Class-I/III differences concern position 48 (typically Ser in class I, Thr in class III), position 93 (Phe versus Tyr), position 141 (branch-chained aliphatic residue versus methionine), position 57 (hydrophobic residue versus Asp), position 115 (Asp versus Arg), position 116 (Leu or Ile versus Val), position 306 (Met or Leu/Ile versus Phe), position 309 (Phe or Leu/Ile versus Val) and position 318 (Val or Ile versus Ala). In contrast, coenzyme binding is more conserved. A characteristic coenzyme-binging motif, covering only a 50-residue stretch, is defined as tVDiK (residues 178, 203, 223, 224, 228; capital letters for residues strictly conserved and small-cases letters for residues nearly so). This motif is class independent and unique to animal alcohol dehydrogenases. Therefore, the novel enzyme structure establishes class-I isozyme relationships, shows characteristic 'constant' residues also in the 'variable' class-I line, and defines residue-specific patterns which may have a predictive value in functional assignments of an increasing number of undefined further forms expected to result from gene projects.

Alcohol Dehydrogenase↗
PubMed1995

Isolation, characterization and structure of subtilisin from a thermostable Bacillus subtilis isolate.

A serine protease has been isolated and characterized from Bacillus subtilis, strain RT-5 (a thermostable soil isolate from the Tharparkar desert of Pakistan) able to grow at 55 degrees C. The primary structure was established by a combination of protein and DNA-sequence analyses. The amino-acid sequence, inhibition pattern and solubility properties identify the enzyme as a subtilisin. It has 43 amino-acid replacements toward subtilisin BPN' and as much as 83 replacements toward another subtilisin, confirming that strain variabilities are extensive between different subtilisin forms. However, the structure is identical to one of unknown functional properties deduced from DNA and is closely related to mesentericopeptidase but that homologue is not thermostable. From comparisons with that form and with subtilisin BPN', it is concluded that replacements of Ala --> Ser at positions 85 and 89, Ser --> Ala at position 88 and Asp or Ser --> Asn at position 259 may promote thermostability.

Amino Acid Sequence↗
PubMed1995

Alcohol dehydrogenase of class III: consistent patterns of structural and functional conservation in relation to class I and other proteins.

Class III alcohol dehydrogenase from the lizard Uromastix hardwickii has been characterized. This non-mammalian, gnathostomatous vertebrate class III form allows correlations of structures and functions of this class, the traditional class I alcohol dehydrogenase, and other well-studied proteins. Catalytically, results show similar recoveries and activities of all vertebrate class III forms independent of source, similar activities also in invertebrates but in lower amounts, and considerably higher specific activities in microorganisms. Structurally, variability patterns are consistent throughout the vertebrate system with a ratio in accepted point mutations versus class I of 0.4. This ratio between different classes of a zinc enzyme is comparable to that between different heme proteins (cytochrome c and myoglobin), suggesting defined but non-identical functions also for the alcohol dehydrogenase classes.

Alcohol Dehydrogenase↗
PubMed1995

Multiplicity of N-terminal structures of medium-chain alcohol dehydrogenases. Mass-spectrometric analysis of plant, lower vertebrate and higher vertebrate class I, II, and III forms of the enzyme.

Ten different alcohol dehydrogenases, representing several classes of the enzyme and a wide spread of organisms, were analyzed for patterns of N-terminal structures utilizing a combination of conventional and mass spectrometric peptide analysis. Results show all forms to be N-terminally acetylated and allow comparisons of now 40 such alcohol dehydrogenases covering a large span of forms and origins. Patterns illustrate roles of acetylation in proteins in general, define special importance of the class I N-terminal acetylation, and distinguish separate acetylated structures for all classes, as well as a common alcohol dehydrogenase motif.

Acetylation↗
PubMed1994

Alcohol dehydrogenase class III contrasted to class I. Characterization of the cyclostome enzyme, the existence of multiple forms as for the human enzyme, and distant cross-species hybridization.

Alcohol dehydrogenases of classes I (the classical liver enzyme) and III (formaldehyde dehydrogenase) constitute a pair of moderately related enzymes (63% residue identity between the human forms) that differ fundamentally in many respects. To elucidate the nature of the differences, we have characterized alcohol dehydrogenase from the most primitive vertebrate line (a cyclostome, Atlantic Hagfish), related that to the multiplicity of the human enzyme, and submitted the enzymes to in vitro hybridization for evaluation of subunit interactions. Three findings illustrate important principles of the enzyme system. First, the alcohol dehydrogenase purified from cyclostomes is a class-III protein, compatible with the facts that cyclostomes constitute the earliest extant vertebrate line and that class III has a distant pre-vertebrate origin. Second, the hagfish enzyme shows multiplicity, with acidic forms in decreasing yield and with amino acid sequences identical between two major isoforms, both aspects constituting properties similar to those of the corresponding human forms. The chemically different subunits are present as homodimers and heterodimers of unmodified and modified subunits, suggesting that the class-III multiplicity derives from modification of a type common to lines as divergent as mammals and cyclostomes. Third, the human enzyme can form cross-species hybrid dimers in vitro with the cod and hagfish or Drosophila class-III enzymes (positional identity with the human form of 82, 76 and 70%, respectively). Hence, the results provide experimental evidence for little class-III divergence in the segments of subunit interactions. The extent of conservation of residues directly involved in the formation of the subunit interface also reveals a clearly different pattern between classes I and III. This highlights separation of divergent forms in an enzyme system, with the constant form (class III) resembling house-keeping enzymes, and exhibiting a correlation between subunit-interacting and substrate-interacting segments.

Alcohol Dehydrogenase↗
PubMed1992

Reptilian alcohol dehydrogenase. Heterogeneity relevant to class multiplicity of the mammalian enzyme.

Liver alcohol dehydrogenase of the ethanol-active type ('class I enzyme') from the lizard, Uromastix hardwickii, was purified and screened for relationships with other vertebrate forms of the enzyme. Two different acetylated N-termini (acetyl-Gly and acetyl-Ser) and further positional differences already in the N-terminal segments establish the presence of two types of protein chain. The multiplicity is different from that hitherto detected within vertebrate class I alcohol dehydrogenase isozymes but typical of that which would be expected for subunits of different classes. In particular, relationships to class II or to class II-related forms appear likely. This may indicate yet further vertebrate alcohol dehydrogenase multiplicity or discovery of a class II non-mammalian enzyme. The results give prospects of defining gene duplications corresponding to more than one alcohol dehydrogenase class split to at an early vertebrate stage.

Alcohol Dehydrogenase↗
PubMed1991

Extensive multiplicity of the miscellaneous type of neurotoxins from the venom of the cobra Naja naja naja and structural characterization of major components.

A multiplicity of miscellaneous type neurotoxins were detected in the venom of the cobra Naja naja naja by use of reverse-phase HPLC and FPLC. The primary structures of major forms were determined, giving 4 novel structures. All four contain 62-65 residues, with 10 half-cystine residues and resemble the miscellaneous type of toxins from other Naja species. Differences within the species are extensive, exchanges occur at 27 positions, giving only 58% residue identity between all forms. However, the differences are largely limited to 3 regions corresponding to structurally important loops where two functional residues participating in receptor binding are exchanged. The four miscellaneous neurotoxins now characterized, together with the minor components of the miscellaneous type, the minimally four neurotoxins reported before, and other related toxins, indicate the existence of an extensive toxin gene multiplicity.

Amino Acid Sequence↗
PubMed1991

Characterization of phospholipase A2 from the venom of Horned viper (Cerastes cerastes).

Phospholipase A2 has been purified from the venom of Horned viper (Cerastes cerastes) by gel permeation chromatography followed by reverse-phase HPLC. The primary structure was established by sequence analysis of the intact protein and its enzymic peptides. The structure has 120 residues, properties like other group IIB phospholipases, but only 45-55% identity with the enzyme from other viperid species, and large variations even within the species (26% residue differences at known positions in another form).

Amino Acid Sequence↗
PubMed1990

Primary structure and functional properties of cobra (Naja naja naja) venom Kunitz-type trypsin inhibitor.

A trypsin inhibitor from the venom of the cobra Naja naja naja has been isolated by a single step of reverse-phase high-performance liquid chromatography. The protein strongly inhibits trypsin (Ki = 3.5 pM). The primary structure was determined by peptide analysis of the [14C]carboxymethylated inhibitor. The 57-residue polypeptide chain belongs to the family of Kunitz-type inhibitors, and exhibits 42% residue identity with bovine pancreatic trypsin inhibitor. The structure shows only 70% identity with the corresponding peptide from the Capa cobra (Naja nevia), establishing that the inhibitor molecule exhibits extensive variations. Functionally, a basic residue at position P3' correlates with strong inhibition.

Amino Acid Sequence↗
PubMed1990

Purification and characterization of a chymotrypsin Kunitz inhibitor type of polypeptide from the venom of cobra (Naja naja naja).

A chymotrypsin Kunitz inhibitor type of polypeptide has been isolated from the venom of Naja naja naja by reverse phase HPLC and cation exchange FPLC. It is present in a considerably lower amount than that of the corresponding trypsin inhibitor. The primary structure, determined by sequence analysis of the whole molecule and its tryptic peptides, has 57 residues with an apparent molecular mass of 6.2 kDa. The main contact site with the protease (P1) has a Phe, showing the specificity of the inhibitor. Of residues considered functionally important in Kunitz-type inhibitors, Gly-36 is replaced by Ser in a segment of weak contacts with the protease.

Amino Acid Sequence↗
PubMed1990

Characterization of a cytotoxin-like basic protein from the cobra (Naja naja naja) venom.

A cytotoxin-like basic protein has been isolated from the venom of the nominate race of cobra (Naja naja naja from Pakistan) by a single step of high-performance liquid chromatography. The primary structure was determined and consists of 62 amino acid residues in a single polypeptide chain. It is highly similar to that of the cytotoxin-like basic proteins isolated from other Naja species, but differs in two of the SS-loop structures from that of cytotoxins.

Amino Acid Sequence↗
PubMed1989

Characterization of two different peptides from the venom of the scorpion Buthus sindicus.

Two disulfide-rich, low-molecular mass peptides (approximately 3 kDa and approximately 4 kDa) have been isolated from Buthus sindicus venom using ion-exchange and reverse-phase HPLC. Peptide I has 35 residues with 8 half-cystine residues and is clearly related to four-disulfide core proteins of the neurophysin type and to toxins of other scorpion species (55-63% residue identity). Peptide II, present in low yield, has 28 residues with 6 half-cystine residues and a structure largely dissimilar from that of peptide I and other characterized toxins, although probably still a member of the disulfide core peptide type. Consequently, scorpion venom contains, in addition to toxins characterized before, toxin-like compounds with distant relationships.

Amino Acid Sequence↗
PubMed1989

Phospholipase A2 from cobra (Naja naja naja) venom. Primary structure and subspecies variation.

The primary structure of phospholipase A2 of the major race of Indian cobra has been determined. Together with previous data on other subforms, it establishes subspecies variations at no less than 20 of the 119 positions in the protein. These variations are large, not only in number but in several cases also regarding properties of the residues involved. Nevertheless, all structures are compatible with largely unaltered enzyme properties.

Amino Acid Sequence↗