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S S Karnik

Publications and source records attributed to S S Karnik.

31 records · Page 2Linked to original sources
PubMed1994

The high affinity state of the beta 2-adrenergic receptor requires unique interaction between conserved and non-conserved extracellular loop cysteines.

A disulfide bond between two extracellular cysteines, conserved in all G-protein-coupled receptors, is believed to be critical for stabilization of the ligand-binding pocket. The beta 2-adrenergic receptor (beta 2-AR) contains two conserved cysteines (Cys106 and Cys184) as well as two other extracellular cysteines (Cys190 and Cys191). The specificity of the interactions between these four cysteines has not yet been clearly established. Mutants encoding alanines for specific extracellular cysteines in the beta 2-AR gene were constructed and expressed in COS-1 and Chinese hamster ovary cells. Ala106, Ala184,190,191, and Ala106,184,190,191 mutants displayed low affinity for the beta-antagonist, 125I-cyanopindolol and insensitivity to dithiothreitol (DTT). The Ala106,191 mutant displayed an intermediate affinity and DTT sensitivity. Mutants Ala184, Ala184,190, and Ala184,191 displayed high affinity and DTT sensitivity, indicating that a solvent-accessible disulfide bond(s) is present in these mutant receptors as in the wild-type beta 2-AR. Additionally, thermal stability studies provided evidence that the extracellular disulfide bonds are essential for stabilization of the high affinity state of the receptor. These studies indicate that the covalent linkage between loops 1 and 2 of the beta 2-AR extracellular domains involves the formation of disulfide bonds, uniquely between Cys106 and Cys191, and Cys184 and Cys190, and is, thus, distinct from that of other G-protein-coupled receptors.

Amino Acid Sequence↗
PubMed1993

Dipeptide processing activates recombinant human prochymase.

Human chymase (h-chymase) is a serine protease that efficiently converts angiotensin I to II. Its structure and homology to other serine proteases suggest that it is synthesized as a zymogen, and is processed to the active form by cleavage of a 19-residue signal peptide and of a dipeptide pro-segment. To evaluate maturational processing of this enzyme, the proteins encoded by three h-chymase cDNA constructs (wild-type, lacking the pro- or lacking the prepro-segment) were characterized after expression in COS-1 cells. These recombinant proteins were not catalytically active. Purification and NH2-terminal sequence analysis of the protein expressed from the wild-type construct revealed processing to the proenzyme. Prochymase activation was achieved by incubation with a B-cell lymphoma homogenate, which apparently contains a heterologous processing enzyme sensitive to thiol protease inhibitors. NH2-terminal sequence analysis of the activated h-chymase revealed cleavage of the pro-segment, and its biochemical characteristics were identical to those of native h-chymase purified from the myocardium. These findings indicate that processing of the dipeptide pro-segment is necessary and sufficient for activation of human chymase. Such processing is probably also required for the activation of related serine proteases, e.g., cathepsin G, which have homologous dipeptide pro-segments.

Amino Acid Sequence↗
PubMed1993

Palmitoylation of bovine opsin and its cysteine mutants in COS cells.

Previously, bovine rhodopsin has been shown to be palmitoylated at cysteine residues 322 and 323. Here we report on palmitoylation of bovine opsin in COS-1 cells following expression of the synthetic wild-type opsin gene and several of its cysteine mutants in the presence of [3H]palmitic acid. Two moles of palmitic acid are introduced per wild-type opsin molecule in thioester linkages. Palmitoylation is abolished when both Cys-322 and Cys-323 are replaced by serine residues. Replacement of Cys-322 by serine prevents palmitoylation at Cys-323, whereas replacement of the latter with serine allows palmitoylation at Cys-322. Opsin mutants that evidently do not contain a Cys-110/Cys-187 disulfide bond and presumably remain in the endoplasmic reticulum are not palmitoylated. Replacement of Cys-140 or Cys-185 reduces the extent of palmitoylation of the opsin. Lack of palmitoylation at Cys-322 and/or Cys-323 does not affect 11-cis-retinal binding, absorption maximum or extinction coefficient of the chromophore, the bleaching behavior of the chromophore, or the light-dependent binding and activation of transducin. Mutants containing serine substitutions at Cys-140 or Cys-323 showed reduced light-dependent phosphorylation by rhodopsin kinase.

Amino Acid Sequence↗
PubMed1990

Assembly of functional rhodopsin requires a disulfide bond between cysteine residues 110 and 187.

Cysteine residues 110 and 187 are essential for the formation of the correct bovine rhodopsin structure (Karnik, S. S., Sakmar, T. P., Chen, H.-B., and Khorana, H. G. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8459-8463). We now show that the sulfhydryl groups of these 2 cysteine residues interact to form a disulfide bond. Rhodopsin mutants containing cysteine----serine substitutions were prepared as follows. In one mutant, CysVII, all the 10 cysteine residues of rhodopsin were replaced by serines. A second mutant, CysVIII, contained only C110 and C185; a third mutant, CysIX, contained only C185 and C187 while the fourth mutant, CysX, contained only C110 and C187. Only mutant CysX formed functional rhodopsin. Mutants CysVIII and CysIX reacted with [3H]iodoacetic acid showing the presence of free sulfhydryl groups while mutant CysX was inert to this reagent. CysX reacted with cyanide ion to form a thiocyanate derivative showing the presence of a disulfide bond. The C110-C187 disulfide bond is buried in rhodopsin because reactions with disulfide reducing agents and cyanide ion require prior treatment with denaturants.

Amino Acid Sequence↗
PubMed1988

Cysteine residues 110 and 187 are essential for the formation of correct structure in bovine rhodopsin.

To investigate the role of different cysteine residues in bovine rhodopsin, a series of mutants were prepared in which the cysteine residues were systematically replaced by serines. The mutant genes were expressed in monkey kidney cells (COS-1) and the mutant opsins were evaluated for their levels of expression, glycosylation patterns, and ability to form the chromophore characteristic of rhodopsin and to activate transducin. Substitution of the three cytoplasmic cysteines (Cys-316, Cys-322, and Cys-323) and the four membrane-embedded cysteines (Cys-140, Cys-167, Cys-222, and Cys-264) produced proteins with wild-type phenotype. Also, single substitutions of Cys-185 gave rise to a wild-type phenotype. In contrast, substitution of the three intradiscal cysteines (Cys-110, Cys-185, and Cys-187) or single substitution of Cys-110 or Cys-187 gave proteins that were expressed at reduced levels, glycosylated abnormally, and unable to bind 11-cis-retinal. Thus, of the 10 cysteines in bovine rhodopsin, only intradiscal Cys-110 and Cys-187 are essential for the correct tertiary structure of the protein.

Amino Acid Sequence↗
PubMed1987

Structure-function studies on bacteriorhodopsin. III. Total synthesis of a gene for bacterio-opsin and its expression in Escherichia coli.

We have chemically synthesized a DNA duplex of 757 base pairs which encodes the entire protein sequence of mature bacterio-opsin of Halobacterium halobium. The main aim of the synthesis was to facilitate site-specific mutagenesis in all parts of the gene by replacement of short restriction fragments by their counterparts containing the required nucleotide changes. Therefore, 30 unique restriction sites that are fairly evenly spaced were introduced in the synthetic DNA. A total of 28 oligonucleotides ranging in size from 21 to 69 nucleotides were synthesized corresponding to both strands. The entire gene was assembled from four synthetic fragments of 25, 268, 219, and 245 base pairs. The correctness of the nucleotide sequence was confirmed by sequencing the fragments as well as the complete gene. When expressed under the control of PL promoter in Escherichia coli, the synthetic and the native genes gave similar amounts of bacterio-opsin. Attempts to increase expression of the synthetic gene by introducing codons that are preferred in E. coli or by introduction of a synthetic transcription terminator were without significant effect.

Bacteriorhodopsins↗
PubMed1987

Structure-function studies on bacteriorhodopsin. II. Improved expression of the bacterio-opsin gene in Escherichia coli.

The aims of this work have been to express bacterio-opsin with minimal variation from the native primary structure and to improve the level of expression in Escherichia coli. We describe the construction of plasmids in which the bacterio-opsin gene contains only an additional methionine residue at the N terminus and in which the C-terminal aspartic acid encoded in the gene has been deleted to conform to the mature protein. In attempts to improve bacterio-opsin expression, a variety of expression plasmids were constructed in which the promoters and the ribosome-binding sequences were varied. Invariably, in these plasmids, translation but not transcription of the bacterio-opsin gene was limiting. A striking increase in expression of the gene occurred when the codons for several of the N-terminal amino acids were changed to increase the A = T content. Bacterio-opsin expressed in E. coli was degraded with a half-life of 8-10 min. The addition of hydrophobic signal sequences at the N terminus increased the half-life and overall yield of the protein. Bacterio-opsin thus produced regenerated the native bacteriorhodopsin-like chromophore and carried out light-dependent proton translocation at a rate comparable to that of the native bacterio-opsin prepared from the purple membrane.

Amino Acid Sequence↗
PubMed1986

Total synthesis of a gene for bovine rhodopsin.

To carry out systematic structure-function studies of bovine rhodopsin by specific amino acid replacements, we have accomplished the total synthesis of its gene, which is 1057 base pairs long. The synthetic gene contains 28 unique restriction sites that are on the average 60 base pairs apart. Replacement of specific restriction fragments by synthetic counterparts containing the desired nucleotide changes permits specific mutagenesis in all parts of the gene. The synthesis of the gene involved enzymatic joining of a total of 72 synthetic oligonucleotides, 15-40 nucleotides long, to form DNA duplexes. The total gene was assembled from three synthetic fragments that were cloned. All synthetic oligonucleotides were characterized by 5'-end analysis, and the accuracy of the joining reactions was confirmed by sequencing the three fragments as well as the complete gene.

Animals↗
PubMed1983

Transfection of Mycobacterium smegmatis SN2 with mycobacteriophage I3 DNA.

Mycobacterium smegmatis SN2 does not exhibit natural competence for the uptake of phage I3 DNA. Competence can artificially be induced by treatment with glycine or CaCl2, and the combination of both is even more effective. The efficiency of transfection can be improved by inclusion of protamine sulphate and heterologous RNA in the system. From 32P DNA uptake studies the major barrier for the entry of DNA has been found to be the complex cell wall. The efficiency of transfection calculated on the basis of fraction of DNA which has entered the cell is comparable to that of other bacterial systems. The phage development takes a longer time (7 h for one cycle) after transfection, as compared to infection (4 h).

Calcium Chloride↗
PubMed1981

Presence of nucleoside triphosphates and calcium associated with mycobacteriophage 13.

The association of nucleoside triphosphate molecules and calcium ions with purified particles of mycobacteriophage 13 has been documented. The content of nucleoside triphosphate has been determined to be 118 molecules per phage particle by equilibrium dialysis against labelled ATP or 148 molecules per phage particle by the direct determination of labelled nucleoside triphosphate. The concentration of bound Ca2+ exhibited a high degree of variation between different batches, which may be due to the nonspecific binding of Ca2+ by the virus particles. However, the tightly bound Ca2+ not removable by dialysis against calcium-specific chelating agent, showed a constant value of 2985 atoms/phage particle.

Adenosine Diphosphate↗
PubMed1980

Possible involvement of a calcium-stimulated ATP-hydrolyzing activity associated with mycobacteriophage I3 in the DNA injection process.

Ca2+ ions are necessary for the successful propagation of mycobacteriophage I3. An assay for the phage DNA release in the presence of an isolated cell wall preparation from the host was established, and in this system Ca2+ ions also stimulated the release of DNA. The inhibition of phage DNA injection caused by Tween 80 (polyoxyethylene sorbitan monooleate), a nonionic detergent routinely used in mycobacterial cultures, was reversed by Ca2+. The presence of a phage-associated ATP-hydrolyzing activity was demonstrated. This enzyme was stimulated by Ca2+ ions and inhibited by Tween 80. From this and the behavior of the two agents at the level of DNA injection, as well as the fact that phage I3 has a contractile tail structure, we conclude that the phage-associated ATPase is involved in the DNA injection process.

Calcium↗