Biomaterial Database

Molecular cloning, splice variants, expression, and purification of phospholipase C-delta 4. 1996

S B Lee, and S G Rhee

Laboratory of Cell Signaling, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.

Complementary DNAs encoding a previously unidentified phosphoinositide-specific phospholipase C (PLC) isozyme were cloned from a rat brain cDNA library by the polymerase chain reaction with degenerate oligonucleotide primers based on sequences common to three known delta-type PLC isozymes. The encoded polypeptide contains 772 amino acids (calculated molecular mass, 88,966 daltons) and is similar in primary structure to delta-type PLC isozymes, with overall sequence identities of 45% to PLC-delta 1, 72% to PLC-delta 2, and 47% to PLC-delta 3. Thus, the new PLC isozyme was named PLC-delta 4. Recombinant PLC-delta 4 was purified from extracts of HeLa cells that had been infected with vaccinia virus containing the corresponding cDNA. The purified protein exhibited an apparent molecular mass of 90 kDa on SDS-polyacrylamide gels. The specific activity of PLC-delta 4 and its dependence on Ca2+ were similar to those of PLC-delta 1. The distribution of PLC-delta 4 in 16 different rat tissues was studied by immunoblot analysis with PLC-delta 4-specific antibodies of fractions obtained after an enzyme-enrichment procedure. The 90-kDa immunoreactive protein was detected unambiguously in only eight tissues and was present at concentrations that were low compared to those of other major PLC isozymes. A 93-kDa immunoreactive protein was also prominent in testis but was not detected in the other seven positive tissues. The 93-kDa enzyme appears to be derived from a splice variant of the mRNA that encodes the 90-kDa PLC-delta 4 and contains an additional 32 amino acids between the X and Y catalytic domains. Splice variants have not previously been detected for delta-type PLC isozymes.

UI	MeSH Term	Description	Entries
D007527	Isoenzymes	Structurally related forms of an enzyme. Each isoenzyme has the same mechanism and classification, but differs in its chemical, physical, or immunological characteristics.	Alloenzyme,Allozyme,Isoenzyme,Isozyme,Isozymes,Alloenzymes,Allozymes
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010738	Type C Phospholipases	A subclass of phospholipases that hydrolyze the phosphoester bond found in the third position of GLYCEROPHOSPHOLIPIDS. Although the singular term phospholipase C specifically refers to an enzyme that catalyzes the hydrolysis of PHOSPHATIDYLCHOLINE (EC 3.1.4.3), it is commonly used in the literature to refer to broad variety of enzymes that specifically catalyze the hydrolysis of PHOSPHATIDYLINOSITOLS.	Lecithinase C,Phospholipase C,Phospholipases, Type C,Phospholipases C
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D006367	HeLa Cells	The first continuously cultured human malignant CELL LINE, derived from the cervical carcinoma of Henrietta Lacks. These cells are used for, among other things, VIRUS CULTIVATION and PRECLINICAL DRUG EVALUATION assays.	Cell, HeLa,Cells, HeLa,HeLa Cell
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D001483	Base Sequence	The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.	DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D012333	RNA, Messenger	RNA sequences that serve as templates for protein synthesis. Bacterial mRNAs are generally primary transcripts in that they do not require post-transcriptional processing. Eukaryotic mRNA is synthesized in the nucleus and must be exported to the cytoplasm for translation. Most eukaryotic mRNAs have a sequence of polyadenylic acid at the 3' end, referred to as the poly(A) tail. The function of this tail is not known for certain, but it may play a role in the export of mature mRNA from the nucleus as well as in helping stabilize some mRNA molecules by retarding their degradation in the cytoplasm.	Messenger RNA,Messenger RNA, Polyadenylated,Poly(A) Tail,Poly(A)+ RNA,Poly(A)+ mRNA,RNA, Messenger, Polyadenylated,RNA, Polyadenylated,mRNA,mRNA, Non-Polyadenylated,mRNA, Polyadenylated,Non-Polyadenylated mRNA,Poly(A) RNA,Polyadenylated mRNA,Non Polyadenylated mRNA,Polyadenylated Messenger RNA,Polyadenylated RNA,RNA, Polyadenylated Messenger,mRNA, Non Polyadenylated