Related Experiment Videos
First apyrase splice variants have different enzymatic properties
A Biederbick1, C Kosan, J Kunz
1Institut für Klinische Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Robert-Koch Strasse 5, D-35033 Marburg, Germany.
The Journal of Biological Chemistry
|June 20, 2000
Summary
This study characterizes the novel lysosomal apyrase-like protein 70 (LALP70) and its splice variant. Discovering differences in substrate specificity and cation dependence, this research enhances understanding of apyrase family enzymes.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- The apyrase protein family consists of enzymes that cleave nucleotide tri- and diphosphates.
- These enzymes are calcium- or magnesium-dependent and are not inhibited by common NTPase inhibitors.
- LALP70 is a newly identified lysosomal membrane protein within this family.
Purpose of the Study:
- To clone and sequence the human LALP70 gene and determine its genomic structure.
- To identify and characterize alternatively spliced variants of hLALP70.
- To compare the enzymatic properties, substrate specificity, and divalent cation dependence of hLALP70 and its splice variant.
Main Methods:
- Gene cloning and sequencing of human LALP70.
- Fluorescence in situ hybridization (FISH) for gene mapping.
- Reverse transcriptase polymerase chain reaction (RT-PCR) for expression analysis.
- Enzymatic assays to compare substrate specificity and cation dependence.
Main Results:
- The human LALP70 gene spans approximately 16 kilobase pairs, organized into 11 introns and 12 exons.
- The hLALP70 gene was mapped to chromosome 8p21.1-p21.3.
- A novel splice variant, hLALP70v, was identified, differing by an 8-amino acid motif (VSFASSQQ).
- Both hLALP70 and hLALP70v exhibit ubiquitous expression, with varying mRNA levels across tissues.
- hLALP70v shows broader substrate specificity (CTP, UDP, CDP, GTP, GDP) compared to hLALP70 (UTP, TTP).
- hLALP70v activity is equally dependent on Ca(2+) and Mg(2+) at 1 mM, while hLALP70 shows differential dependence and saturation kinetics.
Conclusions:
- The discovery of the hLALP70v splice variant is the first reported for the apyrase protein family.
- The 8-amino acid motif in hLALP70v significantly influences substrate specificity and divalent cation requirements.
- These findings provide insights into the functional diversity within the apyrase family and the role of alternative splicing.