ALPP Human, Active
Description
Introduction to ALPP Human, Active
ALPP (Alkaline Phosphatase, Placental) is a glycosylated, dimeric metalloenzyme encoded by the ALPP gene. It catalyzes the hydrolysis of phosphate monoesters, requiring one magnesium ion (Mg²⁺) and two zinc ions (Zn²⁺) for catalytic activity . ALPP is primarily expressed in the placenta during the second and third trimesters, as well as in endometrial tissue and cancerous cells (e.g., ovarian adenocarcinomas, germ cell tumors) . The "Active" designation refers to its enzymatically functional state, characterized by optimal tertiary structure and cofactor binding.
Molecular Structure
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Molecular Weight: 53.9–68 kDa (monomer), 140–160 kDa (homodimer) .
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Subunits: Homodimer with identical subunits (494–535 amino acids) .
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Glycosylation: Contains N-linked and O-linked glycans, critical for stability and membrane anchoring .
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GPI Anchor: A 29-amino acid C-terminal propeptide is cleaved to form a glycophosphatidylinositol (GPI) anchor, attaching the enzyme to cell membranes .
Catalytic Properties
| Property | Value | Source |
|---|---|---|
| pH Optimum | 8.0–10.0 | |
| Specific Activity | >2,500 units/mg (pNPP substrate) | |
| Substrate Specificity | Broad (phospholipids, proteins, DNA) | |
| Inhibitors | Levamisole, EDTA, L-phenylalanine |
Physiological Roles
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Phosphate Regulation: Dephosphorylates nucleotides, lipids, and proteins, influencing cellular signaling and metabolic pathways .
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Placental Function: Maintains fetal-maternal homeostasis during pregnancy .
Disease Associations
Diagnostic Utility
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Biomarker: Elevated serum ALPP levels indicate liver injury, bone disorders, or cancer .
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Therapeutic Target: Anti-ALPP antibodies are explored for cancer immunotherapy .
Experimental Uses
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Western Blotting: Detects target proteins via ALP-conjugated secondary antibodies (e.g., FLAG-BAP control) .
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DNA/RNA Processing: Dephosphorylates 5' termini to prevent self-ligation in cloning .
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Histochemical Staining: Used with substrates like Fast Red TR/Naphthol AS-MX for tissue analysis .
Recombinant ALPP
| Parameter | Value | Source |
|---|---|---|
| Production Host | Sf9 Baculovirus cells | |
| Purity | >95% (SDS-PAGE) | |
| Storage | -20°C (avoid freeze-thaw cycles) |
LPS-Induced Sepsis
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ALPP Transgenic Mice: Showed increased mortality after intraperitoneal LPS injection compared to wild-type controls .
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Mechanism: ALPP may modulate inflammatory cytokines (e.g., G-CSF) or impair phagocytosis .
Skin Graft Rejection
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Female ALPP Transgenic Mice: Delayed rejection of male-to-female skin grafts, suggesting T-cell suppression .
In Vitro Phagocytosis
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THP-1 Monocytic Cells: Exogenous ALPP reduced phagocytosis of E. coli bioparticles, indicating immunomodulatory effects .
Comparative Analysis with Other Alkaline Phosphatases
Product Specs
Placental alkaline phosphatase (PLAP) is a membrane-bound enzyme primarily found in the placenta during the third trimester of pregnancy. While initially thought to be exclusive to the placenta, a variant with significant similarity to PLAP has been identified. PLAP is predominantly expressed in the placenta, cervix, fallopian tubes, and specific tumors, including those in the ovaries, gastrointestinal tract, germ cells, and seminomas.
Human ALPP, produced in Sf9 Baculovirus cells, is a single, glycosylated polypeptide chain with a molecular weight of 53.9 kDa. It consists of 494 amino acids (23-506 a.a.) and includes a 10 amino acid His tag at the C-terminus. Purification is achieved through proprietary chromatographic methods.
The ALPP protein solution is provided at a concentration of 0.5 mg/ml and is prepared in a Phosphate-Buffered Saline (pH 7.4) solution containing 10% glycerol.
For short-term storage (up to 2-4 weeks), the product can be kept at 4°C. For longer storage, freezing at -20°C is recommended. Adding a carrier protein like HSA or BSA (0.1%) is advised for extended storage. To maintain product integrity, avoid repeated freezing and thawing.
SDS-PAGE analysis confirms a purity exceeding 95%.
The specific activity, exceeding 2,500 units/mg, is determined by measuring the enzyme's ability to hydrolyze p-nitrophenyl phosphate (pNPP) at a rate of 1.0 nmole per minute at pH 7.5 and a temperature of 37°C.
3 ALPP, Alkaline phosphatase Regan isozyme, Placental alkaline phosphatase 1, PLAP-1, ALP, PLAP, Alkaline phosphatase placental type, EC 3.1.3.1, PLAP-1, Alkaline phosphatase Regan isozyme.
ADLMIIPVEE ENPDFWNREA AEALGAAKKL QPAQTAAKNL IIFLGDGMGV STVTAARILK GQKKDKLGPE LPLAMDRFPY VALSKTYNVD KHVPDSGATA TAYLCGVKGN FQTIGLSAAA RFNQCNTTRG NEVISVMNRA KKAGKSVGVV TTTRVQHASP AGTYAHTVNR NWYSDADVPA SARQEGCQDI ATQLISNMDI DVILGGGRKY MFRMGTPDPE YPDDYSQGGT RLDGKNLVQE WLAKRQGARY VWNRTELMQA SLDPSVTHLM GLFEPGDMKY EIHRDSTLDP SLMEMTEAAL RLLSRNPRGF FLFVEGGRID HGHHESRAYR ALTETIMFDD AIERAGQLTS EEDTLSLVTA DHSHVFSFGG YPLRGSSIFG LAPGKARDRK AYTVLLYGNG PGYVLKDGAR PDVTESESGS PEYRQQSAVP LDEETHAGED VAVFARGPQA HLVHGVQEQT FIAHVMAFAA CLEPYTACDL APPAGTTDHH HHHH.
Q&A
What is ALPP and what are its key structural characteristics?
ALPP (Alkaline phosphatase placental type; also known as SEAP, PALP1, and ALP) is a 66-68 kDa glycoprotein belonging to the alkaline phosphatase family. Human ALPP is synthesized as a 535 amino acid (aa) preproprecursor containing a 22 aa signal sequence, a 484 aa mature region (aa 23-506), and a 29 aa C-terminal propeptide that undergoes cleavage to generate a GPI linkage at Asp506 . The enzyme utilizes one Mg and two Zn ions for its catalytic activity and functions either as a homodimer or as a heterodimer with related family members, including Intestinal (ALPI) and Germ cell (GCAP) . ALPP shares significant sequence homology with other isozymes, including 86% amino acid identity with human ALPI across residues 23-506 .
What is the enzymatic function of ALPP and how is it measured in research settings?
ALPP functions by cleaving phosphate monoesters into alcohol and phosphate . In research settings, ALPP enzymatic activity is commonly measured using fluorogenic substrates such as 4-Methylumbelliferyl phosphate (4-MUP) . The specific activity of recombinant ALPP has been reported at >7000 pmol/min/μg in quality-controlled tests . For protein detection, Western blot analysis can identify ALPP in human placenta tissue and cell lines like JEG-3 (human epithelial choriocarcinoma), where it appears as a band of approximately 68 kDa under reducing conditions . Simple Western analysis allows detection of ALPP at approximately 76 kDa using more sensitive instrumentation with lower sample concentrations (0.2 mg/mL) .
In which tissues and cell types is ALPP normally expressed?
ALPP is principally expressed by second and third trimester syncytiotrophoblasts in the placenta and represents one of four alkaline phosphatase isozymes in the human body . Beyond normal tissue expression, ALPP is highly expressed in various solid tumors, including ovarian, endometrial, germ cell, non-small cell lung, bladder, and gastric cancers . This tumor-associated expression pattern has made ALPP an attractive target for cancer research and potential therapeutic interventions .
What methodological considerations are important when studying ALPP expression and activity in tumor samples?
When investigating ALPP in tumor samples, researchers should implement several critical methodological considerations:
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Antibody specificity validation: Due to high sequence homology with other alkaline phosphatase isozymes, thorough antibody validation is essential. Antigen affinity-purified polyclonal antibodies have shown good specificity for ALPP/ALPI detection in placental tissue and choriocarcinoma cell lines .
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Optimal sample preparation: For Western blot analysis, reducing conditions with appropriate buffer systems (e.g., Immunoblot Buffer Group 1) have been successfully employed . PVDF membranes probed with 1 μg/mL of specific antibodies followed by HRP-conjugated secondary antibodies provide reliable detection .
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Activity assay calibration: When measuring enzymatic activity, establishing a standard curve with purified recombinant ALPP is recommended for accurate quantification. Recombinant proteins with verified specific activity (>7000 pmol/min/μg) serve as excellent positive controls .
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Storage and handling protocols: ALPP antibodies and proteins require specific storage conditions (-20 to -70°C) with minimal freeze-thaw cycles. For reconstituted antibodies, stability is maintained for approximately 1 month at 2-8°C or 6 months at -20 to -70°C under sterile conditions .
How can researchers differentiate between ALPP and other alkaline phosphatase isozymes in experimental settings?
Differentiating between alkaline phosphatase isozymes presents significant challenges due to their high sequence homology. Researchers can employ the following strategies:
What is the current state of ALPP-targeted cancer immunotherapy research?
ALPP-targeted cancer immunotherapy is actively being investigated, with several approaches under development:
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CAR-T cell therapy: Clinical research includes Phase 2 trials of retroviral vector-transduced autologous T cells engineered to express anti-ALPP chimeric antigen receptors (CARs) .
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Target indications: Current clinical investigations focus on ovarian neoplasms, various solid tumors, and endometrial neoplasms, consistent with the high ALPP expression observed in these malignancies .
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Research challenges: Key methodological challenges include ensuring CAR-T specificity for ALPP versus other alkaline phosphatase isozymes, optimizing delivery to solid tumors, developing reliable monitoring protocols, and addressing potential resistance mechanisms.
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Preclinical development considerations: When designing ALPP-targeted therapies, researchers must account for the 86-98% sequence homology with other isozymes to minimize off-target effects while maintaining therapeutic efficacy .
How do post-translational modifications affect ALPP function and experimental detection?
ALPP undergoes several post-translational modifications with significant implications for function and detection:
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Glycosylation: ALPP is heavily glycosylated, causing it to migrate at 64-66 kDa on SDS-PAGE despite having a calculated molecular weight of 54.7 kDa . This modification affects antibody recognition and must be considered when interpreting Western blot results.
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GPI anchoring: The C-terminal propeptide cleavage generates a GPI anchor at Asp506, localizing ALPP to the cell membrane . This anchoring influences ALPP's accessibility in intact cell assays.
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Oligomerization: ALPP can form homodimers or heterodimers with related family members, affecting enzyme kinetics and detection strategies .
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Metal ion coordination: The enzyme requires one Mg and two Zn ions for catalytic activity, necessitating appropriate buffer conditions for in vitro activity assays .
What experimental approaches can researchers use to study ALPP gene regulation and alternative splicing?
The ALPP gene is highly allelic and generates multiple splice variants, particularly involving the signal sequence region . Researchers can investigate these aspects using:
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RNA-Seq and transcriptome analysis: These approaches identify and quantify different splice variants in various tissues and under different conditions.
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ASpdb database utilization: This comprehensive resource integrates experimentally determined structures and AlphaFold 2-predicted models for human protein isoforms, enabling comparative analyses of ALPP variants .
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Reporter assays: Luciferase or fluorescent protein fusions can monitor promoter activity and splicing efficiency under various experimental conditions.
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CRISPR-based approaches: Genome editing can introduce mutations at splice sites or regulatory regions to study their effects on ALPP expression and processing.
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Single-cell analysis: This technique examines cell-specific expression patterns of different ALPP isoforms within heterogeneous populations.
What are the stability considerations for working with recombinant ALPP in laboratory settings?
When working with recombinant ALPP proteins, researchers should implement specific handling protocols:
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Storage conditions: Lyophilized ALPP should be stored at -20°C or lower for long-term stability .
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Reconstitution protocols: Proteins are typically reconstituted in buffered solutions such as 20 mM Tris, 150 mM NaCl, pH 7.5 with trehalose as a protectant .
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Freeze-thaw management: Repeated freeze-thaw cycles significantly reduce protein activity and should be strictly avoided .
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Working solution preparation: For functional assays, maintaining metal ion concentrations (Mg and Zn) is critical for preserving enzymatic activity .
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Quality control verification: Before experimental use, SDS-PAGE analysis under reducing conditions can confirm protein integrity, with expected migration at 64-66 kDa for glycosylated ALPP .
How can the ASpdb database enhance structural analysis of ALPP isoforms?
The ASpdb database (https://biodataai.uth.edu/ASpdb/) offers powerful tools for ALPP structural research:
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Comprehensive isoform coverage: The database includes over 3,400 canonical isoforms with both experimental and predicted structures, plus more than 7,200 alternative isoforms with AlphaFold 2 predictions .
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Integrated analysis features: Researchers can access detailed splicing events, 3D structures, sequence variations, and functional annotations in a unified platform .
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Comparative visualization: The unique visualization tools allow direct comparison of structural alterations among ALPP isoforms .
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Structure-function correlation: By examining how alternative splicing affects protein structure, researchers can generate hypotheses about functional differences between ALPP variants .
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Disease mechanism insights: The resource connects structural variations to potential disease mechanisms, supporting translational research on ALPP-related pathologies .
Product Science Overview
ALPP is a single, glycosylated polypeptide chain consisting of 494 amino acids, with a molecular mass of approximately 53.9 kDa . The enzyme is expressed with a 10 amino acid His tag at the C-terminus, which aids in its purification through chromatographic techniques . The recombinant form of ALPP is produced in Sf9 Baculovirus cells, ensuring high purity and activity .
The specific activity of ALPP is greater than 2,500 units per milligram, defined as the amount of enzyme that hydrolyzes 1.0 nmole of p-nitrophenyl phosphate (pNPP) per minute at pH 7.5 and 37°C . This high level of activity makes it a valuable tool in various biochemical assays and research applications.
ALPP is provided as a sterile, filtered, colorless solution formulated in phosphate-buffered saline (pH 7.4) with 10% glycerol . For short-term storage, it can be kept at 4°C if used within 2-4 weeks. For long-term storage, it is recommended to store the enzyme at -20°C with the addition of a carrier protein, such as 0.1% human serum albumin (HSA) or bovine serum albumin (BSA), to prevent multiple freeze-thaw cycles .
ALPP is widely used in research due to its role in dephosphorylation processes. It is particularly useful in studies involving placental biology, cancer research, and enzyme kinetics. The enzyme is also employed in diagnostic assays to detect placental and germ cell tumors, as its expression is elevated in these conditions .
