Kwok-Wai Lam

Heterogeneity of hairy cell tartrate-resistant acid phosphatase

The human nonerythrocytic acid phosphatases (AcP) are composed of seven distinct activity bands in nondenaturing polyacrylamide gel electrophoresis (PAGE) when stained using either 1-naphthyl phosphate or naphthol ASBI phosphate as substrate. They are numbered 0, 1, 2, 3, 3b, 4, and 5 according to their increasing mobility toward the cathode in acidic conditions. Of these, only the most cationic band 5 is tartrate resistant (TRAcP). When naphthol ASBI phosphate is used as substrate, AcP activity can also be stained in situ. In the presence of tartrate, activity remains strong in the hairy cells (HC) of hairy cell leukemia (HCL). Thus, the TRAcP stain has remained a reliable marker for HC. To investigate the function of TRAcP in HC, we purified two isoforms of TRAcP from HCL spleen tissue and found them to have similar substrate specificities and inhibitor sensitivities. In this report, we describe in detail the methods for TRAcP purification and compare some of the structural properties of the two isoforms to reinforce the concept that human TRAcP is a heterogeneous group of related enzymes. Band 5 represented only 15-20% of the total TRAcP extracted from HCL spleen. The remaining 80% of TRAcP hydrolyzed p-nitrophenyl phosphate but not naphthol ASBI phosphate and was not detectable in acidic, nondenaturing PAGE gels. Band 5 was solubilized from tissue using 500 mmol/L NaCl after previous extraction with 0.5% (v/v) NP-40 removed most other AcP and TRAcP activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoultrastructural Demonstration of Prostatic Acid Phosphatase Isoenzyme 2 in Prostatic Carcinoma

Human prostatic acid phosphatase isoenzyme 2 (HPAcP-2) was isolated from semen. This purified enzyme was immunized to rabbit to produce polyclonal antibodies. The specificity of the antibodies was tested by Western blot transfer method. Rabbit IgG-peroxidase conjugate was prepared from the antiserum and used to localize HPAcP-2 in prostatic carcinoma. It was found that in the tumor glandular acinus the normal basal cells were replaced by tumor cells containing reaction product. In the tumor cells, the reaction product was seen in the cisternae of rough endoplasmic reticulum (ER) and Golgi apparatus. The secretory vesicles which contained reaction product-stained granules and some amorphous material were seen to fuse with the apical plasma membrane and discharged their content into the glandular lumen. On the other hand, some secretory vesicles in the tumor cells facing to the basement membrane also discharged their similar content into the extracellular spaces. Reaction product-stained granules were found in the interstitial spaces surrounding the tumor cells. These findings suggest that HPAcP-2 is synthesized on the bound ribosomes and discharged into the cisternae of rough ER. The molecules are transported to the Golgi cisternae. After concentration and packaging, HPAcP-2 molecules are then transferred to the secretory vesicles, and discharged into the glandular lumen and to the extracellular spaces. The isoenzyme released in the extracellular space may reach the blood stream through the interstitial spaces or the lymphatic system, resulting in the elevation of serum HPAcPase level in some prostatic cancer patients.

Comparison of tartrate resistant acid phosphatase in a giant cell bone tumor and a spleen infiltrated with hairy cells

Acid phosphatase (E.C.3.1.3.2) in a giant cell bone tumor and a spleen infiltrated with hairy cells was extracted by citrate buffer and then by 0.3 mol/L NaCl. The cationic acid phosphatase in the crude extract was isolated by CM-cellulose chromatography, and further separated by high pressure liquid chromatography. The majority of the tartrate resistant acid phosphatase in the hairy cell spleen was unabsorbed on CM-cellulose and was insensitive to iron. A much larger portion of the acid phosphatase in the bone tumor, than in the spleen, was cationic and was eluted from the column by 0.8 mol/L NaCl. The cationic acid phosphatase was further separated into consecutive peaks of acid phosphatases with different sensitivity to iron. A major portion of acid phosphatase in the giant cell bone tumor was enhanced by iron, while the amounts of iron-enhanced and iron-insensitive acid phosphatase were about the same in the spleen. The differences of the phosphatases in these two types of pathologic specimens indicate the occurrence of two types of enzymes with different biological significance.

Immunohistochemical detection of monocytes by the antiserum specific to monocytic esterase.

An antiserum specific to esterase Ib was produced in a rabbit. The antigen-antibody reaction was visualized by the strong esterase activity in the precipitin band in Ouchterlony double diffusion and immunoelectrophoresis. Immunohistochemical procedure demonstrated strong staining in the monocyte-infiltrated splenic sections and tissue sections of true histiocytic lymphoma. Negative results were observed in T- and B-cell lymphomas, granulocytic sarcoma, and chronic granulocytic leukemia. This antibody may be useful for the identification of monocytes and histocytes in paraffin-embedded tissue sections.

Monoclonal Antibody Specific to Acid Phosphatase Isoenzyme 4

Prostatic acid phosphatase isoenzyme 4 was purified by ion exchange column chromatography, followed by high pressure liquid chromatography. The highly purified enzyme was used to produce monoclonal antibody from immunized BALB/c mice. The antibody was specific to isoenzyme 4, with negligible affinity to isoenzyme 2. The specificity of the monoclonal antibody was evaluated by Western blot analysis and by inhibition of radioimmunoassay. Immunohistochemistry method using the antibody to isoenzyme 2 showed heavy staining on the cell surface in contrast to the even staining throughout the cytoplasm when monoclonal anti-isoenzyme 4 was used. These results reflect the secretory nature of isoenzyme 2 and the non-secretory nature of isoenzyme 4.