Immunoassay and activity of calcium-activated neutral proteinase (mCANP): distribution in soluble and membrane-associated fractions in human and mouse brain.

The millimolar form of calcium-activated neutral proteinase (mCANP) is generally regarded as a cytosolic enzyme in nonneuronal systems, although its subcellular localization in brain is less well established. To resolve conflicting reports on the localization of mCANP based on activity measurements, we developed an immunoassay for CANP and compared the content and activity of the molecule in soluble and membrane fractions of mouse and human brain. Western blot immunoassays, using two different antibodies specific for mCANP, demonstrated that mCANP content is 4.5 ng/g in human or mouse brain, about 0.0005% of the total protein. More than 95% of the total immunoreactive mCANP remained in the soluble fraction after 15,000 g centrifugation of the whole homogenate. mCANP activity was determined with [14C]azocasein as substrate after removing endogenous CANP inhibitor(s) by ion-exchange chromatography on DEAE-cellulose. Caseinolytic activity was detected only in fractions derived from the supernatant extract. The distribution of mCANP content and enzyme activity were unchanged when tissues were extracted with different concentrations of Triton X-100. These findings establish the usefulness and validity of the CANP immunoassay and demonstrate that mCANP in mouse and human brain is localized predominantly within the cytosol.

Biochemical and immunological identification of human neutrophil elastase on nitrocellulose membranes.

Human neutrophil elastase (HNE) was analyzed for protein(s), antibody staining and activity staining, on lithium dodecyl sulfate (LDS) polyacrylamide gel electrophoresis followed by Western blotting. The HNE activity, which was identified with N-acetyl-D,L-alanine alpha-naphthyl ester as substrate, was well preserved in the presence of 0.1% LDS at 4 C during electrophoresis. As little as 0.1 microgram HNE was required for the activity staining. The HNE appeared to be three peptides having a major band at mass ratio 27,000, a second major band at mass ratio 28,000 with a minor protein band at mass ratio 29,000. On transfer to nitrocellulose, the mass ratio 28,000 band displayed poor immunoreactivity. This was the second most dense band with highest enzymatic staining. This procedure is a useful method and analytical tool to determine the correlation of enzymatically active proteins, subunits and immunoreactive protein(s) of elastase from various sources, including neutrophils.

The RIIIS/J inbred mouse strain as a model for von Willebrand disease.

Mice of the RIIIS/J inbred strain have prolonged bleeding times (greater than 15 minutes) after experimental injury when compared with normal C57BL/6J mice (1.8 minutes) and other strains of mice. The prolonged bleeding time was accompanied by normal platelet counts. Platelet aggregation with collagen and agglutination with ristocetin were not significantly altered in RIIIS/J mice. Also, platelets from RIIIS/J mice had normal serotonin content and normal numbers of dense granules by electron microscopy. Thus, the bleeding abnormality is not due to platelet storage pool deficiency as has been found in several other mouse mutants. The activated partial thromboplastin time (APTT) which plasma from RIIIS/J mice was prolonged compared with normal mice, and factor VIII:C activity and von Willebrand antigen levels were one half to one third that of normal mouse plasma. Factor XI activity was also significantly deficient (levels at 42% to 64% of normal). Plasma of RIIIS/J mice contained the full complement of multimers of von Willebrand factor, although each multimer was lower in concentration compared with that in normal mice. Platelet alpha-granule von Willebrand antigen levels were similar to those of normal mice. The prolonged bleeding time of RIIIS/J mice was corrected by treatment with desmopressin. Heterozygous C57BL/6J x RIIIS/J F1 animals had low plasma von Willebrand antigen levels like the RIIIS/J parent and had variable bleeding times. Inheritance of the bleeding tendency was as an incomplete dominant, autosomal trait. These data indicate the RIIIS/J strain is a suitable animal model for type IA von Willebrand disease.

The propeptide of beta-glucuronidase. Further evidence of its involvement in compartmentalization of beta-glucuronidase and sequence similarity with portions of the reactive site region of the serpin superfamily.

A significant portion of murine hepatocyte beta-glucuronidase is maintained within the endoplasmic reticulum (ER) by complex formation with the esterase active site of the protein egasyn. The carboxyl-terminal propeptide of the precursor form of glucuronidase appears important in localization of glucuronidase to the ER since a naturally occurring mutation in it is associated with decreased levels of ER glucuronidase. A sequence similarity was noted between the carboxyl-terminal propeptide and portions of the conserved sequences of the reactive site region of members of the serpin (serine proteinase inhibitor) superfamily. Also, previous studies had shown that a synthetic peptide, corresponding to the propeptide region, was a specific and potent inhibitor of the esterase activity of purified egasyn. Taken together, these results suggest that (a) the egasyn-glucuronidase system may use a novel mechanism related to that of serine proteinases and their inhibitors in complex formation and in subsequent localization of glucuronidase within the ER and that (b) a possible function of ER glucuronidase is to modulate the esterase activity of egasyn.

Inhibitors of elastase and cathepsin G in Chédiak-Higashi (beige) neutrophils.

Previous studies have established that mature neutrophils from the peritoneal cavity, blood, and bone marrow of beige (Chédiak-Higashi syndrome) mice essentially lack activities of two lysosomal proteinases: elastase and cathepsin G. There are, however, significant levels of each enzyme in early neutrophil precursors in bone marrow. In the present experiments, it was found that the addition of extracts from mature beige neutrophils to extracts of normal neutrophils or to purified human neutrophil elastase and cathepsin G resulted in a significant inhibition of elastase and cathepsin G G activities. 125I-Labeled human neutrophil elastase formed high molecular mass complexes at 64 and 52 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis when added to beige neutrophil extracts. The molecular masses of the inhibitor-125I-elastase complexes suggested that the molecular masses of the inhibitors are approximately 36 and 24 kDa, respectively. These results were confirmed by gel filtration on Superose 12 under nondenaturing conditions. Cathepsin G was inhibited only by the 36-kDa component. The inhibitors formed a covalent complex with the active sites of elastase and cathepsin G. No inhibitory activity was present in mature neutrophil extracts of genetically normal mice or in extracts of bone marrow of beige mice. These results thus represent an unusual example of an enzyme deficiency state caused by the presence of excess inhibitors. Inactivation of neutrophil elastase and cathepsin G in mature circulating and tissue neutrophils may contribute to the increased susceptibility of Chédiak-Higashi patients to infection.

Elastase and cathepsin G activities are present in immature bone marrow neutrophils and absent in late marrow and circulating neutrophils of beige (Chediak-Higashi) mice.

Elicited peritoneal neutrophils of beige (Chediak-Higashi) mice essentially lack activities of the neutral serine proteinases elastase and cathepsin G, which may explain the increased susceptibility to infection of beige mice and Chediak-Higashi patients. We have examined neutrophils of beige mice at earlier points in their development to determine if the proteinase genes are never expressed or whether they are expressed and then lost during neutrophil maturation. Surprisingly, bone marrow of beige mice had significant elastase and cathepsin G activity (approximately 60% of normal). The results of several experiments indicate that neutrophils were the sole source of elastase and cathepsin G in bone marrow. Neutral proteinase activity was readily demonstrable by histochemical procedures in beige marrow neutrophil precursors up to and including the metamyelocyte stage. However, mature neutrophils of beige marrow had greatly decreased activity. Also mature neutrophils (PMNs) of the peripheral circulation, like peritoneal neutrophils, had very low elastase and cathepsin C activities. Thus we conclude that beige neutrophil precursors express neutral proteinase activity, which is largely and irreversibly depleted by the time they fully mature in marrow.