Localization of chymotrypsin-like cationic protein, collagenase and elastase in azurophil granules of human neutrophilic polymorphonuclear leukocytes.

The subcellular localization of granulocyte collagenase, elastase and chymotrypsin-like cationic protein was determined using velocity centrifugation of cytoplasmic granules of human polymorphonuclear leukocytes. The proteases were assayed by immunochemical and enzymatic methods. Measurements of lactoferrin and myeloperoxidase distinguish exactly between constituents of specific and azurophil granules. Collagenase, elastase and chymotrypsin-like cationic proteins showed an almost identical sharp and unimodal distribution. They co-sedimented with myeloperoxidase demonstrating that these enzymes are localized exclusively in the azurophil granules.     

Interaction of myosin and paramyosin

The interaction of myosin and paramyosin was investigated by enzymological and ultrastructural techniques. The actin-activated Mg⁺² ATPase of rabbit skeletal muscle myosin can be inhibited by clam adductor paramyosin. Both proteins must be rapidly coprecipitated to form filaments for this inhibition. Slowly formed cofilaments are fully activatable by F-actin. In both cases, the cofilaments possess unique structural characteristics when compared to homofilaments. The mode of inhibition appears to be competitive when different concentrations of paramyosin and F-actin are compared. The apparent affinity of the myosin heads for actin is reduced by the presence of paramyosin within rapidly reconstituted thick filaments. These results suggest that paramyosin may serve as part of a relaxing mechanism within invertebrate muscles. It is unlikely that paramyosin plays a role in the initiation and maintenance of catch within specialized molluscan muscles.     

Identification of a new transthyretin variant (Ile49) in familial amyloidotic polyneuropathy using electrospray ionization mass spectrometry and nonisotopic RNase cleavage assay.

Mutation of the transthyretin (TTR) plasma protein and gene in a Japanese patient with amyloid polyneuropathy was investigated by electrospray ionization mass spectrometry (ESI-MS) and nonisotopic RNase cleavage assay (NIRCA), respectively. ESI-MS analysis showed normal TTR peaks and additionally a variant TTR with 12-dalton-higher molecular weight than normal TTR. NIRCA suggested that the mutation existed near either the 5' or 3' end of exon 3. Direct DNA sequencing revealed both a normal ACC (threonine) and a variant ATC (isoleucine) at codon 49, which was located near the 5' end of exon 3. The molecular weight shift of this mutation was 12 D, consistent with the result of ESI-MS.     

The assay of peroxidases by means of dicarboxidine on enzyme-linked immunosorbent assay level

Dicarboxidine is a low- or noncarcinogenic benzidine-type chromogenic substrate to peroxidases. The color formation is of zero order from the onset of the reaction with a rate proportional to the enzyme concentration. Substrate solution and absorbance are stable for at least 24 h, the latter after an early decrease of 4% in 10 min. The complexity of the benzidine reaction necessitates precautions against side reactions. These are analyzed and suitable assay conditions are presented. Dicarboxidine is a valuable chromogen for studies of a slow generation of hydrogen peroxide, a slowly reacting substituted hydroperoxide, or a low peroxidase activity such as in the enzyme-linked immunosorbent assay or other immunological tests.