Fumihide Inoue

Quantification of myonecrosis and comparison of necrotic activity of snake venoms by determination of creatine phosphokinase activity in mice sera

Serum creatine phosphokinase (CK) values were measured in venom-injected mice. The increase of CK levels was proportional to both the amount of venom injected and the myonecrotic actions determined microscopically. The CK isoenzymes were regarded as due to the predominant increase of CK-MM, which was considered to be of skeletal muscle origin in the venom-injected area. No CK isozyme variants were found by electrophoretic examination. For a comparative study on necrotic intensity, nine snake venoms were examined. The elapid venoms induced higher values of serum CK with a stronger necrotic effect than the crotalid venoms, and the data agreed with the histological findings. On application of the antivenom, an initially elevated CK value was significantly decreased and no severe damage was observed under light microscopy. Since the changes of serum CK activities paralleled necrotic intensity, the CK value is useful as an indicator of myonecrosis due to snake venom.

Total absence of protein 4.2 and partial deficiency of band 3 in hereditary spherocytosis

Unlike previously reported cases with total protein 4.2 deficiency due to mutations in the EPB42 gene, we describe a total deficiency in protein 4.2 with normal EPB42 alleles. Hereditary spherocytosis (HS) was observed in a Japanese woman (unsplenectomized) and her daughter (splenectomized). The mother showed a partial deficiency in band 3 and a proportional reduction in protein 4.2. She was heterozygous for a novel allele of the EPB3 gene, allele Okinawa, which contains the two mutations that define the Memphis II polymorphism (K56E, AAG → GAG, and P854L, CCG → CTG) and, additionally, the mutation: G714R, GGG → AGG, located in a highly conserved position of transmembrane segment 9. The latter change was responsible for HS. In trans to allele Okinawa, the daughter displayed allele Fukuoka: G130R, GGA → AGA, an allele known to alter the binding of protein 4.2 to band 3. The daughter presented with a more pronounced decrease of band 3, and lacked protein 4.2, resulting in aggravated haemolytic features. Although the father was not available for study, heterozygosity for allele Fukuoka has been documented in another individual who showed no clinical or haematological signs, and a normal content of band 3. We suggest that band 3 Okinawa binds virtually all the protein 4.2 in red cell precursors, band 3 Fukuoka being unable to do so, and that the impossibility of band 3 Okinawa incorporation into the membrane leads to degradation of the band 3 Okinawa protein 4.2 complex. In contrast, band 3 Fukuoka, free of bound protein 4.2, could then incorporate normally into the bilayer. Thus, protein 4.2 would not appear in the daughters red cell membrane.

A Serine Proteinase from Cow Snout Epidermis Degrades High Molecular Weight Keratins

This study describes a serine proteinase which degrades high molecular weight polypeptides of keratins. The enzyme was coextracted with 8M urea together with keratins and separated from the keratins by gel filtration chromatography. Molecular weight of the enzyme was estimated at 25,000 daltons by gel filtration. Revealed characteristics of the enzyme were as follows; 1) maximally active at pH 8.5–9.0, 2) requires 0.04% SDS for maximum activity, 3) heat stable in the absence of SDS up to 65°C for 2 min, 4) sensitive against serine protease inhibitor, phenylmethanesulfonyl fluoride and soybean trypsin inhibitor, 5) highly specific for natural protein substrates, such as keratin polypeptides.