M. Koji Owada

Cross-linking of lipocortin I and enhancement of its Ca2+ sensitivity by tissue transglutaminase.

The stimulation of human epidermoid carcinoma A431 cells with the calcium ionophore A23187 resulted in the formation of high-molecular-weight lipocortins I, having apparent molecular weights of 75 kDa and 160 kDa as detected with specific anti-lipocortin I antibody. These immunoreactive proteins were identified to be covalently cross-linked multimers of lipocortin I, since essentially the same cross-linked multimers were observed when purified lipocortin I was incubated with tissue transglutaminase (TGase) in vitro. Classical amine substrates for TGase, such as dansylcadaverine and putrescine, were also incorporated stoichiometrically into lipocortin I. Cross-linking or amine incorporation was not observed with lipocortin II. Des 1-26 lipocortin I did not serve as a substrate for TGase, indicating that the N-terminal region of lipocortin I plays an important role in the formation of lipocortin I multimers. The cross-linking of lipocortin I by TGase resulted in a remarkable enhancement of calcium sensitivity for phospholipid binding; i.e., the free calcium concentration required for the cross-linked lipocortin I to attain 50% maximal binding to phosphatidylserine vesicles was as little as 3 microM, while that required for intact monomeric lipocortin I was 20 microM.

Linear relationship between diphosphorylation of 20 kDa light chain of gizzard myosin and the actin-activated myosin ATPase activity

The addition of large amounts of myosin light chain kinase to the reconstituted gizzard actomyosin shows diphosphorylation of 20 kDa myosin light chain. Accompanying diphosphorylation, the actin-activated myosin ATPase activity was also enhanced. The extent of diphosphorylation and the myosin ATPase activity were clearly demonstrated to be in a linear relationship. From the time course experiment, the conversion of monophosphorylated light chain into one which was diphosphorylated seemed to be a sequential process. Moreover, analyzing phospho-amino acid by using a two-dimensional electrophoresis technique revealed that monophosphorylated light chain contained phosphoserine and diphosphorylated one contained phosphothreonine in addition to phosphoserine.

Isolation and characterization of three forms of 36-kDa Ca2+-dependent actin- and phospholipid-binding proteins from human placenta membrane.

We purified three forms of 36-kDa proteins, two monomeric 36-kDa proteins, which had pIs of 7.5 (36K-I) and 6.4 (36K-II), and one 36-kDa complex (36K-C) consisting of two subunits, 36-kDa (pI 7.5) and 12-kDa (pI 5.8), from human placenta membrane. The 36-kDa subunit of 36K-C was identical to 36K-I as judged by pI, cyanogen bromide peptide mapping and immunological cross-reactivity. The three proteins showed F-actin- and phosphatidylserine-binding abilities dependent on Ca2+ concentrations at millimolar and micromolar levels, respectively. They all had phospholipase A2 inhibitory activity. Only 36K-II was phosphorylated extensively at tyrosine residue in Ca2+- and EGF- dependent manners in the membrane fraction of A431 cells. 36K-I was the best substrate for src kinase, whereas 36K-II was the best for fps kinase. However, 36K-C was not phosphorylated by any kinases used here.

A 32-kDa protein associated with phospholipase A2-inhibitory activity from human placenta

Two monomeric 32‐kDa proteins, termed 32K‐I (pI 5.8) and 32K‐II (pI 5.1), were isolated from human placenta, which was solubilized by a Ca2+‐chelator. Only 32K‐I was associated with PLA2‐inhibitory activity. CNBr peptide mapping indicated that 32K‐I was distinct from 32K‐II and two 36‐kDa proteins, called calpactin I and II or lipocortin II and I, which have been shown to possess PLA2‐inhibitory activity. 32K‐I bound to PS in a Ca2+‐dependent manner. 32K‐I was detected in many tissues except brain, cardiac and skeletal muscle.

A rat mutant cell clone showing temperature-dependent transformed phenotypes with functional expression of the src gene product

The cellular mutant B814 isolated from a Fischer rat cell line shows temperature-sensitivity of focus formation on infection with Moloney murine sarcoma virus (Mo-MSV) and Rous sarcoma virus (RSV). An RSV-transformed clone (S814-2) isolated from B814 cells shows temperature-sensitive transformed phenotypes for morphology, growth in soft agar, and glucose uptake. The expression, phosphorylation, and tyrosine kinase activity of pp60v-src in S814-2 were not affected at the nonpermissive temperature, and virus rescued from this clone had wild-type transforming ability, suggesting that a cellular factor altered in S814-2 is responsible for the cellular steps of transformation after the function of pp60v-src. In addition, the cellular 36K protein, a possible candidate as a target of pp60v-src, was phosphorylated at the nonpermissive temperature in S814-2, indicating that phosphorylation of the 36K protein is not correlated with transformed phenotypes.

Establishment of cell lines derived from ataxia telangiectasia and xeroderma pigmentosum patients with high radiation sensitivity

Four human fibroblast cell lines, three of which were derived from a patient with ataxia telangiectasia and the other from a patient with xeroderma pigmentosum, were established after transfection with cloned SV40 DNA. These 4 cell lines showed some phenotypes characteristic of neoplastically transformed cells, and had a human karyotype with heteromorphisms identical to those of the parental fibroblasts. Their sensitivity to the cytotoxic effects of gamma-rays or ultraviolet irradiation was as high as those of their parental fibroblasts.

A p60v-src-related tyrosine kinase in the acetylcholine receptor-rich membranes of Narke japonica: association and dissociation of phosphatidylinositol kinase activity.

We have isolated a tyrosine-specific protein kinase from the acetylcholine receptor (AChR)-rich membranes of the electric ray Narke japonica. The enzyme is immunologically related to p60v-src, the product of the transforming gene of Rous sarcoma virus. A substantial phosphatidylinositol (PI) kinase activity was associated with this enzyme when it was purified through tyrosine-agarose affinity chromatography used previously for the purification of p60v-src. However, by subsequent chromatography on casein-agarose, most of the associated PI kinase activity was separated from the tyrosine kinase activity. The results suggest that the tyrosine-specific protein kinase in the AChR-rich membranes of N. japonica has no intrinsic PI kinase activity.