M.K. Patterson

Catalytic formation of ϵ-(γ-glutamyl)lysine in guinea pig liver transglutaminase

Abstract Incorporation of putrescine into purified guinea pig liver transglutaminase was observed when the enzyme was incubated under catalytic conditions in the absence of the exogenous acceptor substrate casein. Furthermore, in the absence of exogenous substrates, putrescine and casein, the enzyme monomer of 85–90,000 daltons was converted to polymeric material. ϵ(γ-Glutamyl)lysine isopeptide was isolated from polymeric transglutaminase samples following proteolytic digestion. The results were consistent with the formation of an intermolecular isopeptide, ϵ(γ-glutamyl)lysine, between two or more enzyme monomers apparently by an autocatalytic reaction.

ε-(γ-Glutamyl)lysine isopeptide bonds in normal and virus transformed human fibroblasts

Abstract Normal human lung cells (WI-38) possessed 20–40 times more e-(γ-glutamyl)lysine isopeptide bonds than Simian virus transformed counterparts, WI-38 VA13A and WI-38 VA13-2RA. Normal cells arrested in an essentially nonmitotic state had more isopeptide bonds than proliferating cells. Isopeptide content paralleled the transglutaminase activity of these cells. The results suggest that isopeptide crosslinks contribute to a cellular architecture conducive to a nonproliferating state.

Enhanced transglutaminase activity in transformed human lung fibroblast cells after exposure to sodium butyrate

The low level of transglutaminase activity in virus-transformed human embryonic lung fibroblasts (WI-38 VA13A) increased markedly when cells were exposed to sodium butyrate. The effect of sodium butyrate was time- and concentration-dependent and fully reversible. Transformed cells exposed for 5 days to 1 mM sodium butyrate had fewer cells, showed an 8-10 fold higher transglutaminase activity, and stained more abundantly for transglutaminase and pericellular fibronectin than control cells when examined by indirect immunofluorescence. Non-transformed cells (WI-38) showed only a 2-4-fold increase in transglutaminase activity when treated in a similar manner. Studies with metabolic inhibitors revealed the increase in activity was the result of synthesis of new protein. Kinetic studies showed the affinity of putrescine for the enzyme was essentially unchanged but the number of active sites increased 9-fold following exposure to sodium butyrate. Enhanced transglutaminase activity returned to control levels within 7 days after subculture and sodium butyrate removal. These findings suggest that sodium butyrate offers a potential model system to understand better the role of transglutaminase in cells in culture; particularly growth control in transformed cells.

Induction of cellular transglutaminase biosynthesis by sodium butyrate

Cellular transglutaminase activity was induced in simian virus-transformed human embryonic lung fibroblasts (WI-38 VA13A) by sodium butyrate. The level of enzyme activity approached a maximum by 6 days; 9-11-fold higher in the presence of sodium butyrate (1 mM) than in its absence. The observed increases in cellular transglutaminase activity could be entirely accounted for by equivalent increases in the levels of enzyme protein measured by inhibition enzyme-linked immunosorbent assay. Sodium butyrate also increased the rate of enzyme synthesis, but had no effect on the rate of cellular transglutaminase degradation. The increase in the rate of enzyme synthesis was matched by an increased level of translatable transglutaminase mRNA as measured in a cell-free translation system. Our results suggest that sodium butyrate regulates cellular transglutaminase at the pretranslational level.

In vitro Effects of Guinea Pig Serum on the Jensen, JA-1 and JA-2 Sarcomas

Summary The in vitro growth response of the Jensen sarcoma and its nutritional variants (JA-1 and JA-2) to media containing normal guinea pig serum and partially purified asparaginase and media devoid of asparagine was studied. A comparison of the morphology of the cells grown in the presence of guinea pig serum and media devoid of asparagine showed a remarkable similarity. Further, the growth characteristics of cultures exposed to guinea pig serum and partially purified asparaginase were essentially the same. These results suggested the active component of guinea pig serum was asparaginase which catalyzed the destruction of extracellular asparagine.

An improved method of analysis for glycine using Streptococcus faecalis.

Abstract Streptococcus faecalis A.T.C.C. 6057 can be used satisfactorily to determine glycine in a final medium solution concentration of from 0 to 6 μg./ml./tube. This analytical procedure is more sensitive and is as accurate as previous methods of glycine determinations. Advantages of the method include (a) the small amount of protein required for assay, and (b) the accuracy of the assay. The glycine content of seven purified proteins was reported.

Studies on the Aspartic Acid “Sparing Effect” on the Nutritional Requirement of L-Asparagine for Tumors in Vitro

Summary The L-aspartic acid sparing effect of the nutritional requirement for L-asparagine for the Jensen sarcoma and the Walker 256 carcinoma was reinvestigated. The effect was found only when the aspartic acid contained L-asparagine as a contaminant. Studies on the induction of asparagine synthetase by high concentrations of aspartic acid gave negative results.

The isolation of glycine, alanine, leucine, arginine, and glutamic acid for radiometric assay in tracer experiments

Abstract Some amino acid precipitant methods have been modified as to their applicability to a semimicro scale in tracer experiments. Satisfactory methods have been established for glycine, alanine, leucine, arginine, and glutamic acid. The advantages of these methods, in comparison with conventional carrier techniques, include: ( a ) simplicity of operation; ( b ) individual radioassays of an amino acid and its salt, both isolated under different conditions; ( c ) the requirement of less carrier; ( d ) rapidity with which they can be carried out; and ( e ) better yields (in most cases) are obtained for degradation purposes if contemplated. In the isolation of a few amino acids, these methods have a distinctive advantage over ion-exchange chromatography in that extensive laboratory preparation is not necessary.