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Aldehyde dehydrogenase from bovine liver has been purified to homogeneity. Amino acid composition showed a high content of cysteine of 32 mol/mol enzyme. The enzyme is composed of four identical subunits as judged by sodium dodecyl sulfate gel electrophoresis and end-group analysis. The molecular weight was determined to be 220 000 +/- 10 000 by sedimentation equilibrium analysis in an analytical ultracentrifuge. The Michaelis constants for NAD+, glyceraldehyde and acetaldehyde were found to be 47 micron, 170 micron and 130 micron, respectively.

The comparative studies are continued on the influence of two tertiary amines, procaine and nicotinoyl-procaine, on isolated heart and skeletal muscle cells in culture. A daily addition of these substances to cell cultures produces an increase in the specific activities of glutamate dehydrogenase and glutamate-oxaloacetate transaminase and an inhibition of alteration in the isoenzyme pattern of lactate dehydrogenase. The presence of procaine or nicotinoyl-procaine, resp., inhibits thymidine incorporation by cells in culture, whereas the incorporation of thymidine is increased after pretreatment of cells with these substances. These results support the findings of an influence of said tertiary amines on the metabolism of cells in culture.

The tertiary amines, procaine and nicotinoylprocaine, cause an increase in the specific activities of two glycohydrolases, alpha-fucosidase and beta-N-acetylhexosaminidase, which are involved in membrane metabolism. The specific activity of alkaline phosphatase, a plasma membrane enzyme, is lowered in muscle cells after addition of procaine or nicotinoylprocaine to the culture medium. The specific activities of two transferases, aspartate-amino-transferase and creatine phosphokinase, are increased by 10(-5) mol/l butacaine. A combined addition of butacaine and nicotinoylprocaine causes less effects on the transferases. The specific activities of neutral alpha-glucosidase and beta-N-acetylhexosaminidase are scarcely influenced by butacaine alone. Only butacaine and nicotinoylprocaine together lead to an increase of the activities of these hydrolases. These results suggest two different mechanism of action at least concerning these substances: 1. a specific binding of tertiary amines and 2. a coordinated mechanism on the membrane fluidity.

Treatment of mammalian cells in culture with retinoic acid causes a time- and concentration-dependent increase of the specific activity of alkaline phosphatase. The increase reaches a factor of 15 and more and begins at a concentration of 10(-8)M retinoic acid. The induction is inhibited by cycloheximide or actinomycin D. The same isoenzyme of alkaline phosphatase is expressed in control and in retinoic acid-treated cells as demonstrated by the inhibitions by amino acids and peptides. The enzyme induction occurs in rat heart, skeletal muscle, brain, lung cells and HeLa cells. No induction was found in two lines of human melanoma cells. After treatment of cells with tunicamycin, the induction of alkaline phosphatase is detectable only in the homogenate and no longer detectable by histochemical methods. This shows that the glycosylation of the protein is an important step in the insertion of this enzyme into the plasma membrane.

A comparative study was performed on the influence of a series of chemically defined local anaesthetics such as procaine, nicotinoylprocaine and butacaine on isolated skeletal muscle cells and heart cells in culture. About 30 min after addition of local anaesthetics to cell cultures vesicles appear in the cells. These formations are reversible and need a minimum concentration of 13(-3) mol/l procaine, 10(-4) mol/l nicotinoylprocaine, or 10(-4) mol/l butacaine, respectively. Soon after addition of local anaesthetics a reduction of oxygen consumption is measurable in both cell types. The degree of this reduction depends on the chemical structure and the concentration of local anaesthetics and on the kind of cells. After daily addition of local anaesthetics to cell cultures metabolic parameters were studied. The local anaesthetics, except for butacaine, produce an increase of protein concentration, creatine kinase and lactate dehydrogenase activity per sample and an increase of the specific activity of lactate dehydrogenase. These increases depend also on the concentration of local anaesthetics and on the kind of cells. These results support the hypothesis of local anaesthetics action via membrane lipids.

gamma-Glutamyltransferase is a membrane-bound enzyme widely distributed in animal tissues. This enzyme is involved in glutathione metabolism, but its exact biological function is still an open question. In rat brain cells in culture a depletion of L-cystine and L-glutamine or the addition of diethyl maleate to the culture medium leads to a decrease of intracellular glutathione concentration and to an increase of the specific activity of gamma-glutamyltransferase. The induction of the enzyme is inhibited by the addition of cycloheximide or actinomycin D to the culture medium.

A comparative study was performed on the influence of two similar tertiary amines, procaine and nicotinoyl-procaine, on isolated kidney and cerebellum cells in culture. Shortly after the addition of the test substances vesicles are found in the cells and the cells are more broadly attached to the underground. But the attachment of microfilaments is scarcely adhered. Daily addition of procaine or nicotinoyl-procaine, resp., to cell cultures leads to an increase of protein concentration and enzyme activities. Particularly organotypical enzymes are enhanced, as gamma-glutamyltransferase in kidney cells and creatine kinase in cerebellum cells.