Active SH Groups Research Articles

The importance of SH-groups for enzymic activity. 7. The amino acid sequence around the essential SH-group of pig heart lactate dehydrogenase, isoenzyme I.

1 The amino acid sequence of a dodecapeptide containing the essential SH-group of pig heart l-lactate dehydrogenase, isoenzyme I, was determined as Val-Ile-Gly-Ser-Gly-Cys-Asn-Leu-Asp-Ser-Ala-Arg. This sequence shows three differences with that of an SH-peptide from chicken LDH-I. 2 It is shown that the possible codons for the amino acids occupying equivalent positions in the heptapeptides around the essential SH-groups of glyceraldehyde 3-phosphate-, yeast alcohol-, liver alcohol- and lactate dehydrogenases can be related to one another by a chain of point mutations.

Luciferase has active SH groups

Luciferase has active SH groups

On the glucose-6-phosphate dehydrogenase of Ceratocystis ulmi

The properties of glucose-6-phosphate dehydrogenase from a number of sources have been investigated by various workers, but none have reported the enzyme to be inhibited by iodoacetate. This enzyme from Ceratocystis ulmi, the Dutch elm disease fungus, while possessing many properties similar to those of the enzyme from other sources, is inhibited by iodoacetate; it appears to require free SH groups for enzymic activity. Complete protection against iodoacetate and 5,5′-dithiobis (2-nitrobenzoate) inhibition could be obtained if the enzyme were first allowed to incubate with NADP before adding the inhibitors, and p-chloromercuribenzoate inhibition could be partly reversed by the addition of cysteine.

Mechanism of melanophore dispersion

The mechanism of the melanophore reaction of the skin of Hyla arborea (tree frog) has been studied. Apart from the specific hormones MSH and ACTH which elicit melanophore dispersion, the following substances produced darkening: theophylline and caffeine regularly, theobromine erratically, but not, however, dyphylline. Sodium iodoacetate and mercuric chloride elicited melanophore dispersion, which fact may be connected with their ability to block active SH groups. Melanophore dispersion is not dependent upon temperature (13–20°); at the unusual temperature of 37° frog melanophore dispersion is, however, inhibited. On the other hand, melanophore contraction is speeded up with increases in the ambient temperature; this points to the working theory that darkening is a passive reaction whereas lightening is an active reaction requiring energy. The assay of different inhibitors for their interaction with the energy supply has shown that only sodium benzoate inhibits melanophore contraction. This points to the possibility that the energy source should be sought either in the lipid metabolism or in the flavoproteins. Melanophore contraction (lightening reaction) was elicited by dyphylline and noradrenaline. The effect of adrenaline, melatonin and serotonin was insignificant. This points to a highly specific reaction of the Hyla melanophores. Based upon our experience of the effect of calcium on the melanophore reaction the theory is advanced that the passive stage of melanophore dispersion (darkening) is induced by entrance of Ca 2+ into the cell and exit of K +. The active stage of melanophore contraction (lightening), however, needs an energy pump for either removal of Ca 2+ from the cell or for forcing in of K +. The melanophore reaction is a convenient ‘slow-motion picture model’ for many coupling phenomena occurring in muscles, glands, nerves, etc.

Studies on the Formation of Enzyme-Substrate Complex

The studies on the mechanism of the enzyme action was mainly based on the assumption of the formation of enzyme-substrate complex and on the mathematical treatment of its kinetics to calculate the reaction constants. On the other hand, the complex could not be demonstrated experimentally except for a few enzymes.Nakamura has proposed a method, namely the crossing paper electrophoresis, to detect the enzyme-substrate complex and with many enzymes he and his coworkers have succeeded to demonstrate the complexes on the filter paper. In the experiment described here, the enzyme-substrate complex of papain with benzoyl-L-argininamide and casein was demonstrated by this technique.The complex was formed not only in the optimal pH range (pH 5.0-7.0), but also in the pH (pH 3.0 and 9.0) at which no enzymic activity was detected. Moreover, with inactive papain, as well as mercuripapain and PCMB-inhibited papain, the complex formation could be proved with ease. These findings indicate that pH values, inhibitors and activators influence only the activity state of papain but not the binding with substrate. They stand against the theory, that the active SH-group of papain should play a definite role to bind the substrate covalently.It was concluded, therefore, that there should be two separate centers on the papain molecule: the one is the binding site for substrate, and the other, active site of the enzyme.

Reduction of potassium tellurite by living tissues.

SummaryReducing activity in living tissue can be demonstrated with the aid of potassium tellurite. Under the microscope, the sites of reduction are indicated by insoluble dark tellurium. Various bacteria, including strictly anaerobic ones as well as yeast and leucocytes from human material, reduced posassium tellurite. Among different mammalian tissue examined, kidney gave the most constant result, permitting histochemical localization in certain portions of the nephron. Active SH groups apparently are essential for the reduction of potassium tellurite by living tissues.

THE CHANGE IN STATE OF THE PROTEINS OF MUSCLE IN RIGOR

1. When myosin is exposed to a typical denaturing agent (acid) it becomes insoluble and its SH groups are activated. 2. The same number of active SH groups is found in the soluble myosin of resting muscle as in the insoluble myosin of muscle in rigor. No activation of SH groups accompanies the formation of insoluble protein in rigor. 3. When the insoluble myosin of muscle in rigor is treated with a denaturing agent its SH groups are activated. 4. Protein coagulation as brought about by denaturing agents (heat, acid, alkali, alcohol, urea, salicylate, surface forces, ultraviolet light) is a distinctly different change from the coagulation of myosin brought about by the unknown agent in muscle.

SULFHYDRYL AND DISULFIDE GROUPS OF PROTEINS

1. In the denatured proteins of skeletal muscle, the ratio of SH to S-S groups is higher than in the mixed denatured proteins of other tissues, with a single exception-the proteins of the crystalline lens. 2. The number of active SH groups in the proteins of minced muscle or in any of the protein fractions of muscle is only a fraction of the number found after the proteins have been treated with a denaturing agent. 3. The SH groups of the native proteins of muscle are activated by a rise in pH. 4. The relation between pH and number of active SH groups in the proteins of minced muscle and in the various protein fractions of muscle shows that little, if any, denatured protein is present in minced muscle.

SULFHYDRYL AND DISULFIDE GROUPS OF PROTEINS

Hemoglobin and the proteins of the crystalline lens contain active SH groups while in the native state, the number of active groups increasing as the pH rises. All the SH groups of denatured globin and of the denatured lens proteins are active at a pH so low that practically none of the SH groups of native hemoglobin and of native lens protein are active. The effect of denaturation on the SH groups of a protein is to extend towards the acid side the pH range of their activity. It is possible to oxidize the iron-porphyrin and the SH groups of hemoglobin independently of each other.