The alkaline adenosine triphosphatase activity of 30S dynein after modification of the SH groups. Possible involvement of some of the most reactive SH groups.

An apparent 'triphasic' alteration of 30S dynein ATPase activity was produced by treatment with various amounts of NEM when the modification and susequent ATPase assay were carried out at pH 7.4 and pH 10-10.2, respectively. The Mg-ATPase activity was markedly inhibited by modification of the most reactive SH groups with 10 microM NEM, although the same treatment had no significant effect on the activity when assayed at neutral pH. Increasing the NEM concentration to 0.3 mM largely restored the enzyme activity, but a further increase in NEM concentration inhibited the enzyme activity again. This unusual response of 30S dynein ATPase at pH 10-10.2 was accounted for by the results of Arrhenius plots of the enzyme activity at pH 10.1; the enzyme protein modified with not more than 10 microM NEM was not stable under the assay conditions (pH 10-10.2 at 25 degrees C), whereas modification with 0.3 mM NEM stabilized 30S dynein against the assay conditions. The possible significance of the 10 microM NEM-induced inhibition of the 30S dynein alkaline ATPase activity is discussed in connection with the participation of SH groups of 30S dynein in the enzyme activity.     

The presence of reactive SH groups in the enzymatically active dicyano derivative of creatine kinase

It has been shown that the active dicyano derivative of creatine kinase (ATP:creatine N-phosphotransferase) obtained by cyanolysis of the 5,5'-dithiobis(2-nitrobenzoic acid)-modified and inactivated enzyme contains, as does the native enzyme, two reactive SH groups. Modification of these two SH groups leads to complete inactivation of the dicyano enzyme. Reaction with 4-iodoacetamido-1-naphthol introduces fluorescent labels at these reactive SH groups of the native and the dicyano enzymes. Following tryptic digestion, the respective fluorescent-labelled peptides have been separated by HPLC and the amino acid composition analysis of these peptides has shown that they are consistent with the sequence of the peptide segment containing the active-site SH of Cys-282 of creatine kinase for both the native and the dicyano enzymes, showing that the active SH groups are free in the dicyano enzyme. Upon mild denaturation in 3 M urea, it can be shown that two of the SH groups partially buried in the native enzyme have been cyanylated in the dicyano enzyme. The two reactive SH groups are therefore essential for the activity of creatine kinase and the two cyanylated SH groups are internal groups which probably contributes partially to the stabilization of an active conformation of the enzyme molecule.     

Muscle pyruvate kinase: interaction with substrates and analogues studied by difference spectroscopy. Comparative studies of the substrate-binding sites of various ATP phosphotransferases.

The substrate binding sites of pyruvate kinase have been studied by means of spectrophotometric investigations. Two binding sites, one for the nucleotide substrate and one for the acceptor, have been characterized. The interaction of nucleotide substrates with the enzyme, which is metal-dependent, results in a perturbation of the spectrum of the nucleotide chromophore characterized by hypochromic and red shift effects; the hydrophobicity of the nucleotide site was estimated by using a reporter group reagent, 2-(dansylamino)ethyl monophosphate. The comparison between the binding sites of several ATP phosphotransferases is discussed and some common features are reported.     

Protection of one of the two reactive thiol groups in F-actin by ATP and phalloidin

In F-actin, [A(SH)₅]_n, prepared from rabbit skeletal muscle, two thiol groups react with 2,4-dinitrophenyl-glutathionyl-disulfide, DNPSSG, to form [A(SH)₃(SSG)₂]_n. One of the two thiol groups reacts fast, (20 min), while the reaction of the second is slow (200 min). The fast reacting group has been identified as cysteine-373.In the presence of approximately one equivalent of ATP, only one of the thiol groups is reactive. The reaction product is [A(SH)₄SSG]_n. In comparison, the shielding effect of ADP is about 2 to 3 times smaller than that of ATP, while AMP is ineffective.The mushroom toxin phalloidin, which binds to polymeric actin, exhibits a similar protective activity as ATP and shields one thiol group from reaction with DNPSSG.We conclude from these data that in F-actin a second low affinity binding site for adenosin-nucleotides exists, which can be monitored by the reactivity of one of the two reactive thiol groups.     

Immobilized phenylalanine hydroxylase through the SH groups

Purified rat liver phenylalanine hydroxylase [L-phenylalanine:tetrahydropteridine:oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1] was immobilized with activated thiol-Sepharose 4B via disulfide bond formation, which is expected to immobilize the enzyme in its activated form through the SH modification. This immobilized enzyme was more stable against thermal denaturation than the free enzyme. When tetrahydrobiopterin was used as the natural cofactor, the K(m) value for phenylalanine was decreased and that for the cofactor was increased. Constant conversion from phenylalanine to tyrosine was demonstrated continuously for over 8 h at 25 degrees C.     

Heterogeneity of SH groups in sarcoplasmic reticulum.

The incorporation of [¹⁴C] N-ethylmaleimide reveals fast and slow-reacting sulfhydryl groups in sarcoplasmic reticulum. Two proteins react with the label: a fast-reacting glycoprotein recently isolated (Ikemoto, Cucchiaro and Garcia (1976) J. Cell Biol.$\text{70}$, 290a), and the Ca²⁺-ATPase. Labeling sarcoplasmic reticulum with a maleimide spin label gives a similar pattern. The spectra of maleimide-spin-labeled sarcoplasmic reticulum have both ‘strongly’ and ‘weakly’ immobilized components. Maleimide-spin-labeled purified Ca²⁺-ATPase, or sarcoplasmic reticulum labeled first with N-ethylmaleimide, and then with maleimide spin label, show spectra devoid of the ‘weakly’ immobilized component; the latter is enhanced in partially purified glycoprotein obtained from spin-labeled sarcoplasmic reticulum. This indicates that spectra from maleimide-spin-labeled sarcoplasmic reticulum do not reflect exclusively the state of the Ca²⁺-ATPase enzyme.     

Chemical modifications of SH groups intraerythrocytic hemoglobin.

Chemical modifications of intraerythrocytic hemoglobin have been obtained by reaction of β 93 sulfhydryl groups with disulfides i.e. cystamine and cystine-dimethylester. The respiratory properties of the modified erythrocytes are similar to those of the hemoglobin reacted with the same reagents and the changes observed are the same for both compounds. Although other properties of the erythrocytes are modified, the resistance to hemolysis is not vastly impaired. The results suggest the possibility of using these compounds (or similar ones) to modify red cells for therapeutical purposes.