The arrangement of subunits in cholera toxin.

Cholera toxin consists of five similar B subunits of apparent molecular weight about 10 600 and one A subunit (29 000) consisting of two peptides (A1 23 000-24 000 and A2 about 5500) linked by a single disulfide bond. Each B subunit also contains one internal disulfide bond which is readily reduced but is protected from carboxymethylation unless the reduced subunits are heated in urea. Tyrosine residues in A1 and in B subunits are readily iodinated, but the intact B assembly does not react with iodine. Upon reaction with the cross-linking reagent dimethyl suberimidate, B subunits may be covalently connected to each other, to A1 and to A2. A1 and A2 may also be cross-linked. The B subunits are probably arranged in a ring with A on the axis. A2 is required for the re-assembly of toxin from its subunits and may serve to hold A1 on the B ring. The maximum activity of cholera toxin in vitro is obtained only when the active peptide, A1, is separated from the rest of the molecule. Such separation, and the insertion of A1 into the cytosol, must follow the binding of the complete toxin, through component B, to the exterior of intact cells. This binding increases the effective concentration of the toxin in the vicinity of the plasma membrane. Possible ways in which A1 then crosses the membrane are considered in the Discussion.     

Effect of long photoperiod on the maturation of ovary of the catfish, Mystus tengara (Ham.)

A study has been made of the effect of photoperiodism on the ovarian maturation of fresh water teleost, Mystus tengara (Ham.). The magnitude of ovarian recrudescence is more during maturing virgin phase as compared to during immature virgin and preparatory virgin phases in long photoperiod (14 hours/day) treated fish. Thus, the response of the ovary to long photoperiod increases with the approach of natural spawning period. The mature eggs are more in long photoperiod treated fish than in controls exposed to 12 hours/day photoperiod. In long photoperiod treated fish, prespawning period is reduced considerably. During spawning and post-spawning periods, the ovaries are refractory, since long photoperiod is ineffective in maintaining gravid ovaries beyond spawning period and the ovarian regression during post spawning period sets in regardless of nature of photoperiod treatment.     

A new optical multichannel microspectrofluorometer.

A new microspectrofluorometer has been developed that combines a photometric fluorescence microscope with an optical multichannel analyzer. This instrument provides fluorescence emission spectra of biological materials by detecting the entire spectrum simultaneously in real time. These spectra are subsequently recorded and corrected so as to identify the fluorescent reaction products or to test whether fluorescent cytochemical probes bind to the expected substrate within cells. The procedures and advantages of optical multichannel analysis are described, and an application of microspectrofluorometry to acriflavine-Feulgen cytochemistry is given.     

Nucleotide regulation of a eukaryotic protein synthesis initiation complex;.

Formation of a ternary initiation complex containing Met-tRNAf, GTP and eukaryotic initiation factor 2, is the first step in sequential assembly of the initiation complex. The concentration of GTP required for half maximal formation of the ternary complex is 2.5 with 10(-6) M. GDP is a potent competitive inhibitor of ternary complex formation with Ki = 3.4 with 10(-7) M. The nucleotide binding site on eukaryotic initiation factor 2 demonstrates relative specificity for GDP with KD(GDP) = 3.0 with 10(-8) M; 100-fold higher concentrations of GTP than GDP are required for displacement of either [(3)H]GDP or [(3)h]gtp from the necleotide binding site. An ATP-dependent stimulation of ternary complex formation observed in partially purified initiation factor preparations is due to nucleoside diphosphate kinase (EC 2.7.4.6) which serves to remove inhibitory levels of GDP by phosphorylation with ATP. Since GTP is hydrolyzed to GDP during protein synthesis, this provides a mechanism by which the ATP:ADP ratio may regulate the rate of initiation of protein synthesis.     

The circular dichroism of lysozyme.

The circular dichroism spectra of hen egg white lysozyme, and of lysozyme derivatives in which tryptophan residues 62 or 108, or both, are selectively oxidized, have been measured as a function of pH over the range of 200 to 310 nm. Neither Trp-62 nor Trp-108 is principally responsible for the positive rotational strength in the 280 to 300 nm region. The spectrum in the 200 to 230 nm region is nearly the same in the native protein and in the derivatives, and is little affected by binding of saccharide. These results are used to reinterpret the circular dichroism spectra of the lysozymes and alpha-lactalbumins.     

Affinity labeling of the inhibitory DPNH site of bovine liver glutamate dehydrogenase by 5'-fluorosulfonylbenzoyl adenosine.

A new adenosine analogue has been synthesized, 5'-fluorosulfonylbenzoyl adenosine, which reacts covalently with bovine liver glutamate dehydrogenase with the incorporation of approximately 1 mol of 5'-sulfonylbenzoyl adenosine per peptide chain. Native glutamate dehydrogenase is known to be inhibited by relatively high concentrations of DPNH by binding to a second noncatalytic site; the major change in the kinetic characteristics of the modified enzyme is a total loss of this inhibition by DPNH. The modified enzyme retains full catalytic activity as measured in the absence of allosteric ligands, is still inhibited more than 90% by GTP, and is activated normally by ADP. These results demonstrate that the catalytic as well as the GTP and ADP regulatory sites are distinct from the inhibitory DPNH site. The rate constant for reaction of 5'-fluorosulfonylbenzoyl adenosine is decreased by high concentrations of DPNH alone or by DPNH plus GTP, but not by the substrate alpha-ketoglutarate, the coenzymes DPN or TPNH, or the regulators ADP or GTP alone. These observations are consistent with the postulate that the 5'-fluorosulfonylbenzoyl adenosine attacks exclusively the second inhibitory DPNH site. The DPNH inhibition is abolished when an average of only 0.5 mol of 5'-sulfonylbenzoyl adenosine per peptide chain has been incorporated. The structure of 5'-fluorosulfonylbenzoyl adenosine is critical in determining the course of the modification reaction. The smaller compound p-fluorosulfonylbenzoic acid does not affect the kinetic characteristics of the enzyme, and the isomeric compound 3'-fluorosulfonylbenzoyl adenosine produces a different pattern of changes in the regulatory properties (Pal. P. K., Wechter, W. J., and Colman, R. F. (1975) Biochemistry 14, 707-715). Indeed, enzyme which has combined stoichiometrically with 5'-fluorosulfonylbenzoyl adenosine is still able to react with 3'-fluorosulfonylbenzoyl adenosine; thus, the two adenosine analogues appear to react at distinct sites on glutamate dehydrogenase. It is proposed that 5'-fluorosulfonylbenzoyl adenosine will be complementary to 3'-fluorosulfonylbenzoyl adenosine as a general affinity label for dehydrogenases as well as other classes of enzymes which use adenine nucleotides as substrates or regulators.     

7-Amino-actinomycin D as a cytochemical probe. I. Spectral properties.

The optical absorption and fluorescence characteristics of 7-animo-actinomycin D were determined to evaluate its potential as a fluorescent cytochemical probe. At pH 7.0, the absorption maximum and fluorescence excitation maximum are both at 503 nm; the fluorescence emission is at 675 nm. When this compound forms complexes with DNA in solution, the absorption and fluorescence excitation maxima shift to 543 nm and the fluorescence emission shifts to 655 nm. The fluorescence quantum yield is 0.016 for 7-amino-actinomycin D free in solution and 0.01-0.02 for complexes with native DNA. The 7-amino-actinomycin D also exhibits fluorescence shifts characteristic of binding when put into solution with poly(dG-dC) poly(dG-dC), but not with poly(dI-dC) poly(dI-dC). The spectral characteristics are the same at pH 7.0 whether the solvent is 0.01 M PO4 with 0.0001 M EDTA or Earle's salts with 0.025 M N-2-hydroxyethylpiperazine-N1-2-ethanesulfonic acid.     

Comparative studies on the structural phosphoproteins of mammalian type C viruses.

The major phosphoprotein common to woolly monkey sarcoma virus, gibbon ape lymphosarcoma virus, and type C viruses of the lower mammalian species (mouse, rat, cat), with the exception of the endogenous cat virus (RD-114), is the polypeptide of about 12,000 molecular weight. The protein-phosphate bond in this polypeptide of several viruses is of the phosphoserine variety excepting gibbon ape virus, which contains both phosphoserine and phosphothreonine. The primary phosphoprotein of RD-114 virus and the endogenous baboon type C virus, on the other hand, is the polypeptide of about 15,000 molecular weight which contains phosphothreonine as its phosphoamino acid. A second major phosphoprotein of molecular weight of 10,000 is detected only in viruses genetically related to rat species including those derived from the RPL cell line, from Sprague-Dawley rat embryo cells, and the Kirsten mouse sarcoma virus which was recovered from a mouse erythroblastosis virus after in vivo propagation through rat. These phosphorylated polypeptides of molecular weight 15,000, 12,000, or 10,000 are present in the virion structure in several different but nonrandom phosphorylated states.     

Phosphoproteins: structural components of oncornaviruses.

Oncornaviruses, which contain a virion-associated protein kinase, were found to possess phosphoproteins as virion structural components. One major phosphoprotein common to strains of laboratory and wild mouse oncornaviruses and a strain of feline leukemia virus was shown to be a polypeptide of about 12, 000 mol wt. In addition to this, the Kirsten strain of murine sarcoma virus contained a second major phosphoprotein of about 10, 000 mol wt, and mouse erythroblastosis virus contained a second major phosphoprotein that was either identical to or comigrated with the virion glycoprotein of about 74, 000 mol wt. The major phosphoprotein of RD-114 virus was found to be of about 16, 000 mol wt. The major phosphoamino acid of the 12, 000-mol wt polypeptide of the mouse erythroblastosis virus was identified as phosphoserine, and that of the 16, 000-mol wt polypeptide of the RD-114 virus was identified as phosphothreonine.     

Adrenal Cortical Ribonuclease H (hybrid) (38494).

Ribonuclease Hybrid (RNaseH) from adrenal cortical tissue has been characterized. RNase H specifically degrades the RNA strand of purified RNA:DNA hybrids but is inactive on single- or double-stranded RNA or on DNA. The mode of clevage by RNase H is endonucleolytic, producing oligoribonucleoties with 3'-hydroxyl and 5'- phosphate termini. ACTH administration to guinea pigs results in no detectable change in adrenal cortical RNase H activity at times when changes in DNA synthesis are marked.