Intrinsic Protein Phosphorylation in Synaptosomal Plasma Membrane Fragments: A Comparison of Cerebral Cortex Tissue from Several Species, Including Human Biopsy Specimens

Intrinsic protein phosphorylation was studied in synaptosomal membrane fragments made from cerebral cortex tissue taken from the following species: human (biopsy specimens), ox, rat, rabbit, guinea pig and mouse. Membrane fragments from all species exhibited a qualitatively similar range of protein acceptors phosphorylated by cyclic AMP-dependent protein kinase activity; contrary to a previous report, no evidence for cyclic GMP-dependent protein kinase activity was found in the human material. With the exception of membrane fragments prepared from ox brain, all the preparations exhibited the same range of Ca2+-dependent protein kinase activity. Ox brain obtained from a slaughterhouse yielded membranes containing no Ca2+-dependent protein kinase activity, but this may have been due to unavoidable postmortem losses.     

Epidermal growth factor initiates DNA synthesis after a time-dependent sequence of regulatory events in Swiss 3T3 cells—interactions with hormones and growth factors

Epidermal growth factor (EGF) stimulates the initiation of DNA synthesis in Swiss 3T3 cells after a constant prereplicative period of 14–15 hours. The final rate of initiation follows apparent first-order kinetics and can thus be quantified by a rate constant k. The value of k can be changed by later additions during the prereplicative period: When cells stimulated by a very low concentration of EGF, alone or with insulin, which results in a relatively low value of k, receive a saturating amount of EGF at 15 hours, then k is markedly increased after 4–6 hours. Insulin alone (up to 200 ng/ml) is unable to set the lag phase, but does have a synergistic effect on the value of k given by EGF. When added at 15 hours, insulin also increases k, but after a delay of 4–6 hours. In contrast, both hydrocortisone and prostaglandin E₁ (PGE₁) inhibit the stimulation of DNA synthesis by EGF only during the first 8 hours of the prereplicative period of decreasing the value of k. Prostaglandin F_2α (PGF_2α), which stimulates DNA synthesis in a similar mode as EGF, when added with EGF has a synergistic effect on DNA synthesis. This suggests that EGF and PGF_2α, nevertheless, act through different regulatory events.     

Guanosine-5′-diphosphate-3′-diphosphate inhibits the in, vitro synthesis of β-lactamase from pBR322 DNA

The $\text{in}\text{,}\text{vitro}$ synthesis of β-lactamase directed by pBR322 DNA is inhibited by guanosine-5′-diphosphate-3′-diphosphate.     

Stimulation of the initiation of DNA synthesis and cell division in several cultured mouse cell types : Effect of growth-promoting hormones and nutrients

The ability of prostaglandin F_2α (PGF_2α) and other prostaglandins to stimulate the initiation of DNA synthesis in quiescent cultures of various mouse fibroblastic cell types has been investigated. PGF_2α was found to be more effective than the other prostaglandins. Most cell types, with the exception of BALB/c 3T3, responded to PGF_2α. Addition of PGF_2α in combination with insulin resulted in a synergistic increase in the proportion of cells synthesizing DNA. The effect of nutrients on the stimulation of the initiation of DNA synthesis has been examined in detail; it was found that Swiss 3T3 cells showed a requirement for hypoxanthine and vitamin B₁₂ whereas Swiss 3T6 cells demonstrated a stringent requirement for vitamin B₁₂ only. The effect of prostaglandin precursors, synthetic analogues of the prostaglandin endoperoxides and inhibitors of prostaglandin synthesis was also examined in two cell types. The effect of PGF_2α was compared with that of two polypeptide growth factors, epidermal growth factor (EGF) and fibroblast growth factor (FGF) in Swiss 3T6 cells grown in 0.0025% (v/v) serum. In combination with insulin each of these three growth factors stimulated the initiation of DNA synthesis in approximately the same number of cells.     

Trypanosoma cruzi: defined medium for continuous cultivation of virulent parasites.

A liquid medium was developed for the continuous cultivation of Trypanosoma cruzi. Among the several highly purified macromolecules tested only bovine liver catalase, horseradish peroxidase, lactoperoxidase, and bovine hemoglobin supported the continuous growth, at high yield, of mice-virulent Trypanosoma cruzi; other hemoproteins were inactive. Bovine liver catalase showed optimal Trypanosoma cruzi growth-promoting activity, parasites reaching 20 × 10⁶ parasites/ml (95% epimastigotes) at about 10 days in most of the 45 subpassages to date. Furthermore, this protein in the incubation medium provided all the amino acid requirements of actively growing parasites, thus eliminating the need for exogeneous free amino acids. Additional experiments revealed that the hemoprotein's growth-promoting activity was independent of any enzymatic activity and that reconstituting the exact protein composition by means of exogeneous amino acids did not support parasite multiplication, suggesting the importance of the primary structure of the active proteins for growth-promoting activity. These active macromolecules supported the multiplication of five different strains of Trypanosoma cruzi, but did not support Leishmania brasiliensis or Leishmania mexicana proliferation, suggesting species specificity.     

Comparison of adipose glycerol kinase of hyperglycemic obese mice and lean litter-mates

Summary Glycerol kinase activity found in the epididymal adipose tissue of lean litter-mates of hyperglycemic obese mice exhibits two distinct Km values. The Km(s) obtained graphically by a Hoftsee plot are 40 and 637 m. The glycerol kinase of obese mice showed 29 times more total activity per fat pad or 9 times more activity per mg of protein as compared to that of the lean controls. The increased glycerol kinase activity found in the obese mice predominantly exhibited low Km value. Increase in the activity with the high Km value was minimal. The apparent molecular weight of adipose glycerol kinase is approximately 60,000–65,000. A higher activity of glycerol kinase in the epididymal adipose tissue of the obese mice (ob/ob) has been reported by Treble and Mayer ¹. The significance of this increased enzyme in obesity² has been investigated by many investigators^3–6, using either whole epididymal fat pad or isolated fat cells as the source of glycerol kinase. However, little is known regarding the properties of this enzyme, and even the characterization of the liver glycerol kinase is not yet complete^{7, 8}.We have recently shown that adipose glycerol kinase of Sprague-Dawly rat and of Swiss mice exhibit two Km values, and the apparent molecular weight is approximately 55,000–60,000⁹. The present study was undertaken in order to establish whether the enzyme in ob/+ mice also exhibits two Km values and if so, whether the activity associated with both Km(s) is increased. We found that the specific conditions for enzyme activity determination and the basis on which the activity is expressed are quantitatively important in clarifying the difference in glycerol kinase activity of ob/ob and ob/+ mice. The increased activity in ob/ob mice is predominantly the low Km enzyme, and the apparent molecular weight is approximately 60,000–65,000.Supported in part by the Juvenile Diabetes Foundation. We thank Mr. CHARLES ROSENTHAL for technical assistance.     

Binding of aminoacyl-tRNA to rat liver ribosomal proteins

Rat liver ribosome treatment with ethanol and 1 M NH₄Cl releases some 31–33 ribosomal proteins. This split protein fraction binds Phe-tRNA, Ac-Phe-tRNA, Met-tRNA_M and f-Met-tRNA_F in the absence of K⁺ and Mg⁺⁺ ions. When the split protein fraction is passed through Sephadex G-100 only six proteins are retained in the column: S10, S14, S15, S19, L35, and L36. The aminoacyl-tRNA binding activity of this protein fraction retained in the Sephadex G-100 column is similar to that of the total split protein fraction, suggesting that the above six proteins, or only some of them, are involved in the binding reaction.     

Phenobarbital induction of egasyn: availability of egasyn in vivo determines glucuronidase binding to membrane.

Murine egasyn, a protein which stabilizes the binding of β-glucuronidase to microsomal membranes, was induced 1.9 fold in liver by phenobarbital treatment. Accompanying this increase was an alteration of the subcellular distribution of liver β-glucuronidase, although total glucuronidase activity remained constant. In control mice 32.6 ± 4.6% of the activity was microsomal, while after four days of phenobarbital treatment 50.5 ± 3.1% was microsomal. Thus, the availability of egasyn appears to be an important factor in determining the proportion of glucuronidase distributed to either microsomes or lysosomes.     

Studies on cytochrome c. XI. Circular dichroism studies on synthetic peptides related to the C-terminal region of baker's yeast iso-1-cytochrome c

Circular dichroism studies on synthetic peptides related to the C-terminal region of yeast iso-1-cytochrome c were carried out and compared with conformational studies on horse cytochrome c fragments. Evidence is presented for a weaker predisposition for ordered structure in the former peptides when compared with the corresponding region in horse cytochrome c. These findings agree with theoretical predictions and with observations that yeast and other mammalian type cytochromes c differ in several minor respects.