Determination of the number of cross-links in a protein gel from its mechanical and swelling properties

The relation between the chemical structure of a protein and the physical properties of a heat-set gel of that protein has been investigated. The physical properties of the gel are determined by means of mechanical experiments in which the viscoelastic properties of the gel are determined in terms of the storage shear modulus, the loss modulus and the stress-strain curve. The storage shear modulus defined the solid (elastic) character of the gel. The chemical structure of the protein and the nature of the solvent determine the nature and number of cross-links in the gel. The cross-links in gels formed by heating concentrated solutions of ovalbumin in 6M urea solutions were found to be disulfide bridges and the mechanical properties of these ovalbumin/urea gels approximated those of an ideal rubber. The latter finding enables one to calculate the number of cross-links per ovalbumin molecule from the value of the storage modulus, using the classical theory of rubber elasticity. This theory, together with the Flory-Huggins lattice model, can also be used to calculate the number of cros-links per ovalbumin molecule from the swelling behavior of ovalbumin/urea gels. The number of cross-links per ovalbumin molecule calculated from these two types of experiments are in mutual agreement and correspond with the number of thiol groups in ovalbumin. We conclude, thereforee, that theories of polymer physics can be used to relate the chemical structure of a protein to the physical properties of its gel.     

T1 and T2 lymphocytes in primary and secondary delayed type hypersensitivity of mice. I. Contribution in the response to sheep red blood cells and to allogeneic spleen cells.

Peripheral T lymphocytes can be subdivided into two populations (T₁ and T₂ cells) based upon the short life span of T₁ cells after adult thymectomy (ATx) and sensitivity of T₂ cells to treatment with anti-thymocyte serum (ATS) in vivo. The contribution of the T₁ and T₂ cells to primary and secondary delayed type hypersensitivity (DTH) to sheep red blood cells (SRBC) and to primary DTH to allogeneic cells was studied in mice. T₂ cells were found to account for the development of the state of primary DTH responsiveness after intravenous immunization with SRBC and after subcutaneous immunization with allogeneic cells. No clear cut evidence was found that in the presence of T₂ cells DTH related T effector cells were generated from T₁ cells. In mice selectively depleted for T₁ cells by ATx, the remaining T₂ cells were capable to generate SRBC-specific T memory cells, but not in numbers as large as in non-thymectomized mice. On the other hand, T₁ cells in mice depleted for T₂ cells by ATS treatment, could give rise to normal numbers of SRBC-specific T memory cells. Apparently T₁ cells can compensate for the absence of T₂ cells during generation of T memory cells, but T₂ cells cannot do so for the loss of T₁ cells. From the time curve showing the ATx-induced decline of the population of SRBC-specific T₂ cells, involved in primary DTH responsiveness, the half life was calculated to be 6 to 7 months.     

Accumulation of carnitine esters of beta-oxidation intermediates during palmitate oxidation by rat-liver mitochondria

Rat-liver mitochondria were incubated with [14C]palmitate in the presence of L-malate, fluorocitrate, and L-carnitine. The specific activities of acetyl groups incorporated into citrate, ketone bodies and acetyl-L-carnitine were measured. During state-4 oxidation of [1--14C]palmitate the specific activity of the acetyl-CoA pool was 1.3-times higher than that of the average acetyl group of palmitate, indicating an incomplete breakdown of the palmitate molecule. Accumulation of carnitine esters was observed in this condition. The acyl moieties of carnitine esters formed during the state-4 oxidation of [U-14C]palmitate or [16(-14)C]palmitate were analysed by radioactive gas-chromatography. Substantial amounts of beta-oxidation intermediates were found. The accumulation of carnitine esters of C6-C14 intermediates can quantitatively explain the high specific activity of the acetyl-CoA pool during the state-4 oxidation of [1(-14)C] palmitate. The localization and control of beta-oxidation are discussed.     

Binding of labeled initiation factor IF-1 to ribosomal particles and the relationship to the mode of IF-1 action in ribosome dissociation.

The binding of labeled initiation factor IF-1 to ribosomal particles has been studied in relation to the mode of action of this factor in the dissociation of 70-S ribosomes. It is demonstrated that IF-1 interacts specifically with active 70-S tight couples and free 30-S subunits. The binding of IF-1 to both 70-S and 30-S particles is not influenced by the Mg2+ concentration and the affinity of the factor for both particles is about the same. The interaction of IF-1 with these particles is highest at low Tris-HCl concentrations. Under these conditions IF-1 shows a slight dissociating activity. Using 3H-labeled IF-1 and 14C-labeled IF-3 the formation of a 30-S-subunit.IF-1 . IF-3 complex from 70-S ribosomes is demonstrated. Our studies show that IF-3 enhances the binding of IF-1 to the 30-S subunit. In contrast to IF-1, which binds about equally well to 70-S and 30-S particles in the absence of IF-3, 14C-labeled IF-3 binds predominantly to the 30-S subunit. This finding confirms the view that IF-3 acts as an anti-association factor. On the other hand, IF-1 enhances the supply of 30-S subunits in the presence of IF-3 by acting on the 30-S moiety of the 70-S ribosome.     

The binding of protons and inositol hexakisphosphate to ligated and unligated human des-Arg141alpha-hemoglobin.

The region of Bacillus stearothermophilus strain NCA 1503 23-S ribosomal RNA protected from T1 ribonuclease digestion by the 50-S ribosomal subunit protein L1 from Escherichia coli has been established. The sequence of 115 nucleotides is compared to the analogous region in E. coli. The similar behaviour of the RNA towards the recognition of protein L1 may be explained in terms of secondary base-pairing, even though there exists almost 40% difference between the primary nucleotide sequences.     

Absence of a prostaglandinic mediator in the lateral hypothalamic cardio-vascular inhibition.

The cardio-vascular inhibition elicited by electrical stimulation of the paraventricular nucleus in the lateral hypothalamus of anaesthesized and desafferentiated dogs is not linked with a mediator release like PGE. The effect of this biological agent is not registered in the isolated femoral artery of a receiver, after deviation by a cruised circulation of the arterial blood of a hypothalamic stimulated donor dog. Antipyretics, which are also prostaglandin-synthetase inhibitors, enhance the lateral hypothalamic reactions. As the thermoregulation centre is localized in the same region as the cardio-vascular inhibition centre and because lowering of temperature depends upon vasodilation and decrease in the general cellular metabolism, both functions of the paraventricular nucleus activity, a hypothesis is proposed that thermoregulation and cardio-vascular inhibition centres are a functional and anatomical unity.