Relations between enzymatic and association reactions in the development of bovine fibrin clot structure

Development of fibrin clot structure was examined at pH 7.0, Γ/2 0.15, and 29 °C as a function of thrombin and fibrinogen concentrations. Parameters for the release of Apeptides, to give $\text{?}{}_{\mathrm{<mtext>A</mtext>}}$ were evaluated. Characteristics of time dependencies of development of turbidity, 90 ° light scattering, network, and compactible network were established. Mean mass/length ratios of fibrin in developing and mature networks were determined. Relationships between results combined with an inferred dependence of lateral interaction on release of B-peptides are used to disclose a model in which a protofibril network is formed first and the intrinsic length of this network (i.e., length exclusive of overlap or loose ends) determines network length, thus mean mass/length ratio, at maturity. Statements regarding initial protofibril network are: (i) A dominant group of $\text{?}{}_{\mathrm{<mtext>A</mtext>}}$-protofibrils appears first. With decreasing rate of production of $\text{?}{}_{\mathrm{<mtext>A</mtext>}}$ their average length increases and number decreases. (ii) Slower release of B-peptides produces $\text{?}{}_{\mathrm{<mtext>AB</mtext>}}$ whose fraction $\text{θ}{}_{\mathrm{<mtext>AB</mtext>}}\text{=}\text{?}{}_{\mathrm{<mtext>AB</mtext>}}\text{(?}{}_{\mathrm{A}}\text{+?}{}_{\mathrm{AB}}$) determines the occurrence of protofibril regions capable of contributing to a lateral interaction sufficiently stable for the formation of network. (iii) When dominant protofibrils attain a minimum combination of average length, number concentration, and frequency of occurrence of capable regions, an initial protofibril network is rapidly generated. (iv) Capable regions near protofibril ends are preferentially involved in initial network formation. (v) The initial network mesh size is large compared to average concomitant free protofibril length. (vi) With B-peptide release dependent on prior A-peptide release, protofibrils in the initial network have the highest capable region frequency, and this is maintained as lateral interaction progresses. Then, fibrin which is free at initial network formation and fibrin which is produced subsequently interact mainly to increase the mean mass/length ratio of initial network elements.     

Mechanism of action of thrombin on fibrinogen. Reaction of the N-terminal CNBr fragment from the Aalpha chain of human fibrinogen with bovine thrombin

The 51-residue N-terminal cyanogen bromide fragment from the Aα chain of human fibrinogen was isolated, and the Michaelis-Menten constants, K_m and k_cat, for its hydrolysis by bovine thrombin were determined. The measured values of K_m and k_cat are 4.7 × 10^?5m and 4.8 × 10^?10m [(NIH U/liter) sec]^?1, respectively. Since these values are similar to those for fibrinogen, it appears that the N-terminal CNBr fragment contains all amino acid residues whose interactions with thrombin account for the high specificity of this enzyme for fibrinogen.     

Mechanism of action of thrombin on fibrinogen. The reaction of thrombin with fibrinogen-like peptides containing 11, 14, and 16 residues

The following peptides were synthesized by classical methods in solution: Ac-Gly-Gly- Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH₃ (A), Ac-Ala-Glu-Gly-Gly-Gly-Val- Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH₃ (B), and Ac-Phe-Leu-Ala-Glu-Gly-Gly- Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH₃ (C). The rates of hydrolysis of the Arg-Gly bond of these three peptides by thrombin were measured, and the values of $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ were found to be 0.05 × 10^?7 (A), 0.02 × 10^?7 (B), and 1.6 × 10^?7 (C) [(NIH units/ liter)s]^?1. The value of$\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ for peptide C is less than 1% of that for fibrinogen [although the value of k_cat itself, for peptide C (but not for A or B), is comparable to that for fibrinogen]. These results indicate that phenylanine and leucine at positions P₉ and P₈, respectively, play a key role in the reaction of thrombin with fibrinogen. The data also show that factors outside of the 16 residues of peptide C are important in determining the rate of hydrolysis of fibrogen by thrombin.     

A pulsed N.M.R study of D2O bound to 1,2 dipalmitoyl phosphatidylcholine

Spin lattice relaxation times in both the lab and rotating frame, have been measured for deuterons (²H) in a number of unsonicated dispersions of 1,2 dipalmitoyl phosphatidylcholine in D₂O over a range of resonant frequencies from 13 MHz to 1 MHz for temperatures from ?20°C to 65°C.The proton (¹H) spin lattice relaxation time for the lecithin was measured for resonant frequencies of 8.5 MHz, and 40 MHz over a similar range of temperatures.The results agree with broadline measurements by Salsbury et al. [1], and for the liquid crystal phase are consistent with an anisotropic tumbling model of the water molecules bound to the lecithin headgroup. This tumbling occurs with correlation times of ≤10^?10 sec and ≈ 10^?6 sec about axes parallel to and perpendicular to the bisector of the D-O-D angle within a D₂O molecule, hydrogen bonded to the negatively charged phosphate headgroup.     

A favourable assessment of the O.P.T.--histamine reaction in the method of von Redlich and Glick

During the course of an investigation of the properties of triglyceride lipase in nerve endings of the central nervous system (1) there arose a need for rapid determinations of a lipase in a large number of membrane preparations. This communication reports a procedure for the determination of glyceride lipase in which fatty acid, formed from a radioactive substrate by the lipase, is separated from glycerides on DEAE-cellulose paper.     

Control of the general amino acid permease of Penicillium chrysogenum by transinhibition and turnover

When nitrogen-starved mycelium of Penicillium chrysogenum is incubated with relatively high concentrations of labeled hydrophobic amino acids, influx is followed by efflux of the corresponding labeled α-ketoacid. In spite of the efflux, further transport activity is suppressed. Cell-free extracts contain a transaminase that accepts all those amino acids exhibiting α-ketoacid efflux. Transaminase activity is constitutive but is induced to a 2- to 3-fold higher level during a 2-hr preincubation period with a hydrophobic amino acid. Cycloheximide prevents efflux and also the induction of the transaminase. Cycloheximide itself stimulates a partial decay in transport activity but mycelium preincubated with l-leucine and cycloheximide together retain a greater fraction of the original transport activity than mycelium preincubated with l-leucine alone. The results suggest that transport is regulated partially by transinhibition but a significant part of the substrate-induced decay of transport activity is caused by either (a) the degradation of a permease component (perhaps facilitated by transinhibition), or (b) the induction by the substrate of a regulator protein (perhaps the transaminase).The uptake of labeled substrates by nutrient sufficient mycelium correlates well with lipid solubility of the substrates. This suggests that the nonsaturable uptake observed in these mycelia results from free diffusion of the uncharged species.     

Mutagenesis by 4-nitrobenzofurazans and furoxans.

15 nitrobenzofurazans and 10 nitrobenzofuroxans synthesized primarily for testing as potential anti-rheumatic drugs were also tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100. The method used involved placing each compound in a "well" cut out of a plate of selective medium previously seeded with the appropriate tester strain, and then adding a rat liver microsome/cofactor mixture to one of the two wells on each plate. This method is considerably cheaper and more convenient than the conventional agar overlay technique, but in the present series of experiments failed to detect 4 compounds which could be detected by the overlay technique. Using a combination of the two techniques, 21 of the 25 compounds tested were found to be mutagenic. All 10 benzofuroxans and 9 of the 15 benzofurazans were detected as direct-acting mutagens with at least one of the two tester strains. Two of the benzofurazans gave positive results only in the presence of rat liver microsomes, and hence are pro-mutagens. One of the 4 benzofurazans which gave negative results for mutagenicity in these tests was found to be an efficient inhibitor of a neutral protease activity found in rheumatic synovial fluid, and may therefore have some potential as an anti-rheumatic drug.     

The ontogeny of social relationships with group companions among free-ranging infant rhesus monkeys II. Differentiation and attractiveness

Infants are frequently with their mothers when troop members initiate interaction with them. To what extent are the infants' companions attracted specifically to them rather than to the mother? A simple method is presented for approaching this question and for assessing the extent to which the relationships of 11 free-ranging rhesus (Macaca mulatta) infants on Cayo Santiago have acquired qualities independent of (differentiated from) those of the mother. Companions of all ages tend to be attracted specifically to infants between 1 and 14 weeks of age and not simply to their mothers. However, this tendency is less marked for sibling companions than for more distantly related companions. No differences are found between infants of high- and low-ranking mothers. Infantile attractiveness is not necessarily sustained when infants are 17 to 30 weeks of age. The degree to which relationships with infant companions and with older female companions have, differentiated by 17 to 30 weeks is negatively related to the companion's age. Infants show early and increasingly marked tendencies to develop differentiated relationships with one another; however, they show no signs of doing so with adult female companions by 30 weeks of age.     

The ontogeny of social relationships with group companions among free-ranging infant rhesus monkeys I. Social networks and differentiation

Social networks of infant rhesus monkeys (Macaca mulatta) in a free-ranging, lineage-based group on Cayo Santiago are described by assessing the extent to which four measures of positive interaction between infants and finely-divided categories of companions are associated with (1) degree of relatedness through maternal lines; (2) sex of the companion; (3) age of the companion; and (4) dominance rank of the infant's lineage. The results suggest that the infant's social network mirrors that of its mother both in the first weeks of life and as late as 30 weeks of age. Infants have more positive social interaction with close kin than with distant kin or with unrelated individuals, and thus function as members of their lineage from the beginning. They associate more with female companions than with male companions, and more with younger immatures than with older immatures. Finally, infants in the top-ranking lineage spend more time with their own relatives than do infants in other lineages. The fact that these patterns change little as the infant gains independence from the mother supports suggestions that early maternal influence serves to pass on aspects of the mother's social network. It is suggested that the ontogeny of early social relationships resembles a process of differentiation.     

Analyzing the joint effects of two antibodies and the design of molecularly engineered vaccines

A new analytical approach to determine how antibodies relate to each other in producing immunity is presented. One purpose of this analysis is to decide on the antigenic composition of vaccines. Regression analyses and single discrete analyses are shown to be inappropriate to this task. The proposed analyses makes multiple cut points on two pre-exposure antibody levels. At each resulting discrete classification, additive and multiplicative interaction parameters are calculated. Various models of joint antibody action are shown to produce two general patterns of interaction terms with this type of analysis. One pattern characterizes situations where each antibody, or something substituting for the action of each antibody, is always needed to eliminate all infections. This may be because the antigens to which the antibodies are directed are on separate microbes or because microbes can only be neutralized by the joint action of both antibodies. A second pattern characterizes situations where both antibodies act cooperatively but given high enough levels only one may be needed. This may be because the antibodies have the same molecular effect or because they act by separate means against the same organisms in an innoculum.     

Structural aspects of thrombin specificity.

Computer-assisted comparisons were made of the X-ray coordinates of all homologous atoms in the serine protease derivatives tosyl chymotrypsin Aα, tosyl elastase, and diisopropylphosphoryl trypsin. The results provided further quantitative support for the belief that sequence homology in proteins results in close similarity of conformation. On this basis, inferences were drawn about the three-dimensional structure of the serine protease thrombin, for which atomic coordinates have not yet been determined experimentally. Further, it was concluded that the unique specificity of thrombin, i.e., its selective cleavage of certain ArgGly bonds in fibrinogen, is unlikely to be due to the insertions in the amino acid sequence of thrombin or to differences in sequence in the region of the active site and binding pocket. It is possible, however, that the elongated A chain appended to thrombin may be a source of this specificity.     

Conditions for the various types of co-operativity allowed by the adair,equation and their relation with the algebraic character of binding polynomials

A convenient way to obtain for any number, n, of sites, the functions of the constants of the Adair equation that decide the type of co-operativity of ligand binding to a non-dissociating protein is given and is illustrated by the examples n = 4 and n = 5. These functions are invariants of the binding polynomial and various of its derivatives.Although there are some simple sufficient conditions (inequalities relating successive Adair constants) for some co-operativity types, the full necessary and sufficient conditions even for uniform positive and negative co-operativity depend on very complicated functions of the constants for n > 4.However there are alternative ways of writing binding polynomials known as canonical forms. Up to at least n = 5, and probably beyond, the conditions that are complicated in terms of Adair constants are very simple in terms of the constants of canonical forms. For instance any fourth-degree polynomial can be written in the form p(x - α)⁴ + q(x - β)⁴ + 6μ (x - α)²(x - β)² although in three different ways. For one of these ways, the sign of μ distinguishes between mixed and uniform co-operativity. For any kind of mixed co-operativity μ > 0, while μ < 0 corresponds to uniform co-operativity. Advantages of the use of canonical forms are briefly commented on.     

Mössbauer studies on protoporphyrin IX iron(II) frozen solutions containing ligands that cause the iron to be in a five coordinate high spin iron(II) environment

Mössbauer parameters are presented for a number of protoporphyrin IX iron(II) complexes containing ligands that allow the iron to be in a five coordinate high spin iron(II) electronic environment. Such environments are characterised by large quadrupole splittings in the range 4.0 to 4.4 mm s^?1. These compounds have characteristic electronic spectra.The implications of catechol type ligands binding protoporphyrin IX iron II/III moieties are discussed.     

On the identity of the 640-nm component observed in chlorophyll b absorption spectra in vivo

Mathematical analysis of the results of mild acetone extraction of chloro-plast-fragment preparations from Ulva lactuca demonstrates that the 640-nm shoulder on the short-wave side of the red chlorophyll b absorption band is not due to this chlorophyll.

The analysis furthermore provides rather strong evidence that the 640-nm shoulder is correlated with an absorption band peaking around 682nm. It is suggested that the 640-nm component is due to the reaction center pigment of Photo-system II.     

Kinetics of photosynthetic membrane protein assembly in Rhodopseudomonas spheroides

Glycogen phosphorylase in cell-free extracts of Neurospora crassa is activated 10- to 15-fold by incubation with MgATP^2?. When the MgATP^2? is removed, the active form (a form) reverts to the inactive form (b form). The inactivation requires Mg²⁺ and is inhibited by NaF. The results confirm that Neurospora crassa glycogen phosphorylase exists in two interconvertible forms and strongly suggests that the interconversion is catalyzed by a kinase and phosphatase. The a form was partially purified. The enzyme has a molecular weight of 320,000. Uridine diphosphate glucose is a linear competitive inhibitor with respect to glucose-1-phosphate and a linear non-competitive inhibitor with respect to glycogen. Glucose-6-phosphate is a hyperbolic (partial) noncompetitive inhibitor with respect to all substrates in both directions. The b form of the enzyme in crude cell-free extracts is stimulated 2- to 3-fold by 5′-AMP. As the b form is purified, the 5′-AMP activation is diminished. The molecular weight of the partially purified “b” form was also 320,000.