Intramolecular catalysis. VII: The nature of side chain shielding of the 12alpha-hydroxyl group of steroids

A series of 12α-hydroxy steroids with varying side chains was prepared, and their 24-hour acetylation yields were compared, l2α-Hydroxy-5β-pregnan-20-one ($\text{lb}$) was prepared from 3α, 12α-diacetoxy-5β~pregnan-20-one ($\text{2}$) and also by side chain degradation of 12α-acetoxy-5β-cholanoic acid ($\text{5d}$). 21-Benzyl-5β-pregnan-12α-ol ($\text{1g}$) was synthesized by hydrogenation of the 21-benzylidine derivative of ketone $\text{1b}$. 23-Pheny1-5β-norcholan-12α-ol ($\text{1k}$) was obtained by the Grignard reaction of 2-phenyl-ethylmagnesium bromide and ketone $\text{1b}$, dehydration, hydrogenation and hydride reduction; a similar sequence produced 20-methyl-5β-pregnan-12α-ol ($\text{lm}$). The acetylation results (Table 11) imply that branching at C-20 may be more significant for 12α-hydroxyl reactivity than side chain length or type. An additional compound with an unbranched side chain, 21-nor-5β-cholan-12α-ol ($\text{14}$), was synthesized by a Grignard reaction on the 21-bromo intermediate $\text{11b}$. Acetylation rates determined by glc indicate (Table 111) That compounds with unbranched side chains have 12α-hydroxyl groups about ten times as reactive as their analogs with 20-methyl groups.     

Flash-induced absorption changes of the primary donor of photosystem II at 820 nm in chloroplasts inhibited by low pH or tris-treatment

A comparative study is made, at 15 °C, of flash-induced absorption changes around 820 nm (attributed to the primary donors of Photosystems I and II) and 705 nm (Photosystem I only), in normal chloroplasts and in chloroplasts where O₂ evolution was inhibited by low pH or by Tris-treatment.At pH 7.5, with untreated chloroplasts, the absorption changes around 820 nm are shown to be due to P-700 alone. Any contribution of the primary donor of Photosystem II should be in times shorter than 60 μs.When chloroplasts are inhibited at the donor side of Photosystem II by low pH, an additional absorption change at 820 nm appears with an amplitude which, at pH 4.0, is slightly higher than the signal due to oxidized P-700. This additional signal is attributed to the primary donor of Photosystem II. It decays ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ about 180 μs) mainly by back reaction with the primary acceptor and partly by reduction by another electron donor. Acid-washed chloroplasts resuspended at pH 7.5 still present the signal due to Photosystem II ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ about 120 μs). This shows that the acid inhibition of the first secondary donor of Photosystem II is irreversible.In Tris-treated chloroplasts, absorption changes at 820 nm due to the primary donor of Photosystem II are also observed, but to a lesser extent and only after some charge accumulation at the donor side. They decay with a half-time of 120 μs.     

Comparative analysis of the sequences of the three collagen chains α1(I), α2 and α1(III): Functional and genetic aspects

The amino acid sequences of type I collagen containing α1(I) and α2 chains at a ratio of 2:1, and of type III collagen consisting of α1 (III) chains are known. A statistical analysis of the sequences of these α chains is presented. The inter-chain comparison showed a high level of homology between the three α chains. The interactive amino acids, such as the polar charged and part of the hydrophobic residues responsible for the assembly of the molecules, are strongly conserved. The intra-chain analysis revealed that the α chains are divided into four related D units, each with a length of 234 residues. Between the D units within a chain the polar residues show a higher variability than the hydrophobic amino acids.Besides the D units, other periodicities such as $\text{D}\text{3}$ (78 residues), $\text{D}\text{6}$ (39 residues), $\text{solD}\text{11}$ (21 residues) and $\text{solD}\text{13}$ (18 residues) were observed, particularly in α1 (I) and α1 (III). The D unit is a functional repeat that is formed by the interactive polar charged and hydrophobic residues and which determines the aggregation of the molecules. The $\text{solD}\text{3}$ unit is mainly pronounced by the non-interactive residues such as proline and alanine and appears to be a reminiscence of a primordial gene. The smaller periodic repeating units may be considered as additional genetic units or as structural units, which determine the triplehelical pitch and thus the lateral aggregation of the molecules.In contrast to α1 (I) and α1 (III), the α2 chain shows less regularity in its internal structure.     

Heterogeneous amino acid transport rate changes in an E. coli unsaturated fatty acid auxotroph.

Amino acid transport rates in an $\text{E. coli}$ unsaturated fatty acid auxotroph were non-uniformly affected by enrichment of membrane lipids in various unsaturated fatty acids. Proline and threonine transport rates were depressed much more than lysine and asparagine rates by trans unsaturated acids. Myristoleate and linolenate enrichment also produced non-uniform but lesser rate reductions. Although changes in the relative numoer of effective transport catalysts could account for these findings, comparisons of proline and lysine transport rates over a broad temperature range indicated that non-uniform alterations in transport catalyst reaction rates account at least partly for the activity changes associated with membrane lipid alterations.     

Alkyl alkanethiolsulfonate sulfhydryl reagents: β-Sulfhydryl-modified derivatives of l-cysteine as substrates for trypsin and α-chymotrypsin

Derivatives of l-cysteine and the A chain of bovine insulin have been chemically modified at the cysteinyl β-sulfhydryl by certain sulfhydryl-specific alkyl alkanethiolsulfonate reagents. The alkanethiolation products possess mixed-disulfide side chains structurally similar to the side chains of lysine and phenylalanine and hence were studied here as substrates for trypsin and α-chymotrypsin, respectively. Kinetic parameters were obtained for the enzyme-catalyzed hydrolyses of the modified l-cysteine analogs and of specific reference amino acids which were derivatized analogously at both the α-amino and α-carboxyl groups and assayed identically. For both enzymes it was found that the specificity constants, $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$, for analog esters compare favorably with those for specific reference esters, whereas specificity constants for analog amides compare much less favorably with those for specific reference amides. This discrepancy is largely a consequence of the k_cat values for the analog amides being relatively much lower than the corresponding values for the reference amides. Consistent with this trend, no detectable enzyme-catalyzed hydrolysis of the amide bonds at the sites of modified cysteine residues in the A chain of bovine insulin was observed. It is proposed that the predominant kinetic consequence of the mixed-disulfide side chains of the alkanethiolated cysteine moieties is a decrease in the acylation rate constants, k₂, arising from an increase in the transition-state free energies of acylation.     

Specificity in the interaction of phospholipids and fatty acids with vesicle reconstituted cytochrome P-450. A spin label study

The association of fatty acids, androstane, phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid with purified and phospholipid-vesicle reconstituted cytochrome $\text{P-450}$ was studied by spin labeling. Spin-labeled fatty acids were found to be motionally restricted by cytochrome $\text{P-450}$ in both phospholipid vesicles and in microsomes to a much greater extent than spin-labeled phospholipids. The equilibrium of spin-labeled fatty acid between the bulk membrane lipid and the protein interface could be shifted towards an increased amount in the bulk phospholipid phase by the addition of oleic acid or lysophosphatidylcholine, but not by sodium cholate. Microsomes from different animals showed a variable extent of motional restriction of fatty acids, independent of pretreatment of the animals with phenobarbital or β-naphthoflavone, of cytochrome $\text{P-450}$ content, of the presence of type I and type II substrates for cytochrome $\text{P-450}$. These differences are attributed to the presence of varying amounts of lipid breakdown products in the microsomal membrane such as lysolipids or fatty acids which compete with the externally added spin-labeled fatty acids, or with spin-labeled androstane for the binding to cytochrome $\text{P-450}$. The negative charge of the fatty acid was found to be involved in its association with the protein. Cytochrome $\text{P-450}$ was shown to interact only with a few spin-labeled phospholipid molecules in such a way that the motional restriction of the spin acyl chains can be detected by electron paramagnetic resonance ($\text{τ}{}_{\mathrm{<mtext>R</mtext>}}\text{> 10}{}^{\mathrm{?8}}\text{s}$). The number of associated lipid molecules per protein probably is too small to form a complete shell around the protein. This lipid-protein interaction could be destroyed by the addition of sodium cholate, in contrast to the fatty acid-protein interaction.     

Horseradish peroxidase. XXV. An electron spin resonance study of the low temperature photochemical reaction of compound I.

The insoluble acrosome granule content of sea urchin sperm consists of a single 30,500 dalton protein named bindin. Bindin mediates species-specific recognition and adhesion of sperm to the egg surface. Bindin from $\text{Strongylocentrotus purpuratus}$ (Sp) and $\text{Strongylocentrotus franciscanus}$ (Sf) have tyrosine as their single N-terminal amino acid. The pI of Sp bindin is 6.62 and of Sf 6.59. Amino acid analysis reveals almost identical composition between the two species for 16 amino acids. Only two (or three) amino acids, Pro and Asx, show large species differences. Tryptic peptide maps of the two species of bindin show very similar patterns with 24 spots of identical correspondence.     

Quantitative relationship between photosystem I electron transport and ATP formation

The Photosystem I-dependent transport of electrons from diaminodurene to methylviologen is linear with reaction time and supports a constant rate of phosphorylation. However, if the diaminodurene is not kept fully reduced by the presence of excess ascorbate, the oxidized diaminodurene accumulates and begins to compete with the methylviologen as the electron acceptor. Thus, although the rate of ATP formation remains unchanged, an increasing proportion of the electron transport becomes cyclic and hence unmeasured. This leads to a rapid increase in the apparent efficiency of phosphorylation which is misleading.In contrast, it is known that the oxidized form of 3,3′-diaminobenzidine polymerizes to form an insoluble substance which should not be available to serve as an electron acceptor. However, 3,3′-diaminobenzidine is not a satisfactory donor of electrons in Photosystem I reactions for two reasons: the rate of electron transport quickly falls with reaction time and the oxidized form of 3,3′-diaminobenzidine seems to be an exceptionally efficient electron acceptor near the beginning of the period of illumination when it is presumably not yet polymerized. Thus in the first 2–3 sec of illumination when the reaction is still rapid much of the electron transport is cyclic and therefore unmeasured, especially in the absence of excess ascorbate. This cycling of electrons, which leads to an inflated apparent efficiency ($\text{P}\text{e}{}_2\text{> 2}$), is particularly pronounced at low donor concentrations.When cyclic electron transport is avoided by the use of ascorbate or by the selection of appropriate reaction times, both diaminodurene and 3,3′-diaminobenzidine support phosphorylation with an efficiency which is approximately half of the efficiency exhibited by the overall Hill reaction. The same is true when 2,5-diaminotoluene, tetrachlorohydroquinone, 4,5-dimethyl-o-phenylenediamine, and reduced 2,6-dichloroindophenol serve as electron donors. With these six substances, the phosphorylation efficiences were 0.57 ± 0.1 molecules of ATP formed for each pair of electrons transferred ($\text{P}\text{e}{}_2$). In the same chloroplasts preparations, the transport of electrons from water to methylviologen-supported phosphorylation with a $\text{P}\text{e}{}_2$ of 1.2.     

Liver alcohol dehydrogenase inhibition by fatty acid amides, N-alkylformamides and monoalkylureas.

With ethanol as substrate, N-n-alkylformamides and mono-n-alkylureas, like fatty acid amides, inhibited horse liver alcohol dehydrogenase uncompetitively, presumably by forming ternary complexes with the enzyme - reduced nicotinamide adeninedinucleotide binary complex. Compounds with 11- or 12-atom chains were better inhibitors than longer or shorter chain compounds. $\text{In vivo}$ (mice), the urea derivatives were ineffective, as were the amides, with the exception of butanamide; the latter compound was less active than iso-butanamide. Formamides with 4- to 12-atom chains were active $\text{in vivo}$ but, unlike $\text{in vitro}$, the shorter chain compounds were the most potent. A variety of branched-chain alkyl-, cyclic alkyl- and arylalkylamides and N-substituted formamides also inhibited alcohol dehyrogenase $\text{in vitro}$.     

Identification of specific amino acids in diabetic glomerular basement membrane collagen subject to non-enzymatic glucosylation in vivo

Glomerular basement membrane was purified from normal and streptozotocin-diabetic rats, and the insoluble collagenous fraction isolated following reduction and alkylation. Amino acid analysis of diabetic samples revealed three unusual peaks eluting near glucosamine. Two of these peaks had respective elution times identical to those of synthetic glucitol-hydroxylysine and glucitol-lysine. These findings identify lysine-derived amino acids in glomerular basement membrane collagen as sites of excess non-enzymatic glucosylation $\text{in vivo}$ in hyperglycemic diabetes.     

Possible role of NADPH-dependent enoyl coenzyme A reductase in -oxidation of unsaturated fatty acids

An 2-enoyl CoA reductase from rat liver mitochondria catalyzes the reduction of both oct-$\text{cis}$-2-enoyl CoA and its $\text{trans}$ isomer in the presence of NADPH as a specific electron donor. This reductase is solubilized from mitochondria by sonication. The possible role of the reductase in the β-oxidation pathway of the unsaturated fatty acids is discussed.     

Cellular binding of benzo(a)pyrene to DNA characterized by low temperature fluorescence

A new approach has been developed to detect ultra low concentrations of benzo(a)pyrene products bound to nucleic acids $\text{in}\text{vivo}$. The binding to DNA of hamster embryo cell cultures was characterized by low temperature fluorescence spectroscopy. The method can detect less than one polycyclic hydrocarbon residue per 50,000 nucleotides. The fluorescence spectra indicate that the benzo(a)pyrene derivative bound to DNA has a pyrene-like chromophore and resembles that obtained when DNA is reacted $\text{in}\text{vitro}$ with the 7,8-diol-9,10-oxide of benzo(a)pyrene. This confirms that metabolism of the 7,8,9,10 ring on benzo(a)pyrene precedes reaction with DNA. The method should be useful for detecting and characterizing the $\text{in}\text{vivo}$ binding of other fluorescent carcinogens to nucleic acids.     

P700 sensitization by low concentration of DCMU in isolated pea chloroplasts

Using steady-state relaxation spectrophotometric technique a P₇₀₀ component ($\text{t}\text{1}\text{2}\text{～20 ms}$) has been detected which was sensitized by low concentration (10^?7M) DCMU in isolated broken chloroplasts of pea. The relative quantum yield of electron flux through DCMU-sensitized P₇₀₀ was similar to that with methyl viologen or NADP as terminal electron acceptor and water as electron donor. Kinetic analysis showed that a small fraction (10%) of the total P₇₀₀ reaction centers was sensitized by low DCMU.     

Sequence regularities and packing of collagen molecules

Fourier analysis of sequences along edges of the type I collagen molecule constructed from two α1(I) and one α2 chains shows that the molecule is two-sided if the supercoil pitch of the α chains along the molecular axis, P, is 39 residues ($\text{D}\text{6}$, where D = 234 residues or 67 nm). One side has alternating charged and hydrophobic regions with spacings of $\text{D}\text{6}$, while the other side has an excess of hydrophobic residues with a spacing of $\text{D}\text{11}$. These characteristics arise from sequence regularities in the α chains and the geometric relationship between the chains. The pattern is marginally strongest with α2 as chain 1. The $\text{D}\text{6}$ sides could form the inside of a helical microfibril where contacts between molecules would fall P apart along the α chains. The $\text{D}\text{11}$ sides could form the outside of the microfibril where contacts between microfibrils would be spaced apart by the α chain supercoil along the microfibril axis, P′. If the microfibril is a 5₄ helix of D-staggered collagen molecules with a left-handed supercoil of pitch $\text{20D}\text{11}$, P′ is close to $\text{2D}\text{11}$ (43 residues). $\text{2D}\text{11}$ subsets in the α chains give rise to the $\text{D}\text{11}$ spacing along the molecule. The microfibril has 4₁ screw symmetry satisfying X-ray diffraction evidence that microfibrils pack in a tetragonal unit cell.This model is the same as proposed previously by us (Trus & Piez, 1976: Piez & Trus, 1977) except that P = 39 rather than 30 residues. Contrary to our earlier assumption, P = 39 residues is within the range allowed by X-ray diffraction measurements. The present results favor P = 39 since it relates regularities in the α chain sequences to helical parameters in a direct way. Furthermore, model studies show that geometric arguments which support P = 30 are equally strong at P = 39 residues.     

Transfer of cultured bovine embryos

A total of 126 bovine embryos were surgically collected from 16 superovulated donor heifers 5 days after estrus and randomly selected for either immediate transfer to synchronized recipients or $\text{in}$$\text{vitro}$ culture at 37°C for 24 hours and subsequent transfer. Twenty-four of 56 (42.8%) embryos maintained for 24 hours in Ham's F10 medium supplemented with 10% heat treated fetal calf serum (HTFCS) and transferred to 32 recipients produced live calves. Survival of 70 noncultured embryos transferred to 35 recipients was 55.7% (39 calves). The percentages of recipients that were diagnosed pregnant at 42 days with cultured and control embryos were 59.4% ($\text{19}\text{32}$) and 74.3% ($\text{26}\text{35}$), respectively. No statistical difference was observed between the $\text{in}$$\text{vitro}$ cultured and control embryos for viability following transfer to recipient females.In a second study, Day 7 embryos maintained in Ham's F10 medium supplemented with 10% HTFC serum for various culture periods were tested for viability following nonsurgical transfer to recipient females. A total of $\text{1}\text{5}$, $\text{1}\text{3}$ and $\text{0}\text{4}$ embryos cultured for 24, 48 and 72 hours, respectively, resulted in pregnant recipients following transfer.     

Limiting laws and counterion condensation in polyelectrolyte solutions: V. Further development of the chemical model

Counterion binding to polyelectrolyte chains is formulated as a chemical reaction M^z(free) → M^z(bound). Expressions for the chemical potentials of free and bound counterions are set equal to obtain the reaction equilibrium. The results are equivalent to those in the previous paper of this series. An additional result obtained here is that a polyion holds its bound counterion layer with a strength on the order of 100 $\text{kcal}\text{(mole cooperative unit)}$. The method is then applied to the calculation of the polarizability along the chain due to the bound (condensed) counterions.     

Fluorescent cholesteryl esters in the core of low density lipoprotein

Fluorescent cholesteryl esters with chromophores placed either on the acyl chain (cholesteryl-$\text{cis}$-parinarate), the sterol ring system (5,7,9-cholesteryl oleate), or the side chain (naphthylcholenamide oleate) have been incorporated into the core of low density lipoprotein. The temperature dependence of several fluorescence parameters has been evaluated. An analysis of the fluorescence lifetime components of cholesteryl-$\text{cis}$-parinarate reveals coexisting environments whose proportion varies and reflects the thermotropic reorganization of the core of the particle. An analysis of the motion by dynamic depolarization suggests that the motions of the acyl chains in the core of the particle are highly restricted.     

Inactivation of aspartate aminotransferase during transamination with chloropyruvate. Evidence against modification of Cys390 in cytosolic isoenzyme.

Both the cytosolic and mitochondrial isoenzyme of aspartate aminotransferase from pig heart were inactivated during transamination with chloropyruvate. Inactivation occurred with L-alanine as the amino group donor in the presence of potassium formate. When L-glutamate or L-aspartate was employed as the amino group donor in the transamination reaction with chloropyruvate, no inactivation occurred. This is in contrast to the case of inactivation by bromopyruvate (Okamoto, M. &; Morino, Y. (1973) J. Biol. Chem. $\text{248}$, 82–90) where these natural dicarboxylic amino acid substrates were effective in the transamination reaction leading to syncatalytic inactivation (Birchmeier, W. &; Christen, P. (1974) J. Biol. Chem. $\text{249}$, 6311–6315). The Cys₃₉₀ in the cytosolic isoenzyme which was modified in the syncatalytic inactivation was not modified under the present condition for inactivation with either chloropyruvate or bromopyruvate.     

Relationship of metabolite inhibition of growth to flow-of-carbon patterns in nature

Various genetic diseases arise from biochemical imbalances that are relatively subtle in the sense that the original mutations are not lethal, that the organism is most vulnerable to damage during certain phases of rapid development, and that in well-managed cases it may be possible to avoid damaging effects through the use of appropriate nutritional manipulations. Analogous imbalances occur in lower organisms. Data obtained with $\text{Pseudomonas}$$\text{putida}$ illustrate that susceptibility to metabolic imbalance is conditionally dependent upon the nutritional regimen.Stereoisomers of leucine, isoleucine and valine, except for L-allo-isoleucine, are metabolized as sole sources of carbon and energy by $\text{P.}$$\text{putida}$. Although the cell yields calculated following utilization of $\text{D}$-leucine and $\text{L}$-leucine were similar, the rate of growth on D-leucine was seven-fold faster than on $\text{L}$-leucine. Slower growth on the $\text{L}$-isomer is not explained as 2-ketoisocaproate limitation since 2-ketoisocaproate production from $\text{L}$-leucine appears to occur more readily than from $\text{D}$-leucine. Spontaneous mutants were obtained which grew 2–10 times more rapidly than wild type on $\text{L}$-leucine, $\text{L}$-isoleucine, or $\text{L}$-valine. It is concluded that the true growth potential (rate) of wild type on any of the branched-chain amino acids is masked by a partial, sustained inhibitory effect produced by the corresponding keto acids or their derivative metabolites. Inhibition of growth rate was only found during utilization of branched-chain amino acids as the sole source of carbon and energy, indicating that the metabolite vulnerability is unique to particular flow-of-carbon patterns during growth. The partial and sustained depression of growth rate by branched-chain amino acids in the absence of other carbon sources cannot be attributed to mis-regulation events localized within the biosynthetic pathway. It is concluded that the catabolism of branched-chain amino acids produces a generalized state of metabolic imbalance owing to the existence of abnormally high levels of degradative metabolites such as keto acids of Coenzyme-A derivatives. Such compounds could (1) interfere with keto acid (e.g. pyruvate) metabolism, (ii) cause feed-forward inhibition of rate-limiting steps in the pathways of branched-chain amino acid catabolism, (iii) perturb fatty acid composition or disrupt the biochemical integrity of membrane material, or (iv) react with substrate-ambiguous enzymes, either slowing essential biochemical reactions to rates that are growth-limiting or producing erroneous products having antimetabolite properties.These effects of branched-chain amino acids in $\text{P.}$$\text{putida}$ may be quite relevant to the molecular events that characterize maple syrup urine disease in man. Metabolite inhibition is probably more common in nature than is generally appreciated, and an appreciation of the molecular basis for anomalous inhibitions of growth in prokaryotic systems should help supply insight into various molecular diseases in man, many of them yet to be described.