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Bovine brain and pituitary fibroblast growth factors (FGF) have been compared with regard to their chemical and biological properties. Pituitary and one preparation of brain FGF (Prep A) contain a basic mitogenic activity, which migrates to the same position on electrophoresis in acid pH gels as detected by incorporation of [methyl-3H]-thymidine into BALB/c 3T3 cells. In contrast, another preparation of brain FGF (Prep B) contains two mitogens, one (20-30%) indistinguishable from the basic components in pituitary and brain (Prep A) FGF preparations and an acidic activity (70-80%), pl 5-6, that migrates more slowly on acid gels, corresponding to the acidic component of brain FGF described previously (Thomas, K. A., M. C. Riley, S. K. Lemmon, N. C. Baglan, and R. A. Bradshaw. 1980. J. Biol. Chem. 255:5517-5520.) In agreement with that report, none of the mitogens comigrates with fragments of myelin basic protein. Pituitary FGF was virtually inactive, brain (Prep A) FGF had a small amount of activity, and brain (Prep B) FGF was highly potent (50% maximal stimulation at 15-30 ng/ml) in stimulating the growth of human umbilical vein endothelial (HUVE) cells. The acidic component of brain FGF, which is much more unstable at pH 8.5 than the basic one, can be protected by reducing agents, whereas the basic constituent of brain FGF as well as pituitary FGF is unaffected by reducing conditions. Thus, brain FGF preparations may contain two distinct mitogenic activities, one that is acidic and contains HUVE cell activity, and a basic mitogen that is similar to and may be identical with pituitary FGF.

The complete amino acid sequence of a lectin from sainfoin ( Onobrychis viciifolia Scop . var. Eski ) has been determined by sequential Edman analyses of the intact protein and peptides derived from digests with trypsin and thermolysin. Peptides were purified by pH fractionation, by gel filtration, and by cation-exchange and reverse-phase high-performance liquid chromatography. Seven segments of continuous sequence, accounting for the entire protein, were aligned through sequence comparison with several homologous leguminous lectins to give the final structure. Sainfoin lectin monomer, a glycoprotein which contains a single polypeptide chain of 236 amino acid residues with a molecular weight of 26 509, has amino- and carboxyl-terminal residues of alanine and threonine, respectively. A single residue of cysteine, located at position 33, is the only sulfur-containing amino acid present. Asparagine-118 is the single oligosaccharide attachment site. At least two apparent allelomorphic forms of the protein, having valine or isoleucine at position 49 in equal amounts, were detected. The amino acid sequence of sainfoin lectin exhibits circular permutation relative to that of the homologous protein concanavalin A.

The amino acid sequence of bovine heart mitochondrial coupling factor 6 (F6) has been determined by automated Edman degradation of the whole protein and derived peptides. Preparations based on heat precipitation and ethanol extraction showed allotypic variation at three positions while material further purified by HPLC yielded only one sequence that also differed by a Phe-Thr replacement at residue 62. The mature protein contains 76 amino acids with a calculated molecular weight of 9006 and a pI of approximately equal to 5, in good agreement with experimentally measured values. The charged amino acids are mainly clustered at the termini and in one section in the middle; these three polar segments are separated by two segments relatively rich in nonpolar residues. Chou-Fasman analysis suggests three stretches of alpha-helix coinciding (or within) the high-charge-density sequences with a single beta-turn at the first polar-nonpolar junction. Comparison of the F6 sequence with those of other proteins did not reveal any homologous structures.

Experiments were carried out to determine if stimuli which augment preganglionic nerve activity to sympathetic neurons, and thereby cause trans-synaptic induction, increase the retrograde transport of nerve growth factor (NGF). It was found that nerve activity had no effect on retrograde transport of [ 125 I]NGF. It was found, however, that reserpine decreased retrograde transport of [ 125 I]NGF and this inhibition was characterized. Reserpine decreased the maximal accumulation of intravenously administered[ 125 I]NGF in superior cervical ganglia (SCG) by about 60%. It also caused a distinct shift in the time course of accumulation so that maximal accumulation was seen 12 h after [ 125 I]NGF injection rather than at 9 h as in control animals. Reserpine had no effect on retrograde transport in sensory neurons. Dose-response curves showed that maximal inhibition occurred with doses of reserpine of 2.5 mg/kg i.p. and that reserpine was not able to completely block transport at any dose. The maximal inhibition of retrograde transport was achieved within 30 min of reserpine administration and inhibitory activity was unchanged for 36 h. The ability of sympathetic neurons to transport [ 125 I]NGF subsequently recovered and was normal 96 h after reserpine administration. The inhibitory effect of reserpine appears to be due to an action at or very near to the nerve terminal since it was effective at reducing NGF transport at very low doses (0.33 μg) when co-administered directly into the eye with [ 125 I]NGF. An action of reserpine at the nerve terminal was further suggested by the inability of reserpine to affect transport if the drug was given 4 h after [ 125 I]NGF administration. Based upon these data, it is suggested that there may be two pools of retrogradely transported NGF and that only more rapidly turning over pool is reserpine-sensitive. This pool may represent the retrogradely moving synaptic vesicles or some derivative of the vesicles.

A distinction can be drawn between primary hormones (messengers) (rapidly acting and using cyclic nucleotides as second messengers) and secondary hormones, which generally act over a longer time period and characteristically provide trophic stimulation to their target cells. Insulin and three related polypeptide growth factors (relaxin, nerve growth factor and insulin-like growth factor) fall into the second category. They share features of primary and three-dimensional structure as well as certain functional similarities and in all probability evolved from a common ancestral gene.

The guinea pig prostate gland contains high levels of nerve growth factor similarly to the mouse submandibular gland. Nerve growth factor from the guinea pig prostate gland cross-reacts weakly with antisera directed against mouse nerve growth factor, is associated with different proteins, and may be processed by a different mechanism. We have isolated a full-length cDNA clone for nerve growth factor from a library prepared from RNA of the guinea pig prostate gland. The guinea pig cDNA contains 1,075 nucleotides and is very similar to the shorter of two predominant nerve growth factor transcripts present in the mouse submandibular gland. The cDNA sequence predicts a precursor protein of 241 amino acids that is 86% identical to the mouse amino acid sequence. Only 10 amino acid changes are present in the C-terminal region corresponding to the mature 118 amino acid β-nerve growth factor of the mouse. Dibasic amino acid processing sites that are present at the N- and C-termini of the mature protein sequence and two other dibasic amino acid sites, representing potential processing sites within the propeptide, are all conserved, suggesting a similar mechanism of processing.

The amino acid sequence of porcine heart mitochondrial malate dehydrogenase (mMDH; L-malate: NAD+ oxidoreductase, EC 1.1.1.37) has been compared with the sequences of six different lactate dehydrogenases (LDH; L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) and with the "x-ray" sequence of cytoplasmic malate dehydrogenase (sMDH). The main points are that (i) all three enzymes are homologous; (ii) invariant residues in the catalytic center of these enzymes include a histidine and an internally located aspartate that function as a proton relay system; (iii) numerous residues important to coenzyme binding are conserved, including several glycines and charged residues; and (iv) amino acid side chains present in the subunit interface common to the MDHs and LDHs appear to be better conserved than those in the protein interior. It is concluded that LDH, sMDH, and mMDH are derived from a common ancestral gene and probably have similar catalytic mechanisms.

The change in cell surface adhesive specificity previously shown to occur between day 7 and 8 of development in the chick optic tectum ]Gottlieb et al. (1974), Proc. Nat. Acad. Sci. USA 71, 1800-1802] can be induced in rotating cultures of tectal cells by the addition of 10(-7) M mouse submaxillary gland nerve growth factor. Insulin, proinsulin, dexamethasone, and performic-acid-oxidized nerve growth factor are individually inactive in this system, but nerve growth factor in which the three tryptophan residues/subunit have been oxidized with N-bromosuccinimide is active. Thus, the specificity of this system for nerve growth factor is different than that observed with embryonic dorsal root or sympathetic ganglia, where the oxidized tryptophan derivative is inactive in stimulating neurite production. It is possible that in this system nerve growth factor serves as an analog of another specific trophic factor, presumably structurally related to nerve growth factor, may be active at much lower concentrations.

The gamma-subunit of the high molecular weight form of mouse submaxillary gland nerve growth factor is known to be electrophoretically heterogeneous. The protein is a mixture of molecules containing either two or three chains, attached covalently by disulfide bonds, that arise from one or two endoproteolytic cleavages. Analysis of the amino acid composition and partial sequence of the resultant fragments has defined the location of the two cleavage sites. Further heterogeneity is generated by partial removal of the carboxyl-terminal basic residues located at the internal cleavage sites, presumably by an endogenous exopeptidase(s) with carboxypeptidase B specificity. Permutations of these endo- and exoproteolytic cleavages generate six possible microheterogeneous forms, which can account for the five major bands observed on electrophoresis at pH 7.05 and the three principal forms seen on isoelectric focusing.

The specific binding of 125I-nerve growth factor (125I-NGF) to the low speed pellet fraction of dissociated 13-day chick embryo heart and brain is a complex, nonsaturable process exhibiting a range of affinities (heart,Ka = 2.9 x 109 to 6 x 106 liters per mole and brain, 2.5 x 109 to 8 x 106 liters per mole). The binding to both tissues at 24° is rapid (k+1 = 7 x 106 m-1 s-1) and reversible (k-1 = 7 x 10-4 s-1) and the dissociation of bound 125I-NGF is accelerated by 8 x 10-8 m native NGF (k-1 = 4 x 10-3 s-1). Insulin and proinsulin inhibit the binding of 125I-NGF 20 to 30% at concentrations of 10-9 to 10-8 m, lower or higher concentrations being less effective. Bovine growth hormone, human placental lactogen and glucagon inhibit 125I-NGF binding only to the extent of 5 to 8%, and this only at high concentrations. The regional distribution of specific 125I-NGF binding in heart correlates with regions of sympathetic innervation. The properties of 125I-NGF binding to these specific sites in embryonic heart and brain are very similar to those of the NGF receptor of sympathetic and dorsal root ganglia, the recognized target tissues for NGF.

The complete amino acid sequence of the Escherichia coli alkaline phosphatase subunit [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1, isozyme 3] has been determined. The monomer contains 449 amino acid residues in a single unglycosylated polypeptide chain having a calculated Mr of 47,029. Isozyme 1 has an additional arginine residue at the NH2 terminus that presumably results from variability in processing of precursor molecules. Sequence data were obtained from both manual and automatic Edman degradation of the tryptic and cyanogen bromide peptides, as well as other peptides derived therefrom. The two disulfide bonds were determined from analyses of the appropriate peptic peptides. This structure confirms earlier reports of the sequence surrounding the active-site serine and both the NH2- and COOH-terminal cyanogen bromide fragments. A secondary structure prediction places nearly half the residues in alpha-helical segments that have 13% and 16%, respectively, in beta-strand and beta-turn orientations.

Crystals of l -3-hydroxyacyl coenzyme A dehydrogenase from pig heart that are suitable for X-ray analysis have been prepared and characterized. The enzyme is a mitochondrial protein important in the beta oxidation of fatty acids. It is of special interest because it requires coenzyme A-related molecules for its catalytic activity. For one of the crystalline forms of the enzyme, the X-ray data tend to confirm chemical observations that the molecule is a dimer of identical subunits.

The amino acid sequence of a proteolytic fragment of Escherichia coli biotin carboxyl carrier protein was determined from the structures of overlapping tryptic, thermolytic, and staphylococcal protease peptides together with automated sequenator analyses on the intact protein. The fragment, 82 residues in length, contains the single residue of biocytin of the protein. The relationship of the Mr = 9100 fragment to the native Mr = 22,500 subunit is discussed.

Two classes of receptors for 125I-labeled nerve growth factor in chick embryonic dorsal root neurons have been observed. One type is associated with the plasma membrane (or microsomal fraction) and can be completely solubilized by Triton X-100. These receptors display the nonsaturable binding isotherms and curvilinear Scatchard plots previously reported for nerve growth factor receptors in whole cells. The second class of binding sites is located in the nucleus, firmly bound to chromatin. These receptors are not solubilized by detergent, show saturable binding, and yield linear Scatchard plots of the type associated with a single class of binding sites of high affinity. The presence of the two receptor types suggests a bimodal mechanism of action for nerve growth factor.

The amino acid sequence of cytoplasmic malate dehydrogenase (sMDH) has been determined by a combination of X-ray crystallographic and chemical sequencing methods. The initial molecular model incorporated an "X-ray amino acid sequence" that was derived primarily from an evaluation of a multiple isomorphous replacement phased electron density map calculated at 2.5-A resolution. Following restrained least-squares crystallographic refinement, difference electron density maps were calculated from model phases, and attempts were made to upgrade the X-ray amino acid sequence. The method used to find the positions of peptides in the X-ray structure was similar to those used for studying protein homology and was shown to be successful for large fragments. For sMDH, X-ray methods by themselves were insufficient to derive a complete amino acid sequence, even with partial chemical sequence data. However, for this relatively large molecule at medium resolution, the electron density maps were of considerable help in determining the linear position of peptide fragments. The N-acetylated polypeptide chain of sMDH has 331 amino acids and has been crystallographically refined to an R factor of 19% for 2.5-A resolution diffraction data.

It has been proposed that the drugs (6-hydroxydopamine, guanethidine, vinblastine) which are known to destroy sympathetic neurons in neonatal animals do so by preventing the accumulation of retrogradely transported nerve growth factor (NGF). It was found, consistent with the proposal, that administration of 6-hydroxydopamine (100 mg/kg s.c.) or vinblastine (0.4 mg/kg s.c.) 16 h prior to the administration of [ 125 I]NGF completely prevented the accumulation of retrogradely transported [ 125 I]NGF in superior cervical ganglia of neonatal rats. Administration of 6-hydroxydopamine or vinblastine to adult rats (where it does not cause sympathetic neuron cell death) did not completely prevent the retrograde transport of NGF, although 6-hydroxydopamine produced an alteration of the time course of accumulation (early times unaffected, later times depressed). The administration of guanethidine to adult rats (50 mg/kg/day) produced a modest decrease in the accumulation of NGF (40–60%). It would appear, however, that this decrease cannot account for the cytotoxic effects of guanethidine since: (1) sub-cytotoxic doses of guanethidine and non-cytotoxic guanidinium blocking agents also produce modest decreases in the retrograde transport in NGF; and (2) the retrograde transport of [ 125 I]NGF is not affected in neonatal animals until after the neurons are clearly damaged. Hence, the data are entirely consistent with the hypothesis that NGF deprivation caused by 6-hydroxydopamine and vinblastine is the mechanism of the cytotoxic effects of these drugs on sympathetic neurons in neonatal animals. Guanethidine destroys sympathetic neurons by some other mechanism.

Porpoise myoglobin has been fully alkylated after removal of the heme group to produce a derivative modified at the methionyl and histidyl residues as well as the α-amino group. Isolation and characterization of the tryptic peptides on Dowex 50 and Dowex 1 have produced a tentative structure analogous to the known sequence of sperm whale myoglobin. By this technique, 81 of 153 residues can be positioned in the sequence establishing that 10 residues are different from the sperm whale structure. In addition, five replacements are indicated from compositional data. These results indicate that the porpoise protein more closely resembles the sperm whale one than does the seal protein described previously, which had a minimum of 25 replacements when compared to the sperm whale protein.

The fully alkylated derivatives of harbor seal and porpoise apomyoglobin have been subjected to chymotryptic hydrolysis. In each case, the products were completely soluble at pH 2.0 in contradistinction to the tryptic hydrolysates. The purified peptides have been subjected to sequence analysis and amide identification and, in conjunction with the tryptic peptides, aligned to produce the complete sequence of each myoglobin. The resulting structures, when compared to the established sperm whale structure, indicate a close similarity among all three proteins. The sperm whale protein differs from that of seal in 26 positions but from that of porpoise in only 15 positions. The seal and porpoise proteins differ at 17 positions. All of the amino acid replacements were confined to 29 positions, leaving 124 of 153 positions in the sequence invariant in the three proteins. The relation of these data to other, partial sequences is discussed.

A method is described for the preparation of homogeneous 3-hydroxyacyl-CoA dehydrogenase from pig heart muscle in amounts sufficient for structural studies. The procedure utilizes ammonium sulfate precipitation, gel filtration, and ion exchange chromatography on CM-cellulose. The homogeneity of the preparation obtained has been established by isoelectric focusing, sedimentation analysis, starch gel electrophoresis, and sodium dodecyl sulfate gel electrophoresis. Crystals of the protein have been obtained with ammonium sulfate in phosphate buffer at pH 6.8. The enzyme exhibits marked substrate inhibition with acetoacetyl-CoA, and to a lesser extent with S-acetoacetyl pantetheine, when assayed in the direction of NADH oxidation. The activity with S-acetoacetyl-N-acetylcysteamine is too low to determine whether high levels of this substrate also inhibit the enzyme. The purified enzyme is a basic protein with an isoelectric point of 8.95 at 4°. It possesses a sedimentation coefficient of 3.88 S and a maximum ultraviolet absorption at 278 nm. The extinction coefficient at this wave length is E1%1 cm = 4.39. The amino acid composition of the enzyme has been determined. The most notable features are 2 residues of tryptophan and 2 residues of half-cystine per mole of protein. Both half-cystinyl residues are present in the native enzyme as cysteine as judged by titration of the protein in 6 m guanidine hydrochloride with p-chloromercuribenzoate.

The original endotoxin assay of Levin et al. ( N. Engl. J. Med. , 283 (1970) 1313) has been improved to allow detection of picogram quantities of lipopolysaccharides and free lipid A. The response of this assay to both alkali-treated and untreated lipopolysaccharides and their derivatives parallels the previously demonstrated effect of these substances on guinea pig complement fixation.

The mixed disulfide of lysozyme and cystine can be prepared by reaction of fully reduced lysozyme and cystine at pH 9.5. The mixed disulfide is enzymatically inactive and contains 16 residues of half-cystine per molecule. On reaction with cysteine, mercaptoethylamine, or mercaptoethanol, the mixed disulfide can be converted to active lysozyme. The initial rate of reactivation varies with pH, temperature, concentration of disulfide, and concentration of mercaptan; under the best conditions, 50% of the original activity is regenerated in 6 to 7 min. The extent of reactivation is generally between 70 and 85% under a variety of conditions. On the basis of kinetic measurements and characterization of the lysozyme formed throughout the reactivation process, a tentative mechanism for re-formation of the correct disulfide bonds in lysozyme has been proposed. Chromatographic analysis of the reactivated enzyme shows that it contains two major components. One chromatographic component is indistinguishable from native lysozyme, but the other has about 50 to 60% of the specific activity of native lysozyme and it is not converted to native lysozyme on further reduction and oxidation.

The amino-acid sequence and three-dimensional structure of a calcium-binding protein prepared from carp muscle has been determined. This protein, designated carp-muscle calcium-binding protein B, is one of three closely related parvalbumins found in this tissue. The electron density map, calculated by heavyatom substitution crystallographic methods to 2.0-Å resolution, reveals the orientation of most of the amino-acid side chains. The calcium coordination site consists of one glutamic- and three aspartic-acid carboxyl groups in a tetrahedral arrangement. The core of this spherical molecule is remarkably hydrophobic, with 8 of its 10 phenylalanine side chains packed in an approximate herringbone pattern. 52 of the 108 residues are in six α-helixes; there is no β-pleated sheet. The acetylated amino-terminal alanine appears not to be accessible to solvent. All of the heavy-atom derivatives are bound at the sole cysteine. The properties of this protein suggest a relationship to troponin A of mammalian tissue.

1. The amino acid sequence of peptide (1–24) obtained from limited peptic digestion of bovine serum albumin has been determined from characterization of its tryptic and chymotryptic peptides. The 3 histidyl residues have been shown to occupy positions 3, 9, and 18. 2. Hydrogen ion titration of this peptide with and without copper(II) ions indicates that at neutral pH there are two copper(II)-binding sites of appreciable strength on the peptide. Of these, one site binds copper(II) ions strongly while the other binds copper(II) ions more weakly. In addition to the differences observed by titration, marked differences between the sites are apparent from absorption spectra, circular dichroism spectra, and optical rotatory dispersion spectra. 3. Since the peptide binds only 2 copper(II) ions strongly, all 3 histidyl residues do not act with comparable effect in separate binding sites. 4. The site which binds the 1st copper(II) ion has been shown by hydrogen ion titration, absorption spectra, circular dichroism spectra, and optical rotatory dispersion spectra to have properties compatible with previous suggestions that it is composed of the α-amino nitrogen, the imidazole nitrogen from the histidine in position 3, and the 2 intervening peptide bond nitrogen atoms. 5. Occupancy of either site by copper(II) leads to absorption in several circular dichroic bands. In addition to the several bands in the visible and near ultraviolet that have some similarities to those observed previously with complexes of small peptides, it has been possible to resolve two bands of opposite sign centered near 300 to 308 mµ and 270 to 275 mµ. The high stability of the complexes permitted measurements at sufficiently low concentrations to avoid excessive total absorption. In some cases optical rotatory dispersion results were obtained at shorter wave lengths, and it was observed that values of [m']233 changed with pH.

The amino acid sequence of carp muscle calcium-binding protein B has been completely determined by a combination of chemical and x-ray data. The protein was subjected to tryptic digestion and the peptides were separated on ion exchange columns. All of the 108 residues of the protein could be accounted for in 15 tryptic peptides. The sequence of each peptide was elucidated by a combination of Edman degradations with dansylation, carboxypeptidase, and aminopeptidase hydrolyses, and high voltage electrophoresis at pH 6.5. One 19-residue tryptic peptide was resistant to Edman degradation and was found by mass spectral analysis to have N-acetylalanine at the NH2 terminus. The sequence of this peptide was determined after digestion with thermolysin. The alignment of both the thermolytic and tryptic peptides to give the over-all sequence was made primarily from the 2.0 A resolution electron density map.

Overlapping tryptic, peptic, thermolytic, and Pronase peptides containing 4'-phosphopantetheine were isolated from rat liver fatty acid synthetase. Characterization of these peptides and a 4'-phosphopantetheine dipeptide obtained after dilute acid hydrolysis permitted the elucidation of the amino acid sequence of 13 residues. Five out of the 13 residues are identical with those in the corresponding region of Escherichia coli acyl carrier protein.

The reaction of fumarase with several alkylating agents has been examined. The structural similarity between these compounds and the natural substrates for the enzyme, fumarate and malate, suggested that reaction might occur with structures in the active sites. Of all the compounds tested, only iodoacetate and 4-bromocrotonate inactivated fumarase by modification of structures other than the thiol groups. Because the thiol groups appear to fulfill a structural role in the enzyme, it is possible that iodoacetate and bromocrotonate react specifically with fumarase. The enzyme is inactivated to an extent of 80 to 90% when only 3 to 6 eq of each compound have reacted with the enzyme. Inactivation by each compound is reduced markedly by substrates and competitive inhibitors, and extensive structural changes do not occur after inactivation. S-Carboxymethylmethionine and 3-carboxymethylhistidine are the major derivatives found after inactivation with iodoacetate. The distribution of these carboxymethyl derivatives among the tryptic peptides of iodoacetate-inactivated fumarase has been examined. From 80 to 85% of the S-carboxymethylmethionine and 3-carboxymethylhistidine present in the modified enzyme have been recovered in three unique peptides.

1. The amino-terminal peptide (1–24) of bovine serum albumin was subjected to carboxymethylation by 0.2 m bromoacetate at pH 6.8 in the presence of 0, 1, and 2 eq of copper(II) ion. The treatment with bromoacetate in the absence of copper(II) caused almost quantitative carboxymethylation of the imidazole groups of the 3 histidyl residues at positions 3, 9, and 18 in the sequences. In addition, the terminal α-amino group was apparently modified. Spectral measurements on the copper-containing complexes showed little or no effect on the copper chelation by the alkylation treatment. 2. Binding of 1 eq of copper(II) protects quantitatively the terminal α-amino group and histidyl residue 3, and appears to reduce somewhat the reactivity of histidyl residue 18. 3. Binding of 2 eq of copper(II) also protects the components of the preferred terminal site, but, in addition, both histidyl residues 9 and 18 are largely, and about equally, protected from alkylation. The binding of the 2nd eq therefore appears to involve both histidyl residues, in keeping with spectral and titration results. An intramolecular chelate structure or intermolecular chelates involving dimers or higher polymers are suggested. 4. The tetrapeptide l-aspartyl-l-threonyl-l-histidyl-l-lysine, which corresponds to the first 4 residues of peptide (1–24), was shown by titration, absorbance, and circular dichroism measurements to form an equimolar complex with copper(II) that has substantially the properties of the complex of peptide (1–24) containing 1 eq of copper(II) ion. Similar measurements on the equimolar complex of bovine serum albumin fill out a continuity that is especially evident from measurements of circular dichroism.

The study of the structure and function of bovine pancreatic carboxypeptidase A has a long historical record which began in 1937 with the isolation of the crystalline enzyme by Anson (1937). Although the literature of this exopeptidase has been reviewed on several occasions (Neurath & Schwert 1950; Smith 1951; Neurath 1960; Vallee 1964; Neurath, Bradshaw, Ericsson, Babin, Petra & Walsh 1968; Vallee & Riordan 1968; Lipscomb, Hartsuck, Reeke, Quiocho, Ludwig, Steitz & Bethge 1968), nevertheless, it seems appropriate at the outset of a conference on carboxypeptidase to review in brief the major contributions that have been made over the years to the study of the structure and function of this enzyme, as summarized in table 1.

It has been previously shown that the fructose-P2 aldolase isolated from Micrococcus aerogenes is a Class I (Schiff base) enzyme. All other prokaryotic organisms so far studied contain Class II (metallo) aldolases. Studies were undertaken to investigate whether or not this bacterial aldolase is structurally related to the Class I aldolases of higher organisms. 1. A comparison of amino acid compositions gives no indication of homology between M. aerogenes and mammalian aldolases. 2. The NH2-terminal residue of this enzyme was found to be methionine in contrast to proline which is found at the NH2 terminus of the rabbit enzymes. 3. The M. aerogenes enzyme contains lysine rather than tyrosine at the COOH terminus. 4. The tryptic peptide containing the active site lysyl residue of M. aerogenes aldolase was isolated and partially characterized. The peptide contains 28 amino acids as do the peptides of rabbit aldolases A and B. The amino acids of the M. aerogenes peptide may be aligned to obtain the maximum of 48% homology with the corresponding rabbit aldolase peptides. 5. Several of the structural distinctions between M. aerogenes and rabbit aldolases are discussed in relation to the proposed functions of tyrosine, and cysteine in the catalytic process of Class I fructose-P2 aldolases. Our results suggest extensive and perhaps fundamental differences between the M. aerogenes enzyme and the Class I aldolases of mammals. The extent of relationship between this prokaryotic enzyme and the fructose-P2 aldolases of higher organisms must await more thorough structural analyses.

Modifications of the automatic amino acid analyzer (Spinco model 120) are described that allow for doubling the output of amino acid analyses by incorporating a second or “satellite” analytical system. The “satellite” system can simultaneously perform an independent analysis (lacking only proline) without impairment of the conventional operation of the analyzer. In addition, an accelerated buffer system has been described that allows for a complete amino acid analysis in a period of 3 hours.

Summary Examination of the two major crystalline forms, type A and type C, of pig heart cytoplasmic malate dehydrogenase (s-MDH) has revealed that type C crystals are composed of enzyme molecules with one mole of nicotinamide adenine denucleotide bound per mole of enzyme, Type A crystals are devoid of bound coenzyme. The NAD+ can be readily removed by dialysis or gel filtration after type C crystals have been dissolved. The binding of 1 mole of NAD+ results in the formation of an asymmetric complex in which one of the two identical polypeptide chains composing native s-MDH undergoes a small but definitive conformational change. These data are consistent with observed difference in crystallographic properties of the s-MDH subunits.

A rapid and simple procedure for the preparation of sperm whale ferrimyoglobin is described. The method involves homogenization in 70% saturated ammonium sulfate at 4° and chromatography on carboxymethyl Sephadex. The major component was judged to be homogeneous by electrophoresis at two pH values and by analytical chromatography on sulfoethyl Sephadex at a third pH value. The method has also been utilized to prepare electrophoretically pure myoglobin from harbor seal and porpoise. The amino acid composition and amino-terminal end groups of harbor seal and porpoise myoglobin have been determined, and the results compared with sperm whale myoglobin. Each protein was found to possess 153 amino acid residues with a single amino-terminal end group. Both harbor seal and porpoise myoglobins have glycine as the amino-terminal residue, in contrast to the sperm whale protein, which contains a single residue of valine. The amino acid compositions indicate a close similarity among all three myoglobins and suggest that only a limited number of amino acid substitutions in the primary structure differentiates the three proteins. The molar extinction coefficients and the reduced mean residue rotation at 233 mµ were found to be essentially identical in the three myoglobins, indicating that the three-dimensional structure in solution is probably very nearly identical for sperm whale, harbor seal, and porpoise ferrimyoglobins.

The amino-terminal fragment, comprising the first 24 residues, has been isolated from rat and human serum albumins after limited peptic digestion. The complete sequence of each fragment has been deduced from the tryptic peptides alone by taking advantage of overlapping peptides resulting from incomplete cleavage. A comparison of the structure of the (1–24) peptides of rat and human albumins with that obtained from bovine serum albumin (J. Biol. Chem., 242, 5451 (1967)) indicates a high degree of similarity. The rat fragment differs from the bovine at three sites, whereas the human fragment differs at four sites. There is a single replacement at Residue 2 common to the rat and human peptides. Because production of this fragment by limited proteolysis must be indicative of certain conformational parameters in the region of Residue 24, it may be concluded that the rat, human, and bovine serum albumin molecules are closely homologous in primary and three-dimensional structure in the amino-terminal region.

An argument is presented in favor of the hypothesis that, bovine carboxypeptidases A and B fit by and large, the concept of homology on the levels of both amino acid sequence and three-dimensional structure. Two peptide sequences from carboxypeptidases A and B, comprising 46 amino acid residues, display structural homology to the extent of 51 per cent. One of the two peptides contains two of the amino acid residues which have been identified by X-ray crystallography as zinc ligands in carboxypeptidase A. The same residues are conserved in carboxypeptidase B in a loop of comparable dimensions. The other homologous pair of amino acid sequences appears to occur in different regions of the two enzymes which include in carboxypeptidase B the cysteinyl residue proposed to function as the third zinc ligand but not the amino acid residue believed to be the analogous ligand in carboxypeptidase A. The uncertainties of this prediction are considered in the light of existing chemical and structural evidence.

A method is described for the preparation of homogeneous D-3-phosphoglycerate dehydrogenase from chicken liver in amounts sufficient for structural studies. The procedure utilizes ammonium sulfate precipitation, blue dextran-Sepharose chromatography, ion exchange chromatography on phosphocellulose, and crystallization. Previous reports of instability of the enzyme have been shown to be due to proteolysis in the crude extract which can be effectively prevented by leupeptin. The purified enzyme is a basic protein with a pI of 8.95 as measured by isoelectric focusing. The extinction coefficient at 278 nm of a 1% solution is 5.3.