Metmyoglobin/azide: the effect of heme-linked ionizations on the rate of complex formation.

The kinetics of formation and dissociation of the horse metmyoglobin/azide complex has been investigated between pH 3.5 and 11.5. The ionic strength dependence of the reaction has been determined at integral pH values between 5 and 10. Hydrazoic acid, HN3, binds to metmyoglobin with a rate constant of (3.8 +/- 1.0) x 10(5) M-1 s-1. Protonation of a group with an apparent pKa of 4.0 +/- 0.3 increases the rate of HN3 binding 6.5-fold to (2.5 +/- 0.8) x 10(6) M-1 s-1. The ionizable group is attributed to the distal histidine, His-64. The azide anion, N-3, binds to metmyoglobin with a rate constant of (4.7 +/- 0.3) x 10(3) M-1 s-1, about two orders of magnitude slower than HN3. Conversion of aquometmyoglobin to hydroxymetmyoglobin slows azide binding significantly. Binding of HN3 to hydroxymetmyoglobin cannot be detected, while N-3 binds to hydroxymetmyoglobin with a rate of 5.7 +/- 3.2 M-1 s-1, almost three orders of magnitude slower than N-3 binding to aquometmyoglobin. Protonation of the distal histidine facilitates HN3 dissociation from the complex. HN3 dissociates from the metmyoglobin/azide complex with a rate constant of 18 +/- 6 s-1, while the azide anion dissociates with a rate constant of 0.16 +/- 0.02 s-1, about 100 times slower. The apparent pKa for His-64 is essentially the same in metmyoglobin and the metmyoglobin/azide complex, 4.0 +/- 0.3 and 4.4 +/- 0.2, respectively. The ionic strength dependence of the observed association rate constant is influenced by both primary and secondary kinetic salt effects. The primary kinetic salt effect is anomalous, with the rate of N-3 binding decreasing with increasing ionic strength above the isoelectric point of metmyoglobin where the protein has a net negative charge. The ionic strength dependence of the dissociation rate constant can be described solely in terms of the ionic strength dependence of the acid dissociation constant for His-64 in the metmyoglobin/azide complex, a secondary kinetic salt effect.

Metmyoglobin/fluoride: effect of distal histidine protonation on the association and dissociation rate constants.

The kinetics of formation and dissociation of the horse metmyoglobin/fluoride complex has been investigated between pH 3.4 and 11. The ionic strength dependence of the reaction has been measured at integral pH values between pH 5 and 10. Hydrofluoric acid, HF, binds to metmyoglobin with a rate constant of (4.7 +/- 0. 7) x 10(4) M-1 s-1. An apparent ionization in metmyoglobin with a pKa of 4.4 +/- 0.5 influences the rate of HF binding and is attributed to the distal histidine, His-64. Protonation of His-64 increases the HF binding rate by a factor of 2.6. The fluoride anion, F-, binds to metmyoglobin with a rate constant of (5.6 +/- 1.4) x 10(-2) M-1 s-1, about 10(6) times slower than HF. Binding of either HF or F- to hydroxymetmyoglobin cannot be detected. Protonation of the distal histidine facilitates HF dissociation from the metmyoglobin/fluoride complex. HF dissociates with a rate constant of 1.9 +/- 0.3 s-1. The fluoride anion dissociates 2000 times more slowly, with a rate constant of (8.7 +/- 1.6) x 10(-4) s-1. The apparent pKa for His-64 ionization in the fluorometmyoglobin complex is 5.7 +/- 0.1. The association and dissociation rate constants are relatively independent of ionic strength with secondary kinetic salt effects sufficient to account for the ionic strength variation of both, consistent with the idea that association and dissociation of neutral HF dominate the kinetics of fluoride binding to metmyoglobin.

Alpha-factor-leader-directed secretion of recombinant human-insulin-like growth factor I from Saccharomyces cerevisiae. Precursor formation and processing in the yeast secretory pathway.

A synthetic gene coding for human-insulin-like growth factor I (IGFI) was fused to the leader sequence of yeast prepro-alpha-factor and expressed in Saccharomyces cerevisiae under the control of a glyceraldehyde-3-phosphate dehydrogenase promoter fragment. Recombinant IGFI was found inside yeast cells and secreted into the medium. The secreted IGFI migrated on SDS gels with the same electrophoretic mobility as authentic IGFI, i.e. at about 7.5 kDa. HPLC analysis of secreted IGFI revealed the presence of the correctly folded, genuine molecule as well as an isomeric byproduct of equal molecular mass but with two of the three disulfide bonds interchanged. Inside exponentially growing cells the 7.5-kDa IGFI was also found, along with up to four additional IGFI-related polypeptides of higher molecular mass. By endoglycosidase F treatment the three polypeptides between 19-26 kDa were converted to a single peptide of 17 kDa. Since this peptide also reacted with an anti-alpha-factor antibody, it represents most likely the unglycosylated alpha-factor--IGFI fusion precursor. Pulse-chase experiments established the precursor nature of the intracellular higher-molecular-mass IGFI species. Conversion of the primary translation product to the differently glycosylated IGFI precursor proteins and into the mature form occurred very rapidly, within 2 min. Rapid maturation was, however, not followed by an equally rapid secretion of the mature form into the medium: only after 30-40 min did IGFI appear outside the cells. We therefore postulate the presence of an as yet undefined Golgi or post-Golgi bottleneck representing a major obstacle in secretion of recombinant IGFI from S. cerevisiae cells.

Isolation from adult human serum of four insulin-like growth factor (IGF) binding proteins and molecular cloning of one of them that is increased by IGF I administration and in extrapancreatic tumor hypoglycemia.

We have isolated four insulin-like growth factor binding proteins (IGFBPs) from adult human serum by insulin-like growth factor (IGF) I affinity chromatography and high performance liquid chromatography. A 36-kDa binding protein (BP), not digestible with N-glycanase, is increased in patients with extrapancreatic tumor hypoglycemia and during IGF I administration in healthy adults. Its 38 NH2-terminal amino acids are identical to those of an IGFBP sequence derived from a human cDNA that cross-hybridizes with the rat IGFBP-2 cDNA. With probes encoding a NH2-terminal, COOH-terminal, and a middle region of this protein we have obtained three cDNA clones from a Hep G2 cDNA library; one encodes human IGFBP-2, and the other two presumably represent unspliced heteronuclear and alternatively spliced mRNA, respectively. A 28-30-kDa IGFBP represents a novel BP species in human serum. Its 30 NH2-terminal amino acids are not homologous to IGFBP-1, -2, or -3. It is not digestible with N-glycanase and does not bind 125I-IGF I. The NH2-terminal sequences of a 42/45- and a 31-kDa IGFBP are identical to that of human IGFBP-3. The 42/45-kDa proteins are two glycosylation variants of BP-3. The 31-kDa protein presumably is a degradation product of BP-3 that lacks the COOH terminus. It is likely that the different IGFBPs modulate auto-/paracrine and endocrine effects of IGFs on growth and metabolism in a different and specific manner.

Yeast mutants conferring resistance to toxic effects of cloned human insulin-like growth factor I.

The production of extracellular human insulin-like growth factor I (IGF-I) in yeast is deleterious to the growth of the host organism. Mutants resistant to the toxic effects of IGF-I production were isolated. A subset of these mutants produced levels of IGF-I greater than the parent strain and were due to chromosomal recessive mutations at a single locus, hpx1. The overproduction of IGF-I was independent of the original promoter and vector expression system. The mutant strains also displayed enhanced extracellular production of other heterologous proteins.

Location of disulphide bonds in human insulin-like growth factors (IGFs) synthesized by recombinant DNA technology.

Natural human insulin-like growth factor (IGF) I is a relatively large single chain peptide (mol. wt 7649) with a known sequence of 70 amino acids. C6----C48, C47----C52 and C18----C61 assignments have been previously proposed for the three disulphide bonds linking six cysteine residues (C6, C18, C47, C48, C52 and C61), on the basis of analogy (and homology) with proinsulin. In this work, IGF I synthesized by recombinant DNA technology (r-IGF I, with identical biological activity and chromatographic behaviour) was subjected to a three-step mass spectrometric analysis in combination with degradation methods for structural verification. Firstly, the correct molecular weight of the intact peptide was determined by high-mass fast atom bombardment (FAB) analysis. Secondly, twofold enzymatic degradation (chymotrypsin followed by V8 protease, 'FAB mapping' of the cleavage products) was employed in order that fragments with 'isolated' S-S bonds would be produced which allow an unambiguous assignment. This immediately established the C18----C61 linkage as it was contained in a singly bridged two-chain peptide. The two other S-S bonds, which cross-link C6 and the 'tight' C47 to C52 segment, remained 'unresolved' within a more complex, doubly bridged triple-chain peptide. Thirdly, further degradation of this structural block, in which cleavage of the C47-C48 bond was required to discern these bonds, was carried out by using FAB tandem mass spectrometry and (for additional corroboration) manual Edman degradation. Both procedures confirmed the original C6----C48/C47----C52 prediction.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystallization of trimeric recombinant human tumor necrosis factor (cachectin).

Crystals of tumor necrosis factor (TNF) have been obtained in two forms. Rhombohedral crystals grow in 1.8 to 2.0 M ammonium sulfite, pH 7.8 at 21 degrees C, and tetragonal crystals grow in 2.6 M magnesium sulfate, pH 5.5 at 25 degrees C. Analysis of TNF by isoelectric focusing under native and denaturing conditions indicates that TNF molecules exist as trimers in solution. The rhombohedral cachectin crystals belong to space group R3 and have unit cell constants a = b = c = 47.65 A and alpha = beta = gamma = 88.1 degrees. Density determinations and the space group indicate that the unit cell contains one 51,000-dalton trimer. These crystals are stable in the x-ray beam and diffract to at least 1.85 A but are apparently twinned by merohedry. The tetragonal crystals are space group P4(3)2(1)2 or its enantiomorph P4(1)2(1)2 and have unit cell constants a = b = 95.08, c = 117.49. The asymmetric unit contains one trimer; the crystals are stable in the x-ray beam and diffract to beyond 3 A.

Cloning and characterization of a cDNA for murine macrophage inflammatory protein (MIP), a novel monokine with inflammatory and chemokinetic properties.

In the course of studies on cachectin/TNF being conducted in our laboratory, a novel macrophage product has been detected and characterized. Termed macrophage inflammatory protein or MIP, this protein appears to be an endogenous mediator of the inflammatory events induced by endotoxin. A cDNA cloned probe for this protein has been isolated from a lambda gt10 phage library prepared from poly(A)+ RNA obtained of endotoxin-induced RAW264.7 cells. The sequence codes for a 92 amino acid-long polypeptide, of which 69 amino acids correspond to the mature product. The sequence predicts a molecular weight of 7,889 and structural analysis of the protein indicates a characteristic signal sequence alpha-helix and a hydrophobic core. Sequence data also confirm no sequence similarity to any other protein listed in the Dayhoff data base.

Characterization of recombinant human epidermal growth factor produced in yeast.

Four different forms of human epidermal growth factor (h-EGF) are found in the culture medium of a recombinant strain of Saccharomyces cerevisiae. These forms were characterized after purification using reverse-phase high-performance liquid chromatography. The most abundant form of secreted recombinant h-EGF has leucine at the carboxyl terminus and is identical with gamma-urogastrone. A second species is identical with the most abundant form except that it lacks the carboxyl-terminal leucine. This form appears to be the product of a carboxypeptidase found in the growth medium. The other two forms of recombinant h-EGF are the respective oxidation products of the above where the single methionine residue has been converted to methionine sulfoxide. These four forms of recombinant h-EGF are fully active; they bind to the EGF receptor of A431 cells as well as stimulate mitotic activity of human foreskin fibroblasts with equal specific activity. The location of the disulfide bonds in the predominant form of recombinant h-EGF was determined following digestion with thermolysin. The amino acid compositions of the resulting peptides showed that the placement of disulfide bonds in recombinant h-EGF is identical with that in murine EGF.

Shock and tissue injury induced by recombinant human cachectin.

Cachectin (tumor necrosis factor), a protein produced in large quantities by endotoxin-activated macrophages, has been implicated as an important mediator of the lethal effect of endotoxin. Recombinant human cachectin was infused into rats in an effort to determine whether cachectin, by itself, can elicit the derangements of host physiology caused by administration of endotoxin. When administered in quantities similar to those produced endogenously in response to endotoxin, cachectin causes hypotension, metabolic acidosis, hemoconcentration, and death within minutes to hours, as a result of respiratory arrest. Hyperglycemia and hyperkalemia were also observed after infusion. At necropsy, diffuse pulmonary inflammation and hemorrhage were apparent on gross and histopathologic examination, along with ischemic and hemorrhagic lesions of the gastrointestinal tract, and acute renal tubular necrosis. Thus, it appears that a single protein mediator (cachectin) is capable of inducing many of the deleterious effects of endotoxin.

Growth restoration of insulin-deficient diabetic rats by recombinant human insulin-like growth factor I.

Insulin-like growth factor I (IGF-I) and insulin stem from a common precursor, are structural homologues, act through similar receptors and elicit insulin-like and growth-promoting effects in vitro and in vivo. Serum IGF-I levels are controlled by growth hormone, insulin and nutrition. Insulin-deficient growth-arrested diabetic animals have reduced serum IGF-I levels which are restored towards normal by insulin but not by growth-hormone treatment. Here we show that normal growth of diabetic rate is restored by infusion of recombinant human (rh)IGF-I without normalization of the blood sugar level and that insulin acts via an increase of IGF-I synthesis on growth of diabetic rats. We describe a new mechanism of endocrine control of growth in which IGF-I is the major stimulator at the cellular level. Growth hormone and insulin act mainly by modulating the hepatic synthesis of IGF-I.

Characterization of the polypeptide composition of human factor VIII:C and the nucleotide sequence and expression of the human kidney cDNA.

Human coagulation factor VIII:C has been purified approximately 5000-fold from commercial preparations with an average activity yield of 35%. Proteins of 92 kD and 77-80 kD enriched during purification are precipitated by a human serum polyclonal antibody which inhibits factor VIII:C activity. Evidence suggests that these polypeptides are linked by a calcium ion bridge. Partial amino acid sequence information from these proteins has been obtained from the intact polypeptides and from products of digestion with thrombin, endoproteinase lysC, or trypsin after citraconylation. An oligonucleotide probe designed from one of the amino acid sequences was used to isolate a partial genomic clone from a human 4X chromosome library in bacteriophage lambda. The genomic segment was used to isolate two cDNA molecules encompassing the entire human kidney factor VIII:C mRNA. Biologically active factor VIII:C has been produced in a mammalian cell line utilizing a complete cDNA construction.

Wound-induced proteinase inhibitors from tomato leaves. I. The cDNA-deduced primary structure of pre-inhibitor I and its post-translational processing.

A cDNA containing the coding region for the complete amino acid sequence of wound-induced proteinase Inhibitor I from tomato leaves was constructed in the plasmid pUC9 and characterized. The open reading frame codes for a protein of 111 amino acids. This deduced amino acid sequence revealed the presence of a 42-amino acid N-terminal sequence that is not found in the native protein. This sequence appears to contain a 23-amino acid segment typical of a signal sequence followed by a 19-amino acid sequence containing 9 charged amino acids. The 42-amino acid sequence is apparently lost during maturation to the native Inhibitor I and represents 38% of the translated protein. The Inhibitor I amino acid sequence contains 71% identity with potato tuber Inhibitor I sequence and 35% identity with an inhibitor from the leech.

Wound-induced proteinase inhibitors from tomato leaves. II. The cDNA-deduced primary structure of pre-inhibitor II.

A cDNA containing the complete amino acid-coding region of wound-induced tomato Inhibitor II was constructed in the plasmid pUC9. The open reading frame codes for 148 amino acids including a 25-amino acid signal sequence preceding the N-terminal lysine of the mature Inhibitor II. The Inhibitor II sequence exhibits two domains, one domain having a trypsin inhibitory site and the other a chymotrypsin inhibitory site, apparently evolved from a smaller gene by a process of gene duplication and elongation. The amino acid sequence of tomato leaf Inhibitor II exhibits homology with two small proteinase inhibitors isolated from potato tuber and an inhibitor from eggplant. The small potato tuber inhibitors are homologous with 33 amino acids of the N-terminal domain and 19 amino acids from the C-terminal domain. Two identical nucleotide sequences of Inhibitor II cDNA in the 3' noncoding region were present that were also found in an Inhibitor I cDNA. These include an atypical polyadenylation signal, AATAAG, and a 10-base palindromic sequence, CATTATAATG, for which no function is yet known.

Primary structure and gene organization of human hepatitis A virus.

The RNA genome of human hepatitis A virus (HAV) was molecularly cloned. Recombinant DNA clones representing the entire HAV RNA were used to determine the primary structure of the viral genome. The length of the viral genome is 7478 nucleotides. An open reading frame starting at nucleotide 734 and terminating at nucleotide 7415 encodes a polyprotein of Mr 251,940. Comparison of the HAV nucleotide sequence with that of other picornaviruses has failed to reveal detectable areas of homology. However, a computer analysis of the putative amino acid sequence of HAV and poliovirus demonstrated the existence of short areas of homology in virion protein 3 (VP3) and throughout the carboxyl-terminal portion of the polyproteins. In addition, extensive protein structural homologies with poliovirus were detected.

Human alpha 2-macroglobulin gene is located on chromosome 12.

A cDNA clone encoding amino acids 809-1451 of the protease inhibitor alpha 2-macroglobulin has been isolated from an adult human liver cDNA library. This cDNA was used to examine DNA samples prepared from a panel of human-mouse somatic cell hybrids with different numbers and combinations of human chromosomes for the presence of the human alpha 2-macroglobulin gene. The cosegregation of this gene and chromosome 12 in the cell hybrid panel indicated that the alpha 2-macroglobulin structural gene (designated A2M) is on human chromosome 12.

Modulation of human natural killer cell activity by recombinant human interleukin 2.

Recombinant human IL-2, secreted by yeast harboring a plasmid containing a synthetic IL-2 gene, is biologically active in augmenting human natural killer (NK) cell activity. A dose-dependent linear stimulation of NK activity was obtained against the chronic myelogenous leukemia cell line K562 over the range 3 to 300 units/ml of IL-2. Enhancement of NK activity was similarly demonstrable against the less NK-sensitive carcinoma cell lines LoVo and SKOSC. IL-2 could also be demonstrated to augment antibody-dependent cellular cytotoxicity (ADCC) against SKOSC targets. IL-2 responsiveness segregated with a non-E-rosetting fraction comprising 11% of postfractionation lymphocytes and containing 94% of the recoverable NK activity, suggesting that IL-2 might operate directly upon the NK cell rather than through an accessory cell. This is believed to be the first demonstration of NK stimulatory activity by the product of a totally synthetic human IL-2 gene. The availability, purity, and NK-enhancing properties of the recombinant IL-2 make it a potentially important agent for clinical trial.

Modulation of human natural killer cell activity by recombinant human interleukin 2.

Recombinant human IL-2, secreted by yeast harboring a plasmid containing a synthetic IL-2 gene, is biologically active in augmenting human natural killer (NK) cell activity. A dose-dependent linear stimulation of NK activity was obtained against the chronic myelogenous leukemia cell line K562 over the range of 3 to 300 units/ml of IL-2. Enhancement of NK activity was similarly demonstrable against the less NK-sensitive carcinoma cell lines LoVo and SKOSC. IL-2 could also be demonstrated to augment antibody-dependent cellular cytotoxicity (ADCC) against SKOSC targets. IL-2 responsiveness segregated with a non-E-rosetting fraction comprising 11% of postfractionation lymphocytes, and containing 94% of the recoverable NK activity, suggesting that IL-2 might operate directly upon the NK cell rather than through an accessory cell. This is believed to be the first demonstration of NK stimulatory activity by the product of a totally synthetic human IL-2 gene. The availability, purity, and NK-enhancing properties of the recombinant IL-2 make it a potentially important agent for clinical trial.

Biosynthetic human EGF accelerates healing of Neodecadron-treated primate corneas.

Topical administration of biosynthetic human epidermal growth factor (h-EGF), given in combination with an antibiotic and synthetic steroid (Neodecadron) accelerated the rate of corneal epithelial regeneration and significantly increased the strength of full-thickness stromal incisions in primates. The regenerated epithelial cells of EGF-Neodecadron-treated corneas appeared normal on histologic examination and showed no evidence of hypertrophy or hyperplasia. The EGF-Neodecadron-treated stromal incisions were characterized by new collagen formation and a smaller epithelial cell plug than Neodecadron-treated control corneas. These results suggest that biosynthetic h-EGF, which lacks the immunologic potential of nonhuman proteins, may be effective in accelerating healing of corneal epithelial defects and stromal incisions in patients whose healing is suppressed by treatment with steroids.

Biologic activities of recombinant human interleukin 2 on murine lymphocytes.

Recombinant human IL-2, produced by yeast cells, was tested in a number of in vitro responses with murine lymphocytes. The responses studied included proliferation of a cloned murine T lymphocyte line, generation of cytotoxic responses, recall of cytotoxic memory, and restoration of responses in spleen cells taken from cyclophosphamide-treated mice. In all cases, the recombinant human IL-2 had the same activity as purified murine IL-2. The recombinant material represents a source of IL-2 free of other lymphokines. The responses described in this work can therefore be ascribed to the direct effects of human IL-2, of known sequence, on the cells of interest.